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Role of Redox State in Mediating Age-Related Changes in Hippocampal Synaptic Transmission, Plasticity and Neuronal Excit...

Permanent Link: http://ufdc.ufl.edu/UFE0041916/00001

Material Information

Title: Role of Redox State in Mediating Age-Related Changes in Hippocampal Synaptic Transmission, Plasticity and Neuronal Excitability
Physical Description: 1 online resource (162 p.)
Language: english
Creator: Bodhinathan, Karthik
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: afterhyperpolarization, aging, hippocampus, redox, synaptic
Neuroscience (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The mechanisms that disrupt normal neuronal function during aging are poorly understood. Due to this fact we do not yet possess a reliable therapeutic strategy to treat age-related memory loss and cognitive dysfunction. Given the central role played by hippocampal CA1 pyramidal neurons in learning and memory, understanding the senescent changes to the biochemical and physiological properties of these neurons has become a necessary first-step in developing effective therapeutic strategies. The hypothesis that forms the basis for this dissertation is that increased oxidative stress or a more oxidative redox state mediates an age-related shift in Ca2+ homeostasis The experiments presented in this dissertation were designed to delineate the age-related changes to the N-methyl D-aspartate receptor (NMDAR) function of CA1 pyramidal neurons. We tested the hypothesis that the age-related decline in NMDAR function was linked to a more oxidative redox state of the neuron. We confirmed that the NMDAR function declines in the CA1 region of aged hippocampus. The results indicate that the intracellular redox state of the aged neurons shifts to a more oxidative environment. The oxidizing agent xanthine/xanthine oxidase (X/XO) decreased the NMDAR mediated synaptic responses at hippocampal CA3-CA1 synapses, in slices from young (3-8 mo), but not aged (20-25 mo) F344 rats. Conversely, the reducing agent dithiothreitol (DTT) selectively enhanced the NMDAR mediated synaptic response in aged but not in young hippocampal slices. The age-dependent sensitivity of the NMDAR function to DTT was associated with facilitated induction of long term potentiation (LTP) in aged but not young animals. Moreover, experiments using membrane impermeable reducing agent L-glutathione (L-GSH) indicated that the NMDAR response was dependent on the intracellular redox state. The effect of DTT was not observed for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (AMPAR). The intracellular redox state dependent effects of DTT on NMDAR function indicated a role for various intracellular signaling cascades. We tested the hypothesis that the DTT-mediated increase in NMDAR function involved intracellular kinases and/or phosphatases. The blockade of DTT effect by H-7 indicated the involvement of Ser/Thr kinase(s) in mediating the increase in NMDAR function. The DTT-mediated increase in NMDAR function was not blocked by Bis-I (a protein kinase C inhibitor), but was blocked by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor - myristoylated autocamtide-2 related inhibitory peptide (myr-AIP), and the general CaM kinase inhibitor KN-62. Furthermore, the inhibition of the activity of protein phosphatases- PP1 and calcineurin had no effect on the DTT-mediated increase in NMDAR function. These results suggest a role for the CaMKII signaling cascade. Our results with CaMKII activity assays established that DTT increases CaMKII activity in CA1 cytosolic extracts from aged but not from young animals. The findings provide a link between intracellular redox state and CaMKII activity during aging, which causes the decline in the NMDAR function, and subsequently impairs synaptic plasticity in the aged hippocampal neurons. Taken together the results provide a link between a hypothesized mechanism of aging (increased oxidative stress) and mechanisms of impaired memory (decreased NMDAR function and impaired synaptic plasticity). We further tested the hypothesis that increased oxidative stress or a more oxidative redox state decreases neuronal excitability of aged neurons by increasing the post burst afterhyperpolarization (AHP). Application of DTT decreased the slow component of afterhyperpolarization (sAHP) in CA1 pyramidal neurons of aged but not young animals. The DTT-mediated decrease in aged-sAHP was blocked by the depletion of intracellular Ca2+ stores (ICS) using thapsigargin or blockade of ryanodine receptor (RyR) by ryanodine. Neither the inhibition of L-type voltage gated calcium channels (L-type VGCC) nor the inhibition of Ser/Thr kinases by H-7 had any effect on the DTT-mediated decrease in aged-sAHP. The results suggest that a more oxidative redox state during aging contributes to RyR oxidation, increases Ca2+ mobilization from the ICS, and increases the sAHP. The results presented in this dissertation link oxidative redox state of aged neurons to decrease in the NMDAR function, and increase in sAHP. These two processes are very potent and functionally significant biomarkers of aging in the hippocampus. Hence the results of this study will have significant impact on the development of therapeutics that can offset senescent changes to the biochemical and physiological properties of hippocampal neurons and provide fundamental insights into the mechanisms that mediate age-related memory loss and neuronal dysfunction.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Karthik Bodhinathan.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Foster, Tom.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041916:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041916/00001

Material Information

Title: Role of Redox State in Mediating Age-Related Changes in Hippocampal Synaptic Transmission, Plasticity and Neuronal Excitability
Physical Description: 1 online resource (162 p.)
Language: english
Creator: Bodhinathan, Karthik
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: afterhyperpolarization, aging, hippocampus, redox, synaptic
Neuroscience (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The mechanisms that disrupt normal neuronal function during aging are poorly understood. Due to this fact we do not yet possess a reliable therapeutic strategy to treat age-related memory loss and cognitive dysfunction. Given the central role played by hippocampal CA1 pyramidal neurons in learning and memory, understanding the senescent changes to the biochemical and physiological properties of these neurons has become a necessary first-step in developing effective therapeutic strategies. The hypothesis that forms the basis for this dissertation is that increased oxidative stress or a more oxidative redox state mediates an age-related shift in Ca2+ homeostasis The experiments presented in this dissertation were designed to delineate the age-related changes to the N-methyl D-aspartate receptor (NMDAR) function of CA1 pyramidal neurons. We tested the hypothesis that the age-related decline in NMDAR function was linked to a more oxidative redox state of the neuron. We confirmed that the NMDAR function declines in the CA1 region of aged hippocampus. The results indicate that the intracellular redox state of the aged neurons shifts to a more oxidative environment. The oxidizing agent xanthine/xanthine oxidase (X/XO) decreased the NMDAR mediated synaptic responses at hippocampal CA3-CA1 synapses, in slices from young (3-8 mo), but not aged (20-25 mo) F344 rats. Conversely, the reducing agent dithiothreitol (DTT) selectively enhanced the NMDAR mediated synaptic response in aged but not in young hippocampal slices. The age-dependent sensitivity of the NMDAR function to DTT was associated with facilitated induction of long term potentiation (LTP) in aged but not young animals. Moreover, experiments using membrane impermeable reducing agent L-glutathione (L-GSH) indicated that the NMDAR response was dependent on the intracellular redox state. The effect of DTT was not observed for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (AMPAR). The intracellular redox state dependent effects of DTT on NMDAR function indicated a role for various intracellular signaling cascades. We tested the hypothesis that the DTT-mediated increase in NMDAR function involved intracellular kinases and/or phosphatases. The blockade of DTT effect by H-7 indicated the involvement of Ser/Thr kinase(s) in mediating the increase in NMDAR function. The DTT-mediated increase in NMDAR function was not blocked by Bis-I (a protein kinase C inhibitor), but was blocked by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor - myristoylated autocamtide-2 related inhibitory peptide (myr-AIP), and the general CaM kinase inhibitor KN-62. Furthermore, the inhibition of the activity of protein phosphatases- PP1 and calcineurin had no effect on the DTT-mediated increase in NMDAR function. These results suggest a role for the CaMKII signaling cascade. Our results with CaMKII activity assays established that DTT increases CaMKII activity in CA1 cytosolic extracts from aged but not from young animals. The findings provide a link between intracellular redox state and CaMKII activity during aging, which causes the decline in the NMDAR function, and subsequently impairs synaptic plasticity in the aged hippocampal neurons. Taken together the results provide a link between a hypothesized mechanism of aging (increased oxidative stress) and mechanisms of impaired memory (decreased NMDAR function and impaired synaptic plasticity). We further tested the hypothesis that increased oxidative stress or a more oxidative redox state decreases neuronal excitability of aged neurons by increasing the post burst afterhyperpolarization (AHP). Application of DTT decreased the slow component of afterhyperpolarization (sAHP) in CA1 pyramidal neurons of aged but not young animals. The DTT-mediated decrease in aged-sAHP was blocked by the depletion of intracellular Ca2+ stores (ICS) using thapsigargin or blockade of ryanodine receptor (RyR) by ryanodine. Neither the inhibition of L-type voltage gated calcium channels (L-type VGCC) nor the inhibition of Ser/Thr kinases by H-7 had any effect on the DTT-mediated decrease in aged-sAHP. The results suggest that a more oxidative redox state during aging contributes to RyR oxidation, increases Ca2+ mobilization from the ICS, and increases the sAHP. The results presented in this dissertation link oxidative redox state of aged neurons to decrease in the NMDAR function, and increase in sAHP. These two processes are very potent and functionally significant biomarkers of aging in the hippocampus. Hence the results of this study will have significant impact on the development of therapeutics that can offset senescent changes to the biochemical and physiological properties of hippocampal neurons and provide fundamental insights into the mechanisms that mediate age-related memory loss and neuronal dysfunction.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Karthik Bodhinathan.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Foster, Tom.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041916:00001


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ROLE OF REDOX STATE IN MEDIATING AGE-RELATED CHANGES IN
HIPPOCAMPAL SYNAPTIC TRANSMISSION, PLASTICITY AND NEURONAL
EXCITABILITY



















By

KARTHIK BODHINATHAN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2010


































2010 Karthik Bodhinathan
































To my family, my friends, and Ramana









ACKNOWLEDGMENTS

I thank my parents Rajeswari Bodhinathan and Bodhinathan Sundarapandian, for

letting me chase my dreams. Special thanks to my little sister Soundharya Pradha, for

her prayers. I thank my mentor Dr Thomas Foster for his constant encouragement and

able guidance. I would also like to thank my committee members, Drs. Christiaan

Leeuwenburgh, Harry Nick, and Charles Frazier, and the Neuroscience program

director Dr. Susan Semple-Rowland for their helpful comments and suggestions

throughout my time at graduate school. My scientific pursuits would not have been

possible without the guidance of past mentors, Drs. V. Ramamurthy and Saumitra Das,

and the words of Ramana Maharishi and Dr. Abdul Kalam. I also express my gratitude

to the past and current members of Foster lab Dr. Ashok Kumar, Dr. Zane Zeier, Dr.

Kristina Aenlle, Travis Jackson, Asha Rani, Wei-Hua Lee, Olga Tchigrinova, Michael

Guidi, and Sylvia. This dissertation would not have been possible without the support of

Sunitha Rangaraju, Emalick Njie, and numerous friends. Special thanks to Ms. Betty J.

Streetman at the Neuroscience office, and Ms. Valerie Cloud-Driver at the IDP program.

Finally, I would like to thank the Alumni Graduate Program for their four year fellowship

that made a doctor and neuroscientist out of a kid who had the dreams but not the

means.









TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ........... ............... ....... ...........................

LIST O F TA BLES ......... ................ ..................... ...... ............... 8

L IS T O F F IG U R E S .......................................................................................................... 9

LIST OF ABBREVIATIONS......................................... ............... 11

A B S T R A C T .............. ..... ............ ................. .................................................. 1 5

CHAPTER

1 INTRODUCTION .................. ...... ......... ......... 18

Learning and Memory ........................ ......... ......... 18
Aging Effects on H ippocam pus ...................................................................... 19
Learning and Memory Systems Dependent on Hippocampus ............................ 20
Neuroanatom y of Hippocam pus ................................ ........ ..... .................... 22
Aging Effects on NMDAR Mediated Synaptic Transmission............................... 23
Synaptic Transmission and Plasticity in CA1 Pyramidal Neurons........................ 25
lonotropic Glutamatergic Transmission................. .. ..................... 26
NMDA Receptor Dependent Synaptic Plasticity: LTP and LTD ........................... 29
Afterhyperpolarization in CA1 Pyramidal Neurons ......................................... 31
Calcium Homeostasis in CA1 Neurons............... ................................. ........... 34
R e d ox S tate a nd A g ing ............... .................................. ........ ............... 36
S u m m a ry ...................................................................... .. .............. 3 9

2 M ATERIALS AND M ETHO DS ................................. ........................................ 41

Drugs, Solutions and Suppliers................. ......................... 41
A nim al Procedures ............... ........................... ....... ......... ............ 41
Hippocampal Tissue Dissection for Electrophysiological Experiments ............... 42
Electrophysiological Recordings: Extracellular Field Potentials........................... 42
Extracellular Field Potentials: Data Analysis .............................. ... ................ 43
Long-Term Potentiation and Paired-Pulse Ratio Recordings........................ 44
Isolation of NMDAR Mediated Extracellular Synaptic Potentials .................. 45
Electrophysiological Recordings: Intracellular Sharp Microelectrode Recording .... 45
Intracellular Synaptic Potentials: Data Analysis.................................. 46
Intracellular Afterhyperpolarization: Data Analysis ................ ............ ....... 47
Measurement of ROS in Hippocampal Slices................ ..... ................. 48
CaMKII Activity Assay........................... .................. .............. 49
Statistical Methods for Analysis of Data..... ................. ..... ........... 49









3 REDOX STATE DEPENDENT CHANGES IN NMDA RECEPTOR MEDIATED
SYNAPTIC TRANSMISSION IN AGED HIPPOCAMPUS.............. ........... 53

Introduction ......... .. .. ................................................................ ... 53
R e s u lts .................... .. ......... ....... .... ... .. .............. ............ ........................ 5 4
NMDA Receptor Function Decreases in the Hippocampus of Aged Animals
at Various Levels of Pre-synaptic Fiber Volley Amplitude............................. 54
Oxidizing Agents Decrease NMDAR Function in Young, but not in Aged,
H ip p o ca m p a l S lice s .................................. ............................. ............ 5 5
NMDAR Function in Young Animals Recovers From Exposure to Higher
Concentrations of Oxidizing Agents ................................. ........ .......... ..... 56
Reducing Agents Increase NMDAR Function Selectively in Aged
H ip p o c a m p u s ............................................................................................. 5 7
Intracellular Location of Redox Sensitive Cysteines Revealed by Differential
Application of Biologically Available Reducing Agent L-Glutathione ............. 58
Reducing Agent Mediated Recovery of NMDAR Function is Reversed by
Oxidizing Agent, and Specific to NMDARs......................... .... ........... 59
D discussion ............. ... .... ................................ ........ ... ..... ......... 61

4 MOLECULAR MECHANISM UNDERLYING RECOVERY OF NMDAR
FUNCTION AND HIPPOCAMPAL SYNAPTIC PLASTICITY IN AGED ANIMALS. 75

Introduction ............... ...... ........................... ........ ......... 75
R e su lts ................ .. ....... .......... ............. .......... ............ .. 76
ROS Sensitive Dye Indicates Redox State of Live Hippocampal Neurons in
in vitro Slices ................ ........... .... ...................... .... 76
Enhanced ROS Production in the CA1 Region of the Hippocampus of Aged
A n im a ls ............ ......... .......... ............................... ... ................ 78
Broad Spectrum Ser/Thr Kinase Inhibitor Blocks DTT-Mediated Recovery of
NMDAR Function in Aged Hippocampal Neurons...................................... 79
CaM Kinase II specific Inhibitors Block DTT-Mediated Recovery of NMDAR
Function in Aged Hippocampal Neurons ................ ....... ............................ 80
DTT-Mediated Recovery of NMDAR Function in Aged Animals is
Independent of Neuronal Protein Phosphatases........................................ 81
Long-Term Potentiation is Enhanced in Aged Hippocampal Slices Exposed
to D T T .......... ........ ........ ................................................ ......................... 8 2
Reducing Agent does not Alter Long-Term Potentiation in Young
H ippocam pal S lices....................................... ...... ............................. 83
CaMKII Activity is Enhanced in Aged Hippocampal CA1 Cytosolic Extracts
Treated with DTT ............... ....... .. ........... ... ........ 83
DTT does not Alter CaMKII Activity in Young Hippocampal CA1 Cytosolic
Extracts .............. ...... ............ ...... .... ........ ... ...... ...... 84
D discussion ............. ... .... ................................ ........ ... ..... ......... 85

5 REDOX MODULATION MEDIATES REDUCTION IN NEURONAL
AFTERHYPERPOLARIZATION OF AGED HIPPOCAMPAL NEURONS............. 100









In tro d u c tio n ................... ...................1...................0.........0
R results ...... .................................... ................................. .............. 10 1
Age Dependent Decrease in the sAHP Following DTT Application............... 101
DTT Mediated Decrease in Aged-sAHP Involves Intracellular Calcium
Stores and Ryanodine Receptors .............................................................. 102
DTT Mediated Reduction in the Aged-sAHP is Independent of L-VGCC ....... 103
D isc u ss io n ............. ......... .. .. ......... .. .. ......... .................................. 10 5

6 CONCLUSION AND FUTURE DIRECTIONS..................... .... .......... ..... 117

C o nclusio n ....................... .. ........................................................... 1 17
Therapeutic Potential of the Current Study .............................................. 124
F u tu re D ire c tio n s ............... ................................................................................. 1 2 5

APPENDIX

A DRUGS, SOLUTUIONS, AND SUPPLIERS................ ..... ............... 131

B DRUG CONCENTRATIONS USED IN THE EXPERIMENTS ........................... 133

LIST OF REFERENCES .......... ............ ......... ................ ............... 135

BIOGRAPHICAL SKETCH .............. ........... ............................. 162









LIST OF TABLES

Table page

3-1 The NMDAR-fEPSPs from hippocampus of young and aged animals ............ 74

4-1 Paired-pulse ratios from aged animals ............... ............................................ 99

5-1 Physiological properties of CA1 neurons from young and aged animals......... 116









LIST OF FIGURES


Figure page

1-1 C calcium hom eostasis in the neuron.............................................. ... .................. 40

2-1 Hippocampal dissection and setup for electrophysiological recordings ............. 51

2-2 Analysis of electrophysiological signals from hippocampal slices................... 52

3-1 NMDAR mediated synaptic potentials (NMDAR-fEPSP) are reduced in area
CA 1 of the hippocam pus during aging................................... ..................... 65

3-2 The oxidizing agent X/XO decreases NMDAR mediated synaptic potentials in
young animals but not in aged animals ..... ................ .................. 66

3-3 Effect of maximal concentrations of X/XO on NMDAR mediated synaptic
potentials in young animals ........................................ ..................... 67

3-4 The reducing agent DTT increases NMDAR mediated synaptic responses to
a greater extent in aged than in the young animals........................... ......... 68

3-5 Extracellular application of reduced L-glutathione does not affect NMDAR
fu nctio n ................ ................................... ........................... 6 9

3-6 Intracellular application of reduced L-glutathione enhances intracellular
NMDAR mediated synaptic potentials ................................................ .......... 70

3-7 Glutathione mediated recovery of NMDAR function in aged animals does not
involve L-type VG CC ......... ......... ................................................. ...... 71

3-8 Redox modification of cysteine residues underlies NMDAR specific effect of
D T T ................ .................................. ........................... 7 2

3-9 DTT does not affect the AMPAR function of aged animals.............................. 73

4-1 Detection of ROS in live hippocampal slices ................................................. 89

4-2 Detection of auto-fluorescence from dye-unexposed hippocampal slices.......... 90

4-3 Enhanced ROS production is observed in hippocampal tissue from aged rats.. 91

4-4 A Serine/Threonine (Ser/Thr) kinase, but not protein kinase C, mediates DTT
mediated increase in NMDAR function in aged hippocampus.......................... 92

4-5 CaMKII involvement in the DTT mediated enhancement of NMDAR synaptic
responses in aged anim als ......... ............................................. ............... 93









4-6 Calcineurin/PP2B and PP1 are not involved in the DTT mediated
enhancement of NMDAR synaptic responses in aged animals....................... 94

4-7 DTT enhances LTP in hippocampal area CA1 of aged animals...................... 95

4-8 DTT does not alter the LTP in hippocampal area CA1 of young animals ......... 96

4-9 DTT enhances CaMKII activity in aged hippocampal CA1 cytosolic extracts..... 97

4-10 DTT does not enhance CaMKII activity in young hippocampal CA1 cytosolic
extracts ................ ................................... ........................... 98

5-1 Age-dependent reduction in the sAHP by DTT................................... ......... 109

5-2 Intracellular calcium stores underlie DTT-mediated decrease in aged-sAHP... 110

5-3 RyR blockade inhibits DTT mediated decrease in aged-sAHP...................... 111

5-4 RyR blockade inhibits the DTT-mediated decrease in aged-sAHP when the
AHP is increased by increasing calcium in the recording medium.................. 112

5-5 DTT mediated decrease is independent of L-type calcium channel function.... 113

5-6 DTT effects on aged-sAHP are independent of BK channel function .............. 114

5-7 Ser/Thr kinase activity does not mediate DTT effects on aged-sAHP .............. 115

6-1 The biochemical model of brain aging and hippocampal dysfunction............... 128

6-2 Conceptual framework for age-related neuronal dysfunction based on
intracellular calcium levels ...... ............................... .................... 129

6-3 Integrative model of the impact of aging on the calcium handling mechanisms
and physiological processes.............................. ............... 130











C

xg

+

AC

ACSF

ADP

ad lib

AHP

ANOVA

AMPAR



AP-5

APamp

ATP

BCA assay



Ca2+

[Cai]



CA1

CA3

CaMKII

CaN


LIST OF ABBREVIATIONS

Degree Celsius (unit for expressing temperature)

g-force (unit for expressing relative centrifugal force)

plus-minus sign (symbol for variability around a value)

Alternating current

Artificial Cerebro Spinal Fluid

Adenosine diphosphate

ad libitum

Afterhyperpolarization

Analysis of Variance

Alpha-amino-3-hydroxy-5-Methyl-4-isoxazole Propionic Acid

Receptor

2-Amino-5-Phosphonovaleric acid

Action Potential Amplitude (expressed in milli volts)

Adenosine-5'-triphosphate

Bicinchoninic acid assay (a method to determine total protein levels

in a sample)

Calcium (ionic form)

Intracellular Calcium concentration; usually expressed in

nanomoles to micromoles

Cornu Ammonis Area 1

Cornu Ammonis Area 3

Ca 2/Calmodulin-Dependent Protein Kinase II

Calcineurin









Cat

DC

DG

DNA

DTNB

DTT

EC

EPSP

F(x, y)

F344

fEPSP

Fisher's PLSD

GABA

GSSG

H202

HFS

Hz

ICS

kHz

L-GSH

L-type VGCC

LTD

LTP


Catalase (Enzyme)

Direct current

Dentate Gyrus

Deoxyribonucleic acid (genetic material of living cells)

5, 5'-dithiobis (2-nitrobenzoic acid)

Dithiothreitol

Entorhinal Cortex

Excitatory Post Synaptic Potential

F-test statistic value of F-distribution

Fischer 344 (strain of rat commonly used in aging studies)

Field Excitatory Post Synaptic Potential

Fisher's Protected Least Significant Difference

Gama Amino butyric acid

Glutathione Disulfide (a dimer of two glutathione molecules)

Hydrogen peroxide

High Frequency Stimulation

Hertz (unit for representing frequency of periodic events)

Intracellular Calcium Stores

Kilo Hertz (1000 Hertz)

L-Glutathione

L-type Voltage gated calcium channel

Long-Term Depression

Long-Term Potentiation









pm

pM

Ps

M

MO

mg

Mg2+

min

mL

mm

mM

mo

mV

mU

myr-AIP

N

nA

Na+

nm

NMDAR

NMDAR-fEPSP

02-

OA


Micro Meter (1/1000000 of a meter; unit of length)

Micro Molar (unit for representing concentration of solutions)

Microsecond (1/1000000 of a second; unit of time)

Molarity (unit for representing concentration of solutions)

Mega Ohms (1000000 Ohms; unit of electrical resistance)

Milli Grams (1/1000 of a gram; unit of mass)

Magnesium (ionic form)

Minutes (unit of time)

Milli Liters (1/1000 of a liter; unit of volume)

Milli Meter (1/1000 of a meter; unit of length)

Milli Molar (1/1000 of a Mol; unit for representing concentration)

Months Old (Ex. 24 mo means 24 months old)

Milli Volt (1/1000 of a Volt; unit of representing potential difference)

Milli Units (1/1000 of a Unit of enzymatic activity)

Myristoylated Autocamtide-2 Related Inhibitory Peptide

Normality (unit for representing concentration of solute in solution)

Nano Ampere (1/1000000000 of an Ampere; unit for current)

Sodium (ionic form)

Nano meters (1/1000000000 of a meter; unit of length)

N-methyl D-aspartate Receptor

NMDAR Mediated Field Excitatory Post Synaptic Potential

Superoxide anion

Okadaic Acid









OH- Hydroxyl Radical

PFV Pre synaptic Fiber Volley

pH Measure of acidity or basicity of solutions

PKC Protein Kinase C

post hoc Post Hoc Ergo Propter Hoc (Latin for "after this")

PP1 Protein Phosphatase Type 1

PP2B Protein Phosphatase Type 2B

Rin Input Resistance

RNA Ribonucleic acid

ROS Reactive Oxygen Species

RyR Ryanodine Receptor

S.E.M Standard Error of the Mean

s.or Stratum Oriens

s.pyr Stratum Pyramidale

s.rad Stratum Radiatum

SC Schaffer collateral

sec Seconds (unit of time)

Ser/Thr Serine/Threonine

SOD Superoxide Dismutase (Enzyme)

Vm Resting Membrane Potential (expressed in milli volts)

X/XO Xanthine/Xanthine Oxidase









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ROLE OF REDOX STATE IN MEDIATING AGE-RELATED CHANGES IN
HIPPOCAMPAL SYNAPTIC TRANSMISSION, PLASTICITY AND NEURONAL
EXCITABILITY

By

Karthik Bodhinathan

August 2010

Chair: Thomas C Foster
Major: Medical Sciences Neuroscience

The mechanisms that disrupt normal neuronal function during aging are poorly

understood. Due to this fact we do not yet possess a reliable therapeutic strategy to

treat age-related memory loss and cognitive dysfunction. Given the central role played

by hippocampal CA1 pyramidal neurons in learning and memory, understanding the

senescent changes to the biochemical and physiological properties of these neurons

has become a necessary first-step in developing effective therapeutic strategies. The

hypothesis that forms the basis for this dissertation is that increased oxidative stress or

a more oxidative redox state mediates an age-related shift in Ca2+ homeostasis The

experiments presented in this dissertation were designed to delineate the age-related

changes to the N-methyl D-aspartate receptor (NMDAR) function of CA1 pyramidal

neurons. We tested the hypothesis that the age-related decline in NMDAR function was

linked to a more oxidative redox state of the neuron. We confirmed that the NMDAR

function declines in the CA1 region of aged hippocampus. The results indicate that the

intracellular redox state of the aged neurons shifts to a more oxidative environment. The

oxidizing agent xanthine/xanthine oxidase (X/XO) decreased the NMDAR mediated









synaptic responses at hippocampal CA3-CA1 synapses, in slices from young (3-8 mo),

but not aged (20-25 mo) F344 rats. Conversely, the reducing agent dithiothreitol (DTT)

selectively enhanced the NMDAR mediated synaptic response in aged but not in young

hippocampal slices. The age-dependent sensitivity of the NMDAR function to DTT was

associated with facilitated induction of long term potentiation (LTP) in aged but not

young animals. Moreover, experiments using membrane impermeable reducing agent

L-glutathione (L-GSH) indicated that the NMDAR response was dependent on the

intracellular redox state. The effect of DTT was not observed for the alpha-amino-3-

hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (AMPAR).

The intracellular redox state dependent effects of DTT on NMDAR function

indicated a role for various intracellular signaling cascades. We tested the hypothesis

that the DTT-mediated increase in NMDAR function involved intracellular kinases and/or

phosphatases. The blockade of DTT effect by H-7 indicated the involvement of Ser/Thr

kinase(s) in mediating the increase in NMDAR function. The DTT-mediated increase in

NMDAR function was not blocked by Bis-I (a protein kinase C inhibitor), but was

blocked by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor -

myristoylated autocamtide-2 related inhibitory peptide (myr-AIP), and the general CaM

kinase inhibitor KN-62. Furthermore, the inhibition of the activity of protein

phosphatases- PP1 and calcineurin had no effect on the DTT-mediated increase in

NMDAR function. These results suggest a role for the CaMKII signaling cascade. Our

results with CaMKII activity assays established that DTT increases CaMKII activity in

CA1 cytosolic extracts from aged but not from young animals. The findings provide a

link between intracellular redox state and CaMKII activity during aging, which causes









the decline in the NMDAR function, and subsequently impairs synaptic plasticity in the

aged hippocampal neurons. Taken together the results provide a link between a

hypothesized mechanism of aging (increased oxidative stress) and mechanisms of

impaired memory (decreased NMDAR function and impaired synaptic plasticity).

We further tested the hypothesis that increased oxidative stress or a more

oxidative redox state decreases neuronal excitability of aged neurons by increasing the

post burst afterhyperpolarization (AHP). Application of DTT decreased the slow

component of afterhyperpolarization (sAHP) in CA1 pyramidal neurons of aged but not

young animals. The DTT-mediated decrease in aged-sAHP was blocked by the

depletion of intracellular Ca2+ stores (ICS) using thapsigargin or blockade of ryanodine

receptor (RyR) by ryanodine. Neither the inhibition of L-type voltage gated calcium

channels (L-type VGCC) nor the inhibition of Ser/Thr kinases by H-7 had any effect on

the DTT-mediated decrease in aged-sAHP. The results suggest that a more oxidative

redox state during aging contributes to RyR oxidation, increases Ca2+ mobilization from

the ICS, and increases the sAHP.

The results presented in this dissertation link oxidative redox state of aged

neurons to decrease in the NMDAR function, and increase in sAHP. These two

processes are very potent and functionally significant biomarkers of aging in the

hippocampus. Hence the results of this study will have significant impact on the

development of therapeutics that can offset senescent changes to the biochemical and

physiological properties of hippocampal neurons and provide fundamental insights into

the mechanisms that mediate age-related memory loss and neuronal dysfunction.









CHAPTER 1
INTRODUCTION

Learning and Memory

The mammalian brain is endowed with the amazing capacity for learning new

information that can be stored as memories. It is fascinating that the brain possesses

the unique capacity for learning and memory, in addition to being the seat of a wide

variety of human faculties (Crick, 1995). Certain regions of the brain are designed to

take part in distinct forms of learning and memory, for example, the hippocampus

located in the medial temporal lobe of the brain is involved in the formation and retrieval

of declarative/explicit memory. The two main classes of cells in the hippocampus that

possess unique properties (Kupfermann et al., 2000), and enable the learning and

memory function of the brain are the neurons and the glial cells. The neurons are the

predominant type of signaling cells, which communicate through chemical

neurotransmitters and receptors. The glial cells are the supporting cells, which provide

nutrition and recycle neurotransmitters released by the neurons (Kandel, 1991, 2000a;

Alberts et al., 2002). The learning and memory function of the hippocampus is

accomplished by the utilization of a complex array of molecules and signaling

mechanisms present in these cells.

The studies presented in this dissertation were designed to test the hypothesis

that an increased oxidative stress or a more oxidative redox state mediates age-related

shift in Ca2+ homeostasis and contributes to neuronal dysfunction. Neuronal function is

defined by the neuron's synaptic transmission, plasticity and excitability (described in

detail in the following sections). Our experiments were designed to test the effects of

redox state modulators on important markers of neuronal function, namely the synaptic









transmission mediated by the N-methyl D-aspartate receptors (NMDARs), synaptic

plasticity and neuronal excitability. Before describing the results in the following

chapters, an overview of the key components of the hypothesis is provided in the

following sections of this introduction.

Aging Effects on Hippocampus

The hippocampal function is particularly vulnerable to dysfunction during aging.

The National Institute on Aging (NIA) has identified cognitive impairment due to memory

dysfunction as a normal part of aging. In particular, the hippocampal function is impaired

in aged animals such that they learn slower and forget easily (Barnes, 1979; Foster,

1999). In this context it is worthwhile to differentiate the effects of normal aging on

hippocampus-dependent memory function and the effects arising from

neurodegenerative processes. The pattern of changes to hippocampus during normal

aging is different from that observed in individuals suffering from neurodegenerative

disorders like Alzheimer's disease (AD). The primary difference seems to be the

absence of major neuron loss in people with normal aging. Although initial data

suggested neuronal loss during aging (Ball, 1977; Brizzee et al., 1980; Coleman and

Flood, 1987), subsequent studies have conclusively proved that no significant neuron

loss is observed (West et al., 1994; Rapp and Gallagher, 1996; Rasmussen et al., 1996;

Gazzaley et al., 1997; Morrison and Hof, 1997; Pakkenberg and Gundersen, 1997;

Merrill et al., 2001). Furthermore, the loss of memory function, due to neuron loss, is

associated with neurodegenerative disorders but not normal aging (Rapp and

Gallagher, 1996; Rasmussen et al., 1996). In other words, the basic elements required

for hippocampus dependent learning and memory seems to be intact during normal

aging; however these systems are progressively weakened in their function.









The absence of neuron loss indicates that the hippocampus does not exhibit an

"anatomical lesion" during aging. Nevertheless, the memory systems dependent on

hippocampus become dysfunctional during aging, such that aged animals learn slower

and forget rapidly, as noted above (Barnes, 1979; Dunnett et al., 1990; Mabry et al.,

1996; Oler and Markus, 1998; Foster, 1999; Norris and Foster, 1999). Based on the

evidence presented in the following chapters, it is becoming increasingly clear that the

neurobiological correlates of memory loss during normal aging are subtle physiological,

biochemical and posttranslational changes accumulated in the hippocampal neurons.

These lines of evidence support the idea of a "functional lesion" of hippocampus during

aging characterized neuronal dysfunction.

Learning and Memory Systems Dependent on Hippocampus

In chapters 3, 4 and 5 we have presented results that suggest that increased

oxidative stress or more oxidative redox state contributes to the physiological and

biochemical changes in the hippocampal CA1 pyramidal neurons during aging. In order

to better understand the functional significance of these results, a brief overview of the

memory systems in the brain and a detailed overview of the role of CA1 pyramidal

neurons in hippocampus dependent learning and memory are provided in the following

section.

Although several memory systems are thought to utilize the neural networks of

hippocampus (Riedel et al., 1999), the declarative/explicit memory system is particularly

dependent on the intact functioning of the hippocampus (Eichenbaum, 1997; Mingaud

et al., 2007). Declarative memory includes semantic and episodic memory (Squire and

Zola, 1998). Semantic memory is the capacity to store general knowledge and recollect

factual information (Squire and Zola, 1998; Kapur and Brooks, 1999; Holdstock et al.,









2002; Manns et al., 2003) and episodic memory is the capacity to store and recollect

information about time, places and their context in a temporal order (Vargha-Khadem et

al., 1997; Tulving and Markowitsch, 1998). Models of memory impairment in nonhuman

primates (Mishkin, 1982; Squire et al., 2004), combined with extensive behavioral

characterization (Zola-Morgan et al., 1994) has indicated that hippocampal pyramidal

neurons are necessary for the acquisition and consolidation of declarative memory;

while long-term storage occurs in the neocortical regions of the brain (McClelland et al.,

1995; McClelland and Goddard, 1996; Eichenbaum, 2000; Fell et al., 2001; Kali and

Dayan, 2004). The hippocampal CA1 pyramidal neurons mediate memory consolidation

(Shimizu et al., 2000; Remondes and Schuman, 2004; Frankland and Bontempi, 2005;

Ji and Wilson, 2007; Takehara-Nishiuchi and McNaughton, 2008), and the retrieval or

recollection of recently formed memories (Gabrieli et al., 1997; Roozendaal et al., 2001;

Smith and Squire, 2009). All these observations support the idea that the CA1

pyramidal neurons of the hippocampus play a crucial role in the formation, consolidation

and retrieval of declarative/explicit memories in the mammalian brain. The hippocampal

formation is also important for the acquisition of spatial memory, which denotes the

capacity to store and retrieve information regarding the spatial location and relative

orientation of objects (O'Keefe, 1993; O'Keefe and Burgess, 1996; Nakazawa et al.,

2004; McNaughton et al., 2006). Spatial information is represented in the hippocampus

through alterations in the firing properties of the CA1 pyramidal neurons in the

hippocampus (O'Keefe and Speakman, 1987; Foster et al., 1989).

The intricate anatomy of the hippocampus enables its learning and memory

function. One of the ideas used in constructing the hypotheses in chapters 3, 4, and 5









has been that age-related changes to the learning and memory function of the

hippocampus arises, in part, from the physiological and biochemical changes to the

CA1 pyramidal neurons. Since all studies presented in this dissertation concern the CA1

pyramidal neurons, a brief overview of the hippocampal neuroanatomy and the

organization of the CA1 pyramidal neurons within the hippocampal CA1 subfield are

provided below.

Neuroanatomy of Hippocampus

The hippocampus is a sea horse shaped structure located in the medial temporal

lobe of the brain. It is part of the hippocampal formation in the medial temporal lobe,

which includes the entorhinal cortex (EC), the subiculum, the presubiculum and the

parasubiculum. The hippocampus is divided into three major subfields: the CA1 region,

the CA3 region, and the dentate gyrus (DG). The abbreviation CA stands for cornu

ammonis, due to its semblance to a ram's horn. The experiments described in the

following chapters were all designed to study the synaptic transmission and plasticity in

the CA3-CA1 synaptic contacts, which are part of the tri-synaptic pathway. In the tri-

synaptic pathway, the first set of synaptic contacts occur between the axonal afferents

from EC onto the DG pyramidal neurons, the second set of synaptic contacts between

the afferents from DG pyramidal neurons onto the CA3 pyramidal neurons, and the third

set of synaptic contacts between the afferents from the CA3 pyramidal neurons onto the

CA1 pyramidal neurons (Kandel, 2000b; Amaral and Lavenex, 2007). In addition to the

tri-synaptic connection between the principal pyramidal cells of the hippocampus, there

are many recurrent and interneuronal connections in all the major subfields of the

hippocampus thus providing a massive, yet organized, network of neurons.









The subfields of hippocampus are further distinguished into various layers which

reflect the underlying laminar organization, orientation and location of the principal

pyramidal cells. The CA1 subfield is distinguished into stratum lacunosum molecular

(s.l.m), stratum radiatum (s.r), stratum pyramidale (s.p or the pyramidal cell layer), and

stratum oriens (s.o) (Amaral and Lavenex, 2007). The dendrites of the CA1 pyramidal

neurons are located in s.l.m and s.r; the cell body is located in s.p; and axon passes

through s.o. In addition, the DG subfield is distinguished into the molecular layer, the

granule cell layer and the polymorphic cell layer, and the CA3 subfield is distinguished

into stratum radiatum, stratum lucidum, stratum pyramidale (or pyramidal cell layer) and

stratum oriens.

The function of the CA1 pyramidal neurons is defined by the neuron's synaptic

transmission, plasticity and excitability. In chapters 3, 4, and 5 we tested the hypothesis

that a shift in the redox state to a more oxidative environment contributes to neuronal

dysfunction by altering synaptic transmission, plasticity and excitability. In order to

clarify the key components of this hypothesis, a brief description of the above

mentioned parameters of CA1 pyramidal neurons is provided in the following sections.

Aging Effects on NMDAR Mediated Synaptic Transmission

The decline in NMDAR function is thought to be one of the critical biomarkers of

aging in CA1 pyramidal neurons (Foster, 2006), which is also supported by previous

reports (Barnes et al., 1997; Billard and Rouaud, 2007). In chapter 3, we tested the

hypothesis that age-related decline in NMDAR function is caused by increased oxidative

stress or a more oxidative redox state. In order to better understand the hypothesis and

results, a brief description of the properties and function of NMDARs in the CA1

pyramidal neurons and their role in synaptic transmission and plasticity is provided in









the following sections. But first, an evaluation of the age-related changes to NMDAR

function is discussed.

The NMDAR mediated synaptic transmission is critical for acquisition and

consolidation of hippocampus dependent spatial learning and memory (Bannerman et

al., 1995). As noted above, there is considerable evidence to indicate that aging is

associated with a decline in NMDAR function within regions involved in processing and

performing higher brain function including learning and memory (Gonzales et al., 1991;

Pittaluga et al., 1993; Barnes et al., 1997; Magnusson, 1998; Eckles-Smith et al., 2000;

Gore et al., 2002; Liu et al., 2008; Zhao et al., 2009). Perhaps the strongest evidence

for a reduction in NMDAR function comes from physiological studies which indicate that

the NMDAR mediated excitatory post synaptic potentials in the CA3-CA1 synapses of

the hippocampus are reduced by approximately 50% in aged animals (Barnes et al.,

1997; Eckles-Smith et al., 2000; Bodhinathan et al., 2010). However, age-related

changes in the amplitude of NMDA-evoked responses were not observed in dissociated

cortical neurons suggesting the possibility of regional specificity in the loss of NMDAR

function (Kuehl-Kovarik et al., 2003). Several studies indicate a decrease in the level of

NMDAR protein expression in the hippocampus during aging (Bonhaus et al., 1990; Kito

et al., 1990; Miyoshi et al., 1991; Tamaru et al., 1991; Wenk et al., 1991; Magnusson,

1995; Magnusson et al., 2006; Billard and Rouaud, 2007; Das and Magnusson, 2008;

Liu et al., 2008; Zhao et al., 2009). Moreover, the decrease has been localized to area

CA1 of the hippocampus (Magnusson and Cotman, 1993; Gazzaley et al., 1996;

Magnusson, 1998; Wenk and Barnes, 2000); wherein the studies report reduced

binding of [3H] glutamate (agonist site), [3H] glycine (NR1 site), [3H] CPP (a competitive









antagonist to the L-glutamate binding site), and [3H] MK-801 (an open channel blocker)

in the hippocampus of aged rats. However, others have reported no age-related change

in antagonist binding (Kito et al., 1990; Miyoshi et al., 1991; Araki et al., 1997; Shimada

et al., 1997), or an increased MK-801 binding in animals with learning and retention

deficits (Ingram et al., 1992; Topic et al., 2007). It is important to note that MK-801 binds

to the hydrophobic channel domain of NMDAR, exclusively labeling open channels.

Thus, an apparent increase in NMDAR channel open time may act as a compensatory

mechanism for the decrease in receptor number (Serra et al., 1994). However, the

majority of reports, including our recent findings, indicate that the net function of the

NMDARs decreases at CA3-CA1 hippocampal synaptic contacts during aging

(Bodhinathan et al., 2010). Thus, our working hypothesis is based on the idea that the

age-related changes in the NMDAR function are predominantly posttranslational,

probably involving oxidation/reduction and/or changes in phosphorylation. Before

describing the results which indicate a link between oxidative redox state and decrease

in NMDAR function during aging, a brief overview of the general properties of synaptic

transmission and plasticity involving the CA1 pyramidal neurons is provided in the

following section.

Synaptic Transmission and Plasticity in CA1 Pyramidal Neurons

Information is transmitted between neurons through the synapses (a narrow cleft

between two neurons). Neurotransmitters released by presynaptic neurons bind to and

activate the receptors at the postsynaptic sites. This action constitutes synaptic

transmission. The ability to modify the strength of synaptic transmission between the

neurons is thought to underlie the learning and memory function of the brain, including

hippocampal-dependent learning and memory (Bliss and Collingridge, 1993; Foster et









al., 1996; Malenka and Nicoll, 1999; Bailey et al., 2000; Martin et al., 2000; Dragoi et al.,

2003). In particular, the neurons possess a vast array of signaling molecules that are

responsive to various aspects of learning and memory (Lisman, 1994; Tsien et al.,

1996; Benson et al., 2000; Abel and Lattal, 2001; Genoux et al., 2002; Koekkoek et al.,

2003). Unfortunately the learning and memory functions of the hippocampus are

weakened during aging and disease.

The role of hippocampus in learning and memory, described previously, depends

on the complex synaptic properties of its neurons. The hypothesis presented in chapter

3 is that age-related increase in oxidative stress or oxidative redox state decreases the

NMDAR mediated synaptic transmission. The focus of this hypothesis has been the

CA3-CA1 synaptic contacts located in the CA1 subfield of the hippocampus. A part of

the CA3 axons constitute the Schaffer collateral pathway that release L-glutamate,

which binds to ionotropic and metabotropic glutamate receptors on the CA1 pyramidal

neurons. Synaptic transmission is completed upon the activation of the receptors,

leading to inflow of Na and Ca2+ ions into the neuron, and outflow of K ions from the

neurons. The ability to modify the strength of synaptic transmission between neurons is

termed synaptic plasticity. In this study we have investigated the influence of redox state

on synaptic transmission and plasticity during aging. Specifically we tested whether the

redox agents can modulate NMDAR mediated synaptic transmission and plasticity in an

age-dependent manner.

Ionotropic Glutamatergic Transmission

The experiments described in chapters 3 and 4 were designed to analyze the

effects of redox agents on NMDARs, and we also tested if they had any effect on

AMPARs. The NMDARs and AMPARs are ionotropic glutamate receptors (iGluRs),









which are activated by the amino acid neurotransmitter L-glutamate (Collingridge et al.,

1983) released from the axonal terminals of CA3 pyramidal neurons. The L-glutamate

can also activate metabotropic glutamate receptors (mGluRs) on the CA1 pyramidal

neurons; however all the studies presented in this dissertation involved the iGluRs.

The iGluRs mediate fast synaptic transmission and are classified into three major

subtypes, named after the synthetic agonists that activate them. They are the alpha-

amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (abbreviated as

AMPAR), the kainate receptor and the N-methyl D-aspartate receptor (abbreviated as

NMDAR). The AMPARs and kainate receptors are also categorized as non-NMDARs.

All the iGluRs are non-selective cationic channels that are permeable to both Na and

K (Aidley, 1989; Johnston and Wu, 1995). However the NMDARs are relatively more

permeable to Ca2+ ions (Garaschuk et al., 1996). The ionic fluxes mediated by the

iGluRs in the CA1 pyramidal neurons are measured as the excitatory post synaptic

potential (EPSP). The non-NMDARs generate the early phase of the EPSP (~ 0 to15

ms from stimulation) and the NMDARs contribute to the late phase of the EPSP (greater

than 15 ms time window from stimulation). In this study we have analyzed both the

AMPAR-mediated and NMDAR-mediated synaptic transmission.

Decreased NMDAR function is an important biomarker of aged CA1 pyramidal

neurons, and is also the central aspect of hypotheses presented in chapters 3 and 4.

We tested the hypothesis that increase in oxidative stress or oxidative redox state of

aged neurons contributes to decrease in the NMDAR mediated synaptic transmission

and plasticity. In order to better understand the results in support of this hypothesis, it is

critical to understand the properties and function of NMDAR mediated synaptic









transmission and plasticity. The following section provides a brief overview of the ionic

currents and synaptic mechanism that are associated with NMDAR function in neurons.

The NMDARs are hetero-tetrameric protein complexes composed of two classes

of subunits, the ubiquitously expressed and essential subunit NR1; and a modulatory

subunit NR2A/2B/2C/2D (Moriyoshi et al., 1991; Kutsuwada et al., 1992; Meguro et al.,

1992; Monyer et al., 1992; Cull-Candy et al., 2001). The activation of NMDAR requires

binding of a ligand (glutamate), membrane depolarization (to remove the Mg2+ block on

the channel), and binding of a co-agonist (glycine). These requirements for NMDAR

activation makes it the ideal coincidence detector to integrate presynaptic and

postsynaptic activity. Since NMDAR is a non-selective cation channel, its activation and

opening leads to simultaneous conductance of Na, K, and Ca2+ ions (Chen et al.,

2005). However the NMDARs are at least 19 times more permeable to Ca2+ ions than

the AMPARs, which are the other major subtype of iGluR in the CA1 pyramidal neurons

(Garaschuk et al., 1996), primarily because all the NMDAR subunits carry the polar but

neutral residue arginine in the M2 region of their pore domain (Zarei and Dani, 1994;

Ferrer-Montiel et al., 1996; Premkumar and Auerbach, 1996). The NMDARs also play a

critical role in influencing the properties of AMPARs. For example the AMPARs are

rapidly inserted into recently activated synapses (Hayashi et al., 2000; Zhu et al., 2000;

Song and Huganir, 2002) where NMDARs have been activated (Petralia et al., 1999;

Shi et al., 1999). This feature serves as the molecular basis for the induction and

expression of synaptic plasticity at CA3-CA1 synapses (described in detail in the

following section). Although AMPAR subunits carry an arginine, instead of glutamine in

their pore domain, can generate significant Ca2+ influxes, akin to the NMDARs (Jonas et









al., 1994; Lomeli et al., 1994); the NMDARs are the primary source of iGluR derived

Ca2+ in the CA1 pyramidal neurons.

NMDA Receptor Dependent Synaptic Plasticity: LTP and LTD

As described in more detail in the previous section, the NMDA receptor

component of synaptic transmission is decreased in aged animals. This is important

because, the influx of Ca2+ through the NMDARs is critical to the activation of signaling

cascades in close proximity to the synapses, such that those synapses undergo a

change in the strength of synaptic transmission. A change in the synaptic strength

between the hippocampal neurons constitutes hippocampal synaptic plasticity which is

thought to underlie the formation of memories in the mammalian brain (Morris et al.,

2003). Age-related decline in memory function is associated with altered hippocampal

synaptic plasticity (Foster, 1999). In chapter 4 we tested the hypothesis that the age-

related increase in oxidative stress and decrease in NMDAR function contributes to the

alteration in synaptic plasticity. In order to better understand the results, a brief overview

of the relationship between NMDAR function, and synaptic plasticity is provided below.

One aspect of synaptic plasticity is long term potentiation (LTP), first reported in

1973 (Bliss and Lomo, 1973), as a long lasting enhancement in synaptic transmission

between two neurons following brief high-frequency electrical stimulation. In contrast,

long term depression (LTD) is a long lasting decrease in the strength of synaptic

transmission between two neurons, which is observed after prolonged low-frequency

stimulation. LTD was first reported in 1977 (Lynch et al., 1977), and an integrative

model of the interplay between LTP and LTD has emerged since then. Although LTP

and LTD are processes of synaptic plasticity with opposite outcomes, they are governed

by changes in the Ca2+ dynamics of the postsynaptic neuron (Cummings et al., 1996;









Shouval et al., 2002), involving calcium influx predominantly through the NMDARs. In

this dissertation we have investigated the effects of redox modulators on NMDAR

function.

Experiments have shown that LTP is initiated upon a quick and large amplitude

rise in the intracellular Ca2+ concentration in the postsynaptic neuron, while LTD

induction requires a moderate rise in intracellular Ca2+ concentration over longer

periods of time (Cho et al., 2001; Cormier et al., 2001). Other studies have shown that

elevations in the cytosolic Ca2+ levels in the postsynaptic neuron is sufficient, by itself,

to cause bidirectional changes in synaptic strength without presynaptic activity (Neveu

and Zucker, 1996a, b; Yang et al., 1999). All these studies support the idea that Ca2+

influx into the postsynaptic neuron through the NMDARs is necessary for initiating LTP

and LTD (Bliss and Collingridge, 1993; Bear and Malenka, 1994). The NMDAR-

mediated Ca2+ influx into the neuron activates numerous Ca2+ sensitive signaling

cascades that are involved in the induction and expression of LTP and LTD. The role of

NMDARs in synaptic plasticity, learning and memory is supported by evidence showing

that NMDAR antagonists (used at concentrations that block LTP in vitro) block

acquisition of hippocampus dependent memory (Bolhuis and Reid, 1992; Davis et al.,

1992). In addition, genetic models carrying a CA1 region specific knockout of the

NMDAR subunit NR1 exhibit impaired hippocampal synaptic plasticity, and poor

memory skills (McHugh et al., 1996; Tsien et al., 1996). Subsequent models connecting

synaptic plasticity, learning and memory have proposed a sliding threshold for the

direction of synaptic modification dependent on the frequency of neural activity (Shouval

et al., 2002; Kumar and Foster, 2007).









One of the contributing factors for the NMDAR mediated Ca2+ influx during the

induction of LTP and LTD is postsynaptic membrane depolarization. In the context of

the current studies, neuronal excitability is closely linked to the membrane

depolarization and determines synaptic transmission and plasticity. The depolarization

of the postsynaptic membrane potential is necessary to remove the Mg2+ block on the

NMDARs. Normally K channels in the postsynaptic neurons gate the efflux of K,

through the afterhyperpolarization (AHP) current that maintains the neurons in a

hyperpolarized state. The synaptic inputs from the presynaptic neurons counteracts the

effect of K channels and shifts the postsynaptic membrane potential to more

depolarized potentials, which ultimately aids in the removal of the Mg2+ block on the

NMDARs. Thus a more hyperpolarized state makes it difficult for the neurons to

depolarize and activate the NMDARs. The complex relationship that exists between

processes that regulate membrane potential and processes that modify synaptic

strength ultimately determines the expression of LTP and LTD. Increased AHP

amplitude is a biomarker of aged CA1 pyramidal neurons, and is also the focus of a

separate set of studies presented in this dissertation. We tested the hypothesis that the

oxidative redox state of aged neurons contributes to increase in the amplitude of the

slow component of AHP or sAHP. In order to better understand the results in support of

this hypothesis, it is critical to understand the properties and function of AHP. The

following section provides a brief overview of the ion channels, currents and signaling

mechanisms that mediate the AHP in neurons.

Afterhyperpolarization in CA1 Pyramidal Neurons

The AHP is a post burst hyperpolarization in the membrane potential of the

neurons which lasts over 1-2 seconds after the offset of the depolarizing pulse. The









AHP is divided into three broad phases based in time kinetics into the fast component

(fAHP), the medium component (mAHP), and the slow component (sAHP) (Sah and

Faber, 2002). Each component of AHP is mediated by distinct classes of ion channels

that differ in their pharmacology and time kinetics. The fAHP, which lasts several tens of

milliseconds, is mediated by the BK channels. The BK channels generate large K

currents with single channel conductance reaching 400 pS (Marty, 1981). The BK

channels are dependent on Ca2+ binding and membrane depolarization for their

activation (McManus, 1991; Cui et al., 1997). In the context of Ca2+ signaling in the CA1

pyramidal neurons, the BK channels contain a Ca2+ detection site on their intracellular

domain (Wei et al., 1994; Schreiber and Salkoff, 1997). Although activated by cytosolic

Ca2+, the Ca2+- sensitivity of BK channels is highly dependent on the membrane

potential, which enables it to generate the hyperpolarizing K currents within few

milliseconds of the onset of the depolarizing pulse.

The mAHP, which lasts a few hundred milliseconds, is generated by the SK

channels. The SK channels have small K conductance ranging from 2-20 pS (Blatz and

Magleby, 1986). In contrast to the BK channels, the SK channels are voltage insensitive

(Hirschberg et al., 1998); however, their activation is dependent on rises in cytosolic

Ca2+. In the context of Ca2+ signaling, the SK channels possess unique Ca2+ binding

properties. The SK channels do not directly bind to Ca2+ like the BK channels; however,

reports indicate that they can bind to Ca2+ binding proteins like calmodulin (Xia et al.,

1998; Keen et al., 1999; Schumacher et al., 2001), which leads to activation of the SK

channel through a conformational change. All these properties of SK channels ideally

suit them to generate the relatively late onset K+ current which underlie the mAHP.









The sAHP is the primary focus of a separate set of studies presented in this

dissertation. Unfortunately, the molecular identity of the ion channel underlying the

sAHP is unknown. Nevertheless, numerous observations regarding the current that

underlies the sAHP have given rise to interesting predictions. The current underlying

sAHP has been observed to be voltage independent, but Ca2+ dependent (Sah, 1996),

which is modulated by a range of neurotransmitters including glutamate, acetylcholine

and serotonin (Nicoll, 1988). Although a class of SK channels could possibly mediate

the sAHP current (Marrion and Tavalin, 1998; Bowden et al., 2001), experiments using

clotrimazole analogs, which are highly selective synthetic inhibitors of sAHP current,

have eliminated this possibility (Shah et al., 2001). Recent advances in understanding

the molecular mechanisms underlying the sAHP current suggest that a diffusible

molecule or second messenger system could be operating at the interface between

cytosolic Ca2+ and the channels that mediate the sAHP current. For example, recent

studies indicate that the neuronal Ca2+ sensing protein hippocalcin has been reported to

activate channels that mediate the sAHP current in its membrane bound form

(Tzingounis et al., 2007; Tzingounis and Nicoll, 2008). Another study suggests that the

ionotropic kainate receptors could decrease sAHP currents through a unique

metabotropic action involving protein kinase C (Melyan et al., 2002). A separate set of

studies point to a phosphorylation mechanism for mediating the sAHP involving kinases

like cAMP-dependent protein kinase A (Madison and Nicoll, 1986; Pedarzani and

Storm, 1993), Ca2+/Calmodulin dependent protein kinase II (Muller et al., 1992), and

protein kinase C (Malenka et al., 1986). In summary, numerous slow-activating

secondary systems have been implicated in mediating and modulating the sAHP









current; however the identity of the ion channel that actually conducts the K+ ions

underlying the sAHP current remains to be discovered. Nevertheless, the dependence

of this unknown ion channel on cytosolic Ca2+ is a veritable starting point for

investigations into the mechanisms that modulate sAHP, especially in the context of

aging, because sAHP is reported to increase in aged hippocampal neurons (Landfield

and Pitler, 1984; Moyer et al., 1992; Kumar and Foster, 2004; Matthews et al., 2009).

The cytosolic Ca2+ level, which determines the activation of K channels

underlying the sAHP current, is the outcome of processes involved in maintaining

neuronal Ca2+ homeostasis. The Ca2+ homeostasis is maintained by the complex

interaction between mechanisms that allow Ca2+ entry into the neuron and those

mechanisms that remove Ca2+ from the cytosol and/or buffer them in internal stores. In

addition to the sAHP, the intracellular Ca2+ concentration also modulates synaptic

plasticity. Due to the fundamental role of intracellular Ca2+ homeostasis in the

development of several hypotheses presented in this dissertation a brief discussion of

the key components and regulators of Ca2+ homeostasis in CA1 pyramidal neurons is

provided below.

Calcium Homeostasis in CA1 Neurons

There are three major sources for Ca2+ mobilization in neurons the NMDARs,

the L-VGCC's, and the intracellular Ca2+ stores (ICS). Cytosolic elevation in Ca2+

concentration, from these sources, activates signaling cascades involved in LTP/LTD,

and mediates physiological processes like the AHP. Excessive levels of cytosolic Ca2+

can lead to excitotoxicity and neuronal death, which is offset by pumps and buffering

mechanisms that remove Ca2+. The interplay between the processes that control the

elevation and decrease in cytosolic Ca2+ levels maintains the Ca2+ homeostasis in the









neurons (Fig. 1-1). Age-related neuronal dysfunction is thought to originate, in part, from

the perturbation of Ca2+ homeostatic mechanisms. In aged neurons there is a shift in

the relative contribution of Ca2+ by various sources; such that the NMDARs contribute

less Ca2+, and the L-VGCC's and ICS contribute more Ca2+ (Thibault and Landfield,

1996; Norris et al., 1998a; Thibault et al., 2001; Foster and Kumar, 2002; Kumar and

Foster, 2004; Gant et al., 2006; Foster, 2007; Thibault et al., 2007; Kumar et al., 2009)

for the maintenance of Ca2+ homeostasis. Poor regulation of intracellular Ca2+

concentration contributes to improper activation of the signaling cascades involved in

synaptic plasticity, thus impairing LTP and LTD (Foster, 1999; Burke and Barnes, 2006,

2010). Age-related changes to Ca2+ homeostasis is highlighted in the "calcium

hypothesis of brain aging", which states that the disruption of normal Ca2+ homeostasis

underlies neuronal dysfunction during aging (Landfield and Pitler, 1984; Gibson and

Peterson, 1987; Khachaturian, 1989, 1994). However, the mechanisms that "cause" the

disruption in normal Ca2+ homeostasis are, as yet, poorly understood. The hypothesis

that forms the basis for this dissertation is that increased oxidative stress or a more

oxidative redox state mediates the age-related shift in Ca2+ homeostasis, and

contributes to neuronal dysfunction. The results presented in chapter 3, 4, and 5

delineate the link between oxidative redox state and age-related changes to neuronal

synaptic transmission, plasticity, and sAHP. In order to better understand the

hypotheses and results, it is important to understand the cause and consequence of

increased oxidative stress or an oxidative redox state. Hence an overview of the

neuronal redox state, in the context of aging, is provided in the following section.









Redox State and Aging

Biological aging is thought to be the outcome of accumulation of changes in an

organism over time (Hayflick, 1985, 2007). One school of thought considers aging as a

biological process controlled by the expression pattern of genes (Kennedy et al., 1995;

Martin et al., 1996; Kirkwood and Austad, 2000; Martin, 2007; Budovskaya et al., 2008)

and/or alterations in the structure of chromosomes, specifically the events associated

with the length and state of the ends of chromosomes called telomeres (Harley et al.,

1990; Bodnar et al., 1998; Wright and Shay, 2002; Stewart et al., 2003; Blasco, 2005).

However our current understanding is that accumulation of changes to the biological

molecules (lipids, proteins, DNA, RNA etc) contributes to the process of aging by

altering the structure and function of the biological molecules (Rattan et al., 1992;

Butterfield et al., 1998; Finkel and Holbrook, 2000). In particular, protein oxidation is a

commonly observed age-related change which alters the structure and function of

proteins (Stadtman, 1988, 1992; Yin and Chen, 2005; Widmer et al., 2006; Riemer et

al., 2009), including several neuronal proteins (Smith et al., 1992). Protein oxidation

results in the formation of disulfide bonds on the thiol moieties of cysteine and/or

methionine residues (Shacter, 2000; Davies, 2005). The formation of disulfide bonds

modifies NMDAR function too, which is one of the major focus of this study (Choi and

Lipton, 2000; Choi et al., 2001). Protein oxidation during aging arises from increased

oxyradical production and/or weakened antioxidant capacity of the neurons (Foster,

2006; Poon et al., 2006), which shifts the redox state to a more oxidative environment

and weakens the redox buffering capacity (Parihar et al., 2008). Thus progressive

accumulation of oxidative damage due to increased oxidative stress and a more

oxidative redox state leads to protein dysfunction and contributes, in part, to the age-









related alteration in the structure and function of proteins. One of the central tenets of all

the hypotheses presented in the following chapters is that aging is associated with an

increase in oxidative stress or an oxidative redox state. In order to better understand the

results based on this idea, it is very critical to understand the production and removal of

the free radicals that promote protein oxidation.

One of the widely accepted theories of aging is the Free Radical theory of aging

proposed by Denham Harman in 1956. The theory hypothesized that biological aging is

the consequence of free radical damage to the biological molecules (Harman, 1956,

1972). The free radicals commonly encountered in biological systems are the reactive

oxygen species (ROS) and the reactive nitrogen species (RNS). Among its various

functional roles, the free radicals participate in cellular signaling, immunological

response, neurotransmission, and oxidative metabolism. However, due their toxic

nature, cells have evolved an elaborate detoxification system to remove and neutralize

them. Thus a balance exists between the production and removal free radicals. During

aging there is an excessive production and improper removal of free radicals that leads

to an accumulation of abnormal levels of ROS, and RNS, which leads to oxidative and

nitrosative stress respectively. Although there is insufficient evidence to support the idea

that free radicals determine life-span; there is nevertheless a consensus that increased

oxidative stress has a significant role to play in age-related disorders (Beckman and

Ames, 1998; Migliaccio et al., 1999; Finkel and Holbrook, 2000; Melov, 2000; Schriner

et al., 2005; Muller et al., 2007).

Excessive levels of superoxide are one of the hallmarks of increased oxidative

stress. The increase in the accumulation of free radicals shifts the redox environment of









the neurons to a more oxidative state, and contributes to the free-radical induced

damage proposed by Harman. Normally at the end of the electron transport chain, the

free electrons are absorbed by oxygen, which is reduced to water. However incomplete

reduction of oxygen yields superoxide (denoted by .02-) that contains one free electron.

The enzyme superoxide dismutase (SOD) catalyzes the dismutation of superoxide into

hydrogen peroxide (H202). However H202 is a potent oxidizing agent, which can still

cause oxidative damage to cellular components. The enzyme catalase then converts

H202 into oxygen and water. In addition, cells utilize the enzyme glutathione peroxidase

to convert H202 into water by simultaneous oxidation of reduced glutathione to oxidized

glutathione. The age-related increase in the production of ROS overwhelms these

antioxidant systems and ultimately leads to excessive accumulation of ROS. The

increase in oxidative stress creates an oxidative redox state and weakens the redox

buffering capacity of the aged neurons which is also indicated by lower resting levels of

reduced nicotinamide adenine dinucleotide (phosphate), and reduced L-glutathione (L-

GSH) (Parihar et al., 2008). The results presented in chapters 3, 4, and 5 indicate that

oxidative redox state is one of the critical factors that paves the way for neuronal

dysfunction during aging.

A key component of all the hypotheses presented so far has been neuronal

dysfunction. Our experiments were designed to detect abnormal changes in neuronal

function and understand the mechanisms that cause such dysfunction. As described in

the previous sections, the neuronal dysfunction is closely linked to processes that

regulate intracellular Ca2+ homeostasis. Increased oxidative stress or a more oxidative

redox state during aging can modulate processes that maintain Ca2+ homeostasis by









decreasing NMDAR function (Bodhinathan et al., 2010), increasing the activity of L-type

VGCC (Lu et al., 2002; Akaishi et al., 2004), and increasing Ca2+ mobilization from ICS

(Hidalgo et al., 2004; Kumar and Foster, 2004). Redox state of the aged neuron can

also affect synaptic plasticity in hippocampal slices (Serrano and Klann, 2004;

Bodhinathan et al., 2010). The results presented in the following chapters enhance our

understanding of the link between these diverse processes.

Summary

The overview presented above highlights the complex interaction between

mechanisms that mediate normal functioning of hippocampal CA1 pyramidal neurons,

and mechanism during aging (more oxidative redox state) that contribute to its

dysfunction. At the outset normal aging has significant impact on NMDAR function,

NMDAR-dependent synaptic plasticity, and neuronal excitability. One of the central

themes highlighted in the following chapters is that the CA1 pyramidal neurons express

a broad profile of changes in intracellular Ca2+ homeostasis during aging. This

phenomenon is central to many mechanisms investigated in this dissertation including

changes in synaptic transmission, plasticity, and neuronal excitability. Notably, the ideas

presented in the preceding sections take a reductionist approach (in the context of

aging) to describe age-related changes in learning and memory dependent on

hippocampus, the oxidative redox state arising from increased oxidative stress, and the

subtle age-related biochemical and physiological changes in the CA1 pyramidal

neurons. The following chapters have built upon these ideas and provide novel results

that enhance our understanding of neuronal dysfunction during aging.















-T
Cytoplasm iC DAG .-. ATP ADP+P,
C a 2u rVK -, / -..
O CBP 1 aK PIP2I


IP3R SPCA Complex


NAADP
c- OR __O mNCXr % mPTP
O* r
SERCA Lysosome
(Ca2 ATPase) | .: a Ca .
Endoplasmic Reticulum a I Nucleus Uniportider
(*Nucleus Mitochondrion


Figure 1-1. Calcium homeostasis in the neuron. Model depicting various Ca2+ sources,
sequestrating, buffering mechanisms, and Ca2+ signaling events in a healthy
neuron. Indicated are the voltage dependent Ca2+ channels (VDCC), n-
methyl-d-aspartate receptor (NM DAR), a-amino-3-hydroxy-5-methyl-4-
isoxazolepropionate receptor (AMPAR), and g-protein coupled receptor
(GPCR) involved in Ca2+ (red balls) influx into the cytosol (blue dashed
arrows). The release of Ca2+ into the cytoplasm also occurs from the
intracellular Ca2+ stores (ICS) through inositol (1, 4, 5)-triphosphate receptor
(IP3R) and ryanodine receptors (RyRs). Organelles, including the
endoplasmic reticulum (ER), mitochondria, and lysosomes act as Ca2+
buffering systems, releasing and sequestering Ca2+. Further, the model
depicts Ca2+ buffering and extrusion pathways (red dashed arrows), involving
Na+/Ca2+ exchanger (NCX) and plasma membrane Ca2+ATPase (PMCA),
sarcoplasmic reticulum Ca2+ ATPases (SERCAs), nicotinic acid adenine
dinucleotide phosphate (NAADP), various Ca2+ binding proteins (CBPs).
Mitochondrial permeability transition pore (mPTP) and mitochondrial sodium-
Ca2+ exchanger (mNCX) and secretary pathway Ca2+-ATPases (SPCA)
contribute to Ca2+ regulation (Adapted from Kumar A, Bodhinathan K, and
Foster T C, Front Ag Neurosci 2009).









CHAPTER 2
MATERIALS AND METHODS

Drugs, Solutions and Suppliers

All drugs were prepared according to the manufacturer's specifications and

ultimately dissolved in ACSF prior to bath application on the slices. Appendix A provides

a comprehensive list of all the drugs, solutions and their suppliers, used in this study.

Drugs that need either DMSO or ethanol as the solvent were initially dissolved in DMSO

or ethanol respectively and diluted in ACSF to a final DMSO concentration of less than

0.01% and final ethanol concentration of less than 0.0001%. Appendix B provides a list

of the all the concentrations of various drugs used in this study. Commonly used

laboratory chemicals were acquired from either Sigma-Aldrich (St. Louis, MO) or Fisher

Scientific (Pittsburgh, PA).

Animal Procedures

Procedures involving animals have been reviewed and approved by the

Institutional Animal Care and Use Committee at the University of Florida. All procedures

were in accordance with the guidelines established by the U.S. Public Health Service

Policy on Human Care and Use of Laboratory Animals. Male Fischer 344 rats, young (3-

8 mo) and aged (20-25 mo), were obtained from National Institute on Aging colony at

Harlan Sprague Dawley Inc (Indianapolis, IA). All animals were group housed (2 per

cage), and maintained on a 12:12 hr light schedule, and provided ad libitum access to

food and water. Animal health was regularly monitored with the help of the Animal Care

Services at the University of Florida.









Hippocampal Tissue Dissection for Electrophysiological Experiments

The protocol to prepare live hippocampal slices for electrophysiological

experiments are derived from initial reports by Li and Mcllwain (Li and Mcllwain, 1957),

which has been suitably modified and standardized in our lab (Kumar and Foster,

2004). Animals were deeply anaesthetized using isoflurane (Webster, Sterling, MA) and

decapitated with a guillotine (MyNeurolab, St Louis, MO). The layer of skin covering the

skull was pared open and the skull was removed using bone snips. The brains were

rapidly removed and transferred to a beaker containing ice-cold artificial cerebrospinal

fluid (ACSF) which was calcium free. The hippocampi were dissected out carefully for

slicing. Hippocampal slices (~ 400pm) were cut parallel to the alvear fibers using a

tissue chopper (Mickle Laboratory Engineering Co, Surrey, UK). The slices were

incubated in a holding chamber (at room temperature) with ACSF containing (in mM):

124 sodium chloride (NaCI), 2 potassium chloride (KCI), 1.25 potassium phosphate

monobasic (KH2PO4), 2 magnesium sulfate (MgSO4), 2 calcium chloride dihydrate

(CaCI2), 26 sodium bicarbonate (NaHCO3), and 10 D-glucose. At least 30 min before

recording, slices were transferred to a standard interface recording chamber (Warner

Instrument, Hamden, CT). The chamber was continuously perfused with oxygenated

ACSF (95%-02 and 5%-CO2) at the rate of 2 mL/min. The pH was maintained at 7.4

initially adjusted using 10N hydrochloric acid or 10M sodium hydroxide. The

temperature was maintained at 30 0.5C using the automatic temperature controller

TC-324B (Warner Instrument, Hamden, CT) (Fig. 2-1).

Electrophysiological Recordings: Extracellular Field Potentials

The extracellular field excitatory postsynaptic potentials (fEPSP) represent the net

influx of Na and other positive ions like Ca2+ into the postsynaptic neuron. The net









movement of positive ions from the recording electrode is measured as a negative

deflection on the oscilloscope. This negative deflection is called the fEPSP (Aidley,

1989; Johnston and Wu, 1995; Kandel, 2000b) and is indicated as total fEPSP (black

trace) in Fig. 2-2A. The fEPSPs for studies described here were generated by

stimulating the CA3 afferent fibers onto CA1 pyramidal neurons, known as the Schaffer-

collateral pathway or the CA3-CA1 pathway. The fEPSPs were recorded using glass

micropipette electrodes filled with artificial cerebrospinal fluid as the recording medium.

The glass micropipette electrode resistances ranged from 4-6 MO. The glass

micropipettes were pulled from thin-walled borosilicate capillary glass using a

Flaming/Brown horizontal micropipette puller (Sutter Instruments, San Rafael, CA). The

borosilicate capillary glass had an outer diameter of 1 mm, an inner diameter of 0.75

mm and was about 4 inches in length. Two concentric bipolar stimulating electrodes

(FHC, Bowdoinham, ME) were localized to the middle of the stratum radiatum on either

side of the recording electrode in order to stimulate the afferents onto CA1 pyramidal

neurons. The outer pole of the bipolar electrode was made of stainless steel and was

200 pm in diameter. The inner pole was made of platinum/iridium alloy and was about

25 pm in diameter. Diphasic stimulus pulses of 100 ps duration were delivered by a

stimulator (SD9 Stimulator; Grass Instrument, West Warwick, RI) and alternated

between the two pathways such that each pathway was activated at 0.033 Hz.

Extracellular Field Potentials: Data Analysis

The signals corresponding to the extracellular field potentials were sampled at a

frequency of 20-kHz; filtered and amplified between 1 Hz and 1 kHz using Axoclamp-2A

(Molecular Devices, Sunnyvale, CA) and a differential AC amplifier (A-M Systems,

Sequim, WA) and stored on a computer disk (Dell Inc, Texas) for off-line analysis. A









separate output from the differential AC amplifier was fed into an oscilloscope (Tektronix

2214, Tektronix Inc, Beaverton, OR) and audio monitor (AM8, Grass Technologies,

West Warwick, RI) for real time visualization of the signals. Two cursors were placed to

cover the initial descending phase of the waveform and the maximum negative slope

(mV/ms) of the fEPSP was determined by a Sciworks computer algorithm (Datawave

Technologies, Berthoud, CO) which determined the maximum change across a set of

20 consecutively recorded points between the cursors. To measure the amplitude of the

fEPSP, the cursors were placed to cover the entire waveform of the fEPSP.

Subsequently a Sciworks computer algorithm was used to compute the maximum

amplitude (mV) of the fEPSP at the peak of the waveform (Fig. 2-2B).

Long-Term Potentiation and Paired-Pulse Ratio Recordings

For induction of LTP, the stimulation intensity was set to elicit 50% of the maximal

fEPSP obtained by stimulating the CA3 afferents on CA1 pyramidal neurons. After

stable baseline recording at 0.033 Hz for at least 20 min, high frequency stimulation

(HFS) was delivered to the pathway at 100 Hz for 1 sec (100 pulses) at the baseline

stimulation intensity, and recorded for at least 60 min post-HFS. A simultaneously

recorded control (non-HFS) pathway received the test stimulation but not the HFS. In

some cases the fEPSP was monitored in the presence of drugs to account for changes

on baseline synaptic transmission, before delivering the HFS. The average fEPSP slope

corresponding to the last 5 minutes from each pathway was used to compare changes

in synaptic strength relative to the baseline.

For measuring the paired-pulse ratio, paired pulses were delivered through a

single stimulating electrode at varying inter pulse intervals. The first pulse was set to

elicit 50% of the maximal fEPSP. The various inter pulse intervals between successive









pulses were 50 ms, 100 ms, 150 ms, and 200 ms. The ratio of the maximum negative

slope of the second pulse to the maximum negative slope of the first pulse was

computed as the paired-pulse ratio.

Isolation of NMDAR Mediated Extracellular Synaptic Potentials

To obtain the NMDAR-mediated field excitatory postsynaptic potential (NMDAR-

fEPSP) at the CA3-CA1 synapses, the slices were incubated in ACSF containing low

extracellular Mg2+ (0.5 mM), 6, 7-dinitroquinoxaline-2, 3-dione (DNQX, 30 pM), and

picrotoxin (PTX, 10 pM). Low extracellular Mg2+ was used to facilitate the removal of the

"Mg2+ block" on the NMDAR; DNQX (AMPAR antagonist) was used to block the AMPAR

component of the fEPSP; and PTX (GABAA antagonist) was used to minimize the

GABA-ergic inhibition on the CA1 neurons. In each case the baseline response was

collected for at least 10 min before experimental manipulations (drug application). The

NMDAR-fEPSP is indicated in Fig. 2-2A (blue trace). Successful pharmacological

isolation of the NMDAR-fEPSP was demonstrated by the application of the NMDAR

antagonist AP-5 (100 pm), which is indicated by the red trace in Fig. 2-2A. Changes in

the levels of synaptic transmission, induced by drug application, were calculated as

percentage change from the averaged baseline responses.

Electrophysiological Recordings: Intracellular Sharp Microelectrode Recording

Intracellular excitatory post synaptic potentials were recorded from the CA1

pyramidal neurons using sharp microelectrodes. Sharp microelectrodes were pulled

from thin walled borosilicate capillary glass (1 mm outer diameter; 0.75 mm inner

diameter; 4 inches in length) using the Flaming/Brown horizontal micropipette puller

(Sutter Instruments, San Rafael, CA). All microelectrode tips were filled with 3M

potassium acetate and in some cases were filled with potassium acetate solution









containing the drug (for example L-GSH). The microelectrode resistances typically

ranged from 39-55 MO. Microelectrodes were visually positioned in the CA1 pyramidal

cell layer using a dissecting microscope (SZH10, Optical Elements Corp, Washington,

DC) and a bipolar stimulating electrode was positioned to stimulate the CA3 afferents

onto CA1 pyramidal neurons. On cell entry, positive or negative current was applied to

clamp the neuronal membrane potential at -65 mV. Only neurons with a resting

membrane potential (Vm) more hyperpolarized than -57 mV, and having an input

resistance (Rin) >20 MQ, and an action potential amplitude (APamp) rising 270 mV from

the point of spike initiation were included in the analysis. The resting membrane

potential and holding current was monitored through the entire course of the

experiment. Variations in the resting membrane potential, holding current, input

resistance, action potential amplitude or the microelectrode resistance was also

monitored for those cases in which drugs were included in the pipette and eliminated

accordingly. Diphasic stimulus pulses of 100 ps duration were delivered at 0.033 Hz and

the stimulation intensity was adjusted to elicit an intracellular synaptic response, which

was below the spike threshold. Baseline response recording began within 3 minutes

after cell entry. To obtain the NMDAR-mediated intracellular synaptic potentials from

CA1 pyramidal neurons, slices were incubated in ACSF containing low extracellular

Mg2+, DNQX and PTX as described above. An example of the NMDAR mediated

intracellular synaptic potential is indicated in Fig. 2-2C.

Intracellular Synaptic Potentials: Data Analysis

The signals corresponding to the intracellular synaptic potentials were sampled at

a frequency of 20 kHz; filtered and amplified between 1 Hz and 1 kHz using Axoclamp-

2A (Molecular Devices, Sunnyvale, CA) and a differential AC amplifier (A-M Systems,









Sequim, WA) and stored on a computer disk (Dell Inc, Texas) for off-line analysis. A

separate output from the differential AC amplifier was fed into an oscilloscope (Tektronix

2214, Tektronix Inc, Beaverton, OR) and audio monitor (AM8, Grass Technologies,

West Warwick, RI) for real time visualization of the signals. Two cursors were placed to

cover the entire waveform of the intracellular EPSP; from the pre-stimulus baseline to >

100 ms of the waveform. A Sciworks computer algorithm was used to compute the

maximum amplitude (mV) of the intracellular EPSP at the peak of the waveform.

Intracellular Afterhyperpolarization: Data Analysis

The signals corresponding to the intracellular AHP were sampled at 20 kHz;

filtered and amplified between 1 Hz and 1 kHz and processed as described previously

for the intracellular synaptic potentials. The AHP was recorded from the neurons in the

following manner. Depolarizing current pulses (duration: 100 ms; amplitude: 0.3 to 1.2

nA) were delivered every 20 seconds through the intracellular electrode in order to elicit

a train of action potentials with 5 spikes. Since the AHP amplitude varies with the

number of spikes in a train of action potentials, a train of 5 action potentials were

maintained throughout the recording to study the effect of various treatment conditions

on the AHP amplitude. The AHP recorded during the baseline and under the application

of various drugs were elicited at the same membrane potential (-63 mV), which was

achieved by manually clamping the membrane potential with DC current injection (-1 to

+1 nA). The sAHP amplitude was measured as the difference between the average

membrane potential during the 100 ms period immediately preceding the onset of the

depolarizing current and the average membrane potential 400 to 500 ms after the offset

of the depolarizing current. The sAHP amplitudes were computed in mV and changes

under experimental conditions are assessed as percent change from the average









baseline value over 5 to 10 min period. A representative trace of the intracellular AHP is

indicated in Fig. 2-2D.

Measurement of ROS in Hippocampal Slices

Hippocampal slices were incubated for 30 minutes in ACSF containing 10 pM of

the ROS detection reagent 5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein diacetate

(c-H2DCFDA; Molecular Probes Inc, Eugene, OR). Slices incubated for 30 minutes in

absence of c-H2DCFDA were used to detect background or auto fluorescence. Slices

that were incubated with c-H2DCFDA were imaged to quantify the levels of ROS.

Fluorescent images were obtained with an Axiovert 40 CFL fluorescent microscope and

Axiocam digital CCD camera (Carl Zeiss, Thornwood, NY). Fluorescence intensity was

quantified as follows: fluorescent microscope was used to obtain images under uniform

exposure time (100 ms) and intensity (150%). The images were then converted to

grayscale images in Adobe Photoshop 5.5; the resulting images were quantified by

densitometry analysis using Image J software (http://rsbweb.nih.gov/ij). An area of

about 225 pm along the medial-lateral axis and 187.5 pm along the anterior-posterior

axis, centered on the CA1 pyramidal neurons was selected for analysis of fluorescence

intensity. The mean gray value intensities obtained from the aged and young animals

are represented as the mean fluorescence intensities. The mean fluorescence intensity

from the dye-exposed slices is normalized to the mean fluorescence intensity obtained

from dye-unexposed slices (harvested from the same animal) using the following

relationship:

Mean c-H2DCFDA Fluorescence (% of control) = [(Fe-Fu)/Fu] x 100

Where Fe and Fu were the mean fluorescence intensities obtained from dye-

exposed and dye-unexposed slices respectively.









CaMKII Activity Assay

Hippocampi were isolated from aged F344 rats as described above. CA1 region

was separated from the rest of the hippocampus, collected in an eppendorf tube, flash

frozen in liquid nitrogen and stored at -800C. The frozen CA1 tissue samples were

placed in a dounce homogenizer containing 1 mL of the homogenization buffer

(sucrose, 1M Tris pH 7.5, 1M KCI, protease inhibitor, protein phosphatase 1 inhibitor,

protein phosphatase 2 inhibitor, 100 mg/mL sodium butyrate, 0.1 M phenyl methyl

sulfonyl fluoride; all prepared in distilled H20) and homogenized using at least six

strokes of the pestle. Homogenates were centrifuged at 7700 x g for 10 minutes at 40C.

The supernatant (containing the cytosolic fraction) was carefully isolated and stored at -

800C. Protein concentrations of the cytosolic fractions were determined using the BCA

assay method (Pierce, Rockford, IL). CaMKII activity in the cytosolic fraction was

measured using the CaMKII assay kit (CycLex Co., Ltd, Nagano, Japan). Briefly,

uniform amount of cytosolic extracts (protein concentration = 2.0 pg per well) were

loaded onto micro titer wells coated with a specific peptide substrate for CaMKII -

Syntide-2, along with kinase reaction buffer with or without Ca2+/calmodulin. Purified

CaMKII (30 milli units per reaction; CycLex Co Ltd) was used as positive control and

cytosolic extracts incubated with EGTA + myr-AIP (CaMKII specific peptide inhibitor)

was used as negative control to obtain a measure of comparison for the CaMKII activity

in the DTT treated and untreated cytosolic extracts. CaMKII activity is expressed as

spectral absorbance units at 450 nm, normalized to the control.

Statistical Methods for Analysis of Data

Statistical analyses for purposes of hypothesis testing were performed using Stat

View 5.0 (SAS Institute Inc, NC) and Excel 2003 (Microsoft Corp., Seattle, WA).









Student's t-tests (paired or unpaired as applicable) were used to examine for

differences between data sets. The statistical level of significance was set at p<0.05.

For tests involving more than one factor, analysis of variance (ANOVA) was employed.

In general, ANOVA was followed by Fisher's protected least significant difference

(PLSD) post hoc analysis in order to localize individual differences among various data

sets. Unless otherwise stated, the effects of pharmacological treatments on the synaptic

transmission are represented as percent of baseline (Mean Standard Error of Mean).

Repeated measures ANOVA was used for statistical analysis of data sets obtained

sequentially from the same experimental setup over time. For example, repeated

measures ANOVA was used to compare the NMDAR-fEPSP obtained for consecutively

higher stimulus intensities from a single hippocampal slice and recording setup. Where

stated, n represents the number of slices used in each experiment, an indication of the

statistical power of the analysis.










Dissecting plane


L -\


fEPSP


~Ii


Figure 2-1. Hippocampal dissection and setup for electrophysiological recordings. A).
Depiction of the dissecting plane in the hippocampus. The dissecting plane is
parallel to the alvear fibers. B). Depiction of the hippocampal slice indicating
the position of the recording (R) and stimulating (S) electrodes. The gray
outline indicates the CA1 pyramidal cell layer. C). The fEPSP recorded from
one such arrangement. Calibration Bars: 10 ms (horizontal) and 0.5 mV
(vertical).









A B



NMDAR-fEPSP A
I0 (+AP-5)




Total fEPSP cs o
20 ms 20 ms

D
C
> 10 mV

40 ms
mAHP sAHP
IAHP





Figure 2-2. Analysis of electrophysiological signals from hippocampal slices. A)
Representative traces demonstrating the total field potential (Total fEPSP,
black trace), the pharmacologically isolated NMDA receptor mediated
synaptic response (NMDAR-fEPSP, blue trace) and the NMDAR-fEPSP in the
presence of the NMDAR antagonist AP-5 (NMDAR-fEPSP (+AP-5), red
trace). B) Representation of the measurement of the fEPSP amplitude (blue)
and the fEPSP slope (red) from a representative fEPSP (black trace).
Calibration bars in A and B: 20 ms; 0.5 mV. C) Representation of the
measurement of the amplitude (blue) of the intracellular NMDAR mediated
synaptic potentials obtained from single CA1 pyramidal neurons in the
hippocampus of aged animals. Calibration bars: 40 ms, 2 mV. D)
Representative trace of the intracellular post-burst afterhyperpolarization (red
trace), indicating the fast (fAHP), medium (mAHP), and slow (sAHP)
components. The step current used to elicit a train of 5 action potentials is
indicated beneath the AHP trace. Calibration bars: 100 ms, 10 mV.









CHAPTER 3
REDOX STATE DEPENDENT CHANGES IN NMDA RECEPTOR MEDIATED
SYNAPTIC TRANSMISSION IN AGED HIPPOCAMPUS

Introduction

The NMDAR is a major source of Ca2+ influx into the postsynaptic neurons during

the induction of LTP at hippocampal CA3-CA1 synapses (Bliss and Collingridge, 1993).

The CA1 region specific knockout of the NR1 subunit of the NMDAR abolishes LTP and

impairs spatial learning and memory (Tsien et al., 1996). Similar deficits in LTP and

spatial memory are observed in aged, memory impaired animals. Most importantly,

preliminary studies have indicated that the NMDAR component of the synaptic

transmission at the CA3-CA1 synapses is decreased in aged animals (Barnes et al.,

1997; Billard and Rouaud, 2007). These results have given rise to the "NMDAR

hypofunction" hypothesis in the hippocampus during aging. The NMDAR hypofunction

hypothesis suggests that age-related LTP and memory deficits are due to a decrease in

the NMDAR mediated component of synaptic transmission (Foster, 1999; Rosenzweig

and Barnes, 2003; Foster, 2007). This idea is further supported by reports indicating

that the NMDARs contribute less Ca2+ to the induction of LTP in the CA1 region of aged

hippocampus, when compared to the young hippocampus (Norris et al., 1998a; Shankar

et al., 1998; Boric et al., 2008). However, it is still unclear what age-related mechanism

underlies the NMDAR hypofunction.

Age-related alterations that may contribute to the NMDAR deficits include altered

subunit expression, composition, and splice forms (Magnusson et al., 2005; Magnusson

et al., 2006). However there is a debate concerning whether the NMDAR subunit

expression decreases at the CA3-CA1 synaptic sites (Foster and Kumar, 2002; Kumar

et al., 2009). It is thus possible that NMDAR hypofunction is related to posttranslational









modifications associated with oxidation/reduction and/or phosphorylation state rather

than number and/or type of receptor subunits. Interestingly previous research examining

the ability of reducing and oxidizing redoxx) agents to modulate NMDAR activity in cell

cultures and in tissue from neonates suggests that redox state is an important

determinant of NMDAR function (Aizenman et al., 1989; Aizenman et al., 1990; Bernard

et al., 1997; Choi and Lipton, 2000; Choi et al., 2001).

In this study we tested the hypothesis that the age-related NMDAR hypofunction is

due to increased oxidative stress or a more oxidative redox state of the aged neuron.

The current studies confirm that the NMDAR mediated synaptic potentials are

decreased at CA3-CA1 synapses of the aged hippocampus. The NMDAR responses

were modified by redox agents in an age-dependent manner; such that oxidizing agents

decreased NMDAR responses to a greater extent in young than in aged animals, and

reducing agents increased NMDAR responses to a greater extent in aged than in young

animals. However, using a combination of extracellular and intracellular recordings with

the relatively membrane impermeable reducing agent L-GSH, we found that intracellular

redox state mediates that age-dependent shift in NMDAR responses. Moreover the

intracellular redox state dependent increase in NMDAR function was independent of L-

type VGCC activity. Thus, the results provide a link between oxidative redox state and

decrease in NMDAR mediated synaptic transmission.

Results

NMDA Receptor Function Decreases in the Hippocampus of Aged Animals at
Various Levels of Pre-synaptic Fiber Volley Amplitude

One of the potential mechanisms that might explain the loss of hippocampus

dependent learning and memory function is decrease in the NMDAR function of the









CA1 pyramidal neurons. To study the alterations in the NMDAR function during aging,

the NMDAR mediated field excitatory postsynaptic potentials (NMDAR-fEPSPs) were

pharmacologically isolated (Kumar and Foster, 2004), recorded and analyzed from the

hippocampus of young and aged F344 rats (Fig. 3-1A). The stimulation evoked

presynaptic fiber volley (PFV) served as an indicator of the level of axon activation that

gave rise to the NMDAR-fEPSP; thus enabling the comparison of NMDAR-fEPSP

amplitudes across the age groups at increasingly higher PFV amplitudes (Table. 3-1).

To examine the relationship between PFV and NMDAR-fEPSP across the two age

groups, the PFV amplitude was separated into 0.4 mV bins and plotted against the

corresponding NMDAR-fEPSP amplitude obtained from the aged and the young

animals (Fig. 3-1B). An ANOVA revealed that the amplitude of the NMDAR-fEPSP was

reduced in the aged animals (n = 6 animals) when compared to young animals (n = 5

animals) [F (1, 47) = 27.47, p<0.0001]. In fact, the maximum amplitude of the NMDAR-

fEPSP was 0.73 0.14 mV and 2.87 0.9 mV in aged and young animals, respectively,

approximately exhibiting a fourfold decrease during aging (Table 3-1).

Oxidizing Agents Decrease NMDAR Function in Young, but not in Aged,
Hippocampal Slices

To test the hypothesis that the decrease in the NMDAR response was related to

oxidizing conditions X/XO, an enzyme substrate combination which produces two types

of ROS superoxide anion and hydrogen peroxide, was applied to hippocampal slices

of young and aged animals. X/XO has been previously used in an independent study

that evaluated the effects of ROS on hippocampal synaptic transmission and plasticity

(Knapp and Klann, 2002).









The stimulation intensity was set to evoke a response which was 30% to 50% of

the maximal NMDAR-fEPSP attainable in that pathway, and the slope of the NMDAR-

fEPSP was measured before and after pharmacological manipulations. Paired Student's

t-test revealed that application of X/XO (20 pg/mL/ (0.25 units/mg of xanthine)) for 60

min, significantly decreased the slope of the NMDAR-fEPSP from the baseline levels in

the young (p<0.01) but not in the aged animals. Furthermore, application of X/XO

significantly [F (1, 10) = 15.49, p<0.01] decreased the NMDAR-fEPSP slope to a greater

extent in the young animals (66.37 7.04%, n = 5), when compared to the aged

animals (96.41 6.14%, n = 7) (Fig. 3-2). Paired t-test on the percent change in the

PFV amplitudes (corresponding to the last 5 min of the 60 min recording) from the X/XO

treated files indicated no effect (p > 0.05) of X/XO on the level of presynaptic axonal

activation across the age groups (young: 95.16 + 8.29%; aged: 108.23 + 19.44%).

NMDAR Function in Young Animals Recovers From Exposure to Higher
Concentrations of Oxidizing Agents

The effect of oxidizing agents on the NMDAR function in young animals was

reversible, such that even in the presence of a higher concentration of xanthine oxidase

(X/XO: (20 pg/mL) / (1 unit/mg of xanthine)), the NMDAR mediated synaptic response

decreased to 54.21 5.79%; however the response recovered to 88.11 5.41% of the

baseline (n = 5) following a 50 minute washout (Fig. 3-3). One possibility is that the

redox state of the young hippocampal neurons is comparatively more reduced than

oxidized, thus enabling a quick recovery of the NMDAR-fEPSP in the young animals

upon washout of the oxidizing agent. Furthermore, the recovery of the NMDAR-fEPSP

following washout indicates that young animals possess sufficient antioxidant and/or

redox buffering capacity to absorb the excess oxyradicals due to application of X/XO.









Reducing Agents Increase NMDAR Function Selectively in Aged Hippocampus

The age-dependent sensitivity of NMDAR-fEPSP to oxidizing conditions suggests

that the components of NMDAR signaling system are initially oxidized to a greater

extent in aged animals. To test whether the decline in the NMDAR response in aged

animals might be due to the age-dependent increase in the formation of disulfide

linkages on the cysteine residues, the reducing agent DTT was applied to hippocampal

slices from young and aged animals, and its effect on the NMDAR function in both the

age groups was studied. DTT can reduce the disulfide bonds on cysteine residues of

proteins into free thiols (Ciorba et al., 1997; Cai and Sesti, 2009; Long et al., 2009), thus

partially reversing the effect of increased oxidative stress during aging. Paired t-test

revealed that DTT significantly enhanced the slope of the NMDAR-fEPSP from the

baseline levels in both the aged (p<0.0001) and young (p<0.05) animals. However, bath

application of DTT (0.7 mM, 45 min) significantly [F (1, 19) = 5.49, p<0.05] increased

the slope of the NMDAR-fEPSP to a greater extent in the aged animals (171.38

13.26%, n = 16) when compared to young animals (114.55 4.41%, n = 5) (Fig. 3-4A,

3-4B, 3-4C). Paired t-test on the PFV amplitude before and after application of DTT

confirmed no change (p > 0.05) in the PFV amplitude for aged (102.81 + 3.84) and

young (97.08 + 5.41) animals indicating that the effect of DTT on the NMDAR mediated

response was not due to changes in the number of axons activated.

In a subset of these files, DTT was allowed to washout of the recording chamber,

while its effect on the aged NMDAR-fEPSP was continuously monitored. Interestingly,

the enhancement of the NMDAR-fEPSP in the aged animals was maintained (167.32 +

20.91%, n = 5) following a 45 min washout of DTT (Fig. 3-4D). This finding indicates a

persistent change associated with the NMDAR function upon application of the reducing









agent DTT. It raises the possibility that intracellular signaling cascades that are known

to regulate the NMDAR function could underlie the DTT-mediated increase observed in

aged animals.

Intracellular Location of Redox Sensitive Cysteines Revealed by Differential
Application of Biologically Available Reducing Agent L-Glutathione

The results from the experiments using the membrane permeable reducing agent,

DTT (Susankova et al., 2006), left open the question of the exact location of the redox

sensitive cysteine residues. Sequence comparison with the structurally similar bacterial

periplasmic binding protein predicts that the NMDARs must possess at least 7 cysteine

residues on their extracellular surface (Choi and Lipton, 2000; Choi et al., 2001), which

are redox sensitive and controls the function of NMDARs. In order to delineate the

location of the cysteines that are responsive to the application of DTT, the biologically

available and membrane impermeable reducing agent L-GSH was used to study its

effect on the aged NMDAR-fEPSP. L-GSH is relatively membrane impermeable such

that exogenous application of L-GSH is not effective in increasing intracellular free thiols

when compared to DTT (Mazor et al., 1996; Zou et al., 2001; Susankova et al., 2006).

Moreover previous findings suggest that L-GSH protects hippocampal neurons against

damage due to oxidative stress (Shin et al., 2005; Shih et al., 2006; Yoneyama et al.,

2008). Surprisingly, extracellular application of the reduced form of L-GSH (0.7 mM, 45

min) did not alter NMDAR-fEPSP in the aged animals (104.83 8.39%, n = 6) (Fig. 3-5).

Since extracellular application of the membrane impermeable L-GSH failed to

enhance NMDAR function, L-GSH was delivered into the intracellular compartment of

the aged neurons using sharp microelectrodes. The NMDAR mediated intracellular

EPSP was simultaneously recorded. Inclusion of the reduced form of L-GSH (0.7 mM to









1.4 mM) in the intracellular recording pipette significantly [F(1,6) = 6.87, p<0.05]

enhanced the amplitude of the NMDAR mediated synaptic potentials (203.90 31.38%,

n = 5) in single hippocampal CA1 pyramidal neurons from aged hippocampus when

compared to age matched control cells (91.55 22.94%, n = 3), for which L-GSH was

not included in the intracellular recording pipette (Fig. 3-6A). Application of 100 pM of

NMDAR antagonist AP-5 abolished the intracellular EPSP (Fig. 3-6B, right), suggesting

that the recorded component was specifically due to the activation of the NMDARs in

the aged hippocampal neurons. The fact that intracellular delivery, but not extracellular

application, of L-GSH enhanced the NMDAR response provides strong evidence for

intracellular redox status as a mechanism for the age-associated modulation of NMDAR

function.

One possibility was that intracellular delivery of the reduced form of L-GSH was

increasing the activity of the L- type VGCCs. To test whether L-channel activity was

modified, intracellular NMDAR EPSP was recorded in the presence of L-GSH and the L-

type VGCC antagonist nifedipine. Intracellular delivery of L-GSH in the presence of

nifedipine (10 pM) continued to enhance the amplitude of the NMDAR mediated

intracellular synaptic potentials (223.54 46.56%, n = 3) suggesting that the DTT effect

is not due to differences in L-channel activity (Fig. 3-7A, 3-7B).

Reducing Agent Mediated Recovery of NMDAR Function is Reversed by Oxidizing
Agent, and Specific to NMDARs

To test whether the NMDAR-fEPSP in aged animals was indeed affected by the

redox environment, the oxidizing agent 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) was

applied subsequent to the DTT-mediated enhancement of the NMDAR response in

aged animals. Bath application of DTNB (0.5 mM, 45 min) significantly decreased









(p<0.05) the DTT-mediated increase in NMDAR-fEPSP in aged animals, such that the

NMDAR-fEPSP slope was 86.49 5.54% (n = 4) of the baseline (Fig. 3-8A). A repeated

measures ANOVA comparing the mean NMDAR-fEPSP slope corresponding to the last

5 min under each condition indicated a significant [F(2,9) = 6.24, p<0.05] difference

(Fig. 3-8 B). Post-hoc analysis revealed that the NMDAR-fEPSP slope was significantly

(p<0.05) increased under DTT compared to baseline or DTNB (p<0.05), and no

significant difference was observed between the NMDAR-fEPSP slopes in the baseline

and under DTNB.

In order to delineate the specificity of the DTT effect on the NMDARs, the

NMDAR-fEPSP was isolated and 100 pM AP-5 was applied prior to and during the

application of DTT. With NMDARs blocked, there was no effect of DTT on any residual

component of the field potential, such that the percent change in the residual fEPSP

was 109.43 7.15% (n = 6) of the baseline (Fig. 3-8C). Finally, the NMDAR-fEPSP was

abolished by application of AP-5 (Fig. 3-8D) indicating that the response was generated

by the activation of NMDARs.

To further examine the specificity of the DTT effects, the AMPAR component of

synaptic transmission was studied before and after application of DTT in aged

hippocampal slices. Application of DTT did not affect (p>0.05) the AMPAR component

of the synaptic response such that responses were 101.63 2.89% (n = 10) of the

baseline after application of DTT for 45 minutes (Fig. 3-9A). In this case, the AMPAR

component was recorded as the initial descending phase of the synaptic response

(covering a 15 ms to 20 ms window) indicated in Fig. 3-9B. Finally, the AMPAR

component of the fEPSP was isolated in the presence of 100 pM AP-5, and DTT was









applied to the non-NMDAR or predominantly AMPAR component of the synaptic

transmission. Application of 0.7 mM DTT under these conditions did not significantly

alter (p>0.05) the AMPAR-fEPSP from the baseline levels (94.27 4.42%, n = 10) (Fig.

3-9C, Fig. 3-9D).

Together, the results indicate that oxidation of sulfhydryl groups can rapidly

regulate responsiveness of NMDARs, and that an age-related reduction in the NMDAR

response is linked to an oxidative redox state. Moreover this effect was exclusive to the

NMDARs and had no discernable effect on the AMPAR component of the synaptic

transmission.

Discussion

In this study we have used extracellular and intracellular recording techniques to

understand the alterations in NMDAR responses during aging. The results presented in

this chapter confirmed an age-related decrease in the NMDAR response (or "NMDAR

hypofunction") and demonstrate age-dependent effects of redox modulators on the

NMDAR response. First, the oxidizing agent X/XO selectively decreased NMDAR-

fEPSP in young but not aged hippocampal neurons. The age dependent sensitivity of

NMDAR function to oxidizing condition suggests that the NMDAR signaling system is

oxidized to a greater extent in aged animals. Moreover, the recovery of NMDAR

response under higher concentrations of X/XO in young neurons indicates sufficient

redox buffering capacity in the young animals. Second, application of the reducing

agent DTT increased NMDAR-fEPSP selectively in the aged but not in young

hippocampal slices, suggesting that NMDAR signaling components are relatively less

oxidized in young animals.









The redox state of the extracellular cysteine residues of the NMDARs have been

implicated in regulating the NMDAR function in cell cultures and neonatal animals

(Aizenman et al., 1989; Aizenman et al., 1990; Bernard et al., 1997). However

extracellular application of the membrane impermeable biological reducing agent L-

GSH failed to increase NMDAR-fEPSP. In contrast, intracellular application of L-GSH

increased NMDAR mediated synaptic responses in single aged neurons. The use of

NMDAR antagonist AP-5 confirmed that the intracellular response was mediated solely

by NMDARs. The L-GSH mediated increase in intracellular NMDAR response was

independent of L-type VGCC activity. Taken together, these results indicate that the

age-related decrease in NMDAR function is due to a shift in the intracellular redox state

favoring an oxidative state. This particular result is consistent with recent work in

hippocampal cell cultures indicating a decrease in the intracellular redox ratio during

aging, due in part to a deficit in the reduced form of GSH inside neurons (Parihar et al.,

2008).

Furthermore, in accordance with previous reports (Gozlan et al., 1995; Abele et

al., 1998), the reducing agent DTT had no effect on the AMPAR function of aged

neurons. We have demonstrated this by studying DTT's effect on total fEPSP and

AMPAR-mediated fEPSP, which was isolated by the application of NMDAR antagonist

AP-5. With NMDARs blocked, there was no effect of DTT on any residual component of

the field potential.

One of the consequences of NMDAR hypofunction is that aged neurons could

engage a compensation mechanism in order to maintain Ca2+ homeostasis. Thus during

aging the decreased Ca2+ contribution from the NMDARs is offset by the increased Ca2+









contribution from the L-type VGCCs and ICS, as discussed previously. This situation

poses a problem from a functional standpoint. The NMDARs are located at synaptic

sites that are in close proximity to the Ca2+ sensitive kinases (Ex. CaMKII) and the

postsynaptic density, which contain numerous signaling molecules that are critical for

the induction and expression of synaptic plasticity. NMDAR hypofunction during aging

would decrease the activation of these signaling cascades. Furthermore, L- type

VGCCs are mostly clustered at the base of dendrites (Westenbroek et al., 1990), thus

limiting their ability to activate the Ca2+ sensitive signaling cascades at the distant

synaptic sites.

Our results suggest that age-related NMDAR hypofunction is the consequence of

a shift in the intracellular redox state to a more oxidative state. The various alternative

explanations for NMDAR hypofunction are discussed below. Excessive NMDAR

activation is thought to contribute to excitotoxicity and neuronal death (Waxman and

Lynch, 2005). Thus NMDAR hypofunction could be regarded as a cellular response to

prevent excitotoxicity, and impaired NMDAR-dependent synaptic plasticity and memory

decline may be the consequence of processes that mediate cell preservation (Foster,

1999). Alternatively it is possible that alterations in the NMDAR localization, through the

insertion of receptors into the membrane or recruitment of extra-synaptic receptors into

the synapse, may have important effects on NMDAR function during aging. However, it

remains to be determined whether altered localization of the NMDARs (specifically

extra-synaptic localization) is the mechanism by which the NMDAR function declines

during senescence. Preliminary reports indicate that the NR2B subunit levels, but not

the obligatory NR1 subunit levels of NMDARs, decrease in the synaptic sites (Zhao et









al., 2009). However these findings raise the possibility that the loss in NR2B at the

synaptic sites could possibly be compensated by NR2A/2C/2D subunits that can

combine with the unaltered NR1 subunits and form a normally functioning receptor.

Another likely candidate mechanism extends the idea of posttranslational changes

investigated in this chapter. The NMDAR function is regulated by phosphorylation state

of its cytosolic tail, which is determined by the activity of neuronal kinases and

phosphatases. In the next chapter we have demonstrated the role of one such kinase

(CaMKII) in the DTT-mediated increase in the NMDAR function of aged animals.

In conclusion, the results presented in this chapter demonstrate a link between

age-related decline in NMDAR function and increased oxidative stress or a more

oxidative redox state of the aged neurons. Most importantly we have demonstrated that

the NMDAR function can be recovered in the aged CA1 pyramidal neurons upon

application of DTT. This is a veritable starting point for treatment of NMDAR

hypofunction.









A

Young Aged




~ 1 mV
S10 ms


B
S3.0 O Young
SE Aged
i.. 2.0




04
0.4 0.8 1.2 1.6 2.0 2.4 2.8
Pre synaptic Fiber Volley Amplitude (mV)


Figure 3-1. NMDAR mediated synaptic potentials (NMDAR-fEPSP) are reduced in area
CA1 of the hippocampus during aging. A) Representative traces of NMDAR-
fEPSPs obtained at consecutively higher stimulus intensities from the young
(left) and aged animals (right). Open and filled arrows indicate the PFV and
NMDAR-fEPSP, respectively. As observed in the traces, the aged animals
exhibit a markedly reduced NMDAR mediated synaptic potential. Calibration
bars: 10 ms, 1 mV. B) Plot of the mean NMDAR-fEPSP amplitude versus the
PFV amplitude (at 0.4 mV inning width). The aged animals (filled circles) (n
= 6) exhibited reduced NMDAR-fEPSP when compared to the young animals
(open circles) (n = 5). In this and subsequent figures error bars represent
S.E.M.









X (20 pglmL) I
XO (0.25 Ulmg of X)


0 10 25 40 55 70
Time (minutes)


150


S'VUF i 0,-
2 Q)XO



o.5 mV m?-
4 : 50x
oAGnD 20 ms


Control


Aged Young
XIXO XIXO


Figure 3-2. The oxidizing agent X/XO decreases NMDAR mediated synaptic potentials
in young animals but not in aged animals. A) Time course of the change in
the normalized NMDAR-fEPSP slope in the aged (filled circles) (n = 7) and in
the young animals (open circles) (n = 5) following application of X/XO (20
pg/mL/ (0.25 unit/mg of xanthine)) for 60 minutes. B) Representative traces
(average of 5 traces under each condition) illustrating the change in the
NMDAR-fEPSP in the young (top) and aged animals (bottom) under control
conditions and at the end of a 60 minute application of X/XO. Calibration bars:
20 ms, 0.5 mV. C) Quantification of mean percent change in the NMDAR-
fEPSP slope from baseline (dashed line), corresponding to the last 5 minutes
of a 60 minute application of X/XO in aged (filled bar) and young (open bar)
animals.


A
a.
Co

0




0
z


'I

C;;.
1

0)

S-
50


B. ~ I lb










0 150- X (20 pgml) I
0.
0.- XO (1 U/mg of X)
5 0 ----------



& so 2
z 0
II I I I I
0 10 25 40 55 70 90
Time (minutes)
B
C 150-

2 Z J
IL=100-----------
3 0.5 mV V) T

20 ms < 50-


0
Young Washout
X/XO


Figure 3-3. Effect of maximal concentrations of X/XO on NMDAR mediated synaptic
potentials in young animals. A) Time course of the change in the normalized
NMDAR-fEPSP slope in the young animals (n = 5) following application of
X/XO (20 pg/mL/ (1 unit/mg of xanthine)) for 30 minutes and followed by a
washout for 50 minutes. B) Representative traces (average of 5 consecutive
traces) obtained during the indicated time points: baseline (1), under X/XO
(2), and upon washout (3). Calibration bars: 20 ms, 0.5 mV. C) Quantification
of the mean percent change in the NMDAR-fEPSP slope in young animals
following the application of X/XO for 30 minutes (filled bar) and after washout
for 50 minutes (open bar). In this and subsequent figures dashed lines
represent the baseline level of 100%; pound signs indicate significant
difference (p<0.05) from baseline level of 100%; asterisks indicate significant
difference (p<0.05) between the indicated groups.










A 250

200
I -

p 150

'0 100

r so
I 50

0



C
AGED


| LUU



os
oZ 50.
z


I-


Aged Young
DTT DTT


0 10 25 40 55


Time (minutes)
D


DTT (0.7 mM)


0.-
DTT .


YOUNG
10 ms Q0

| Control

DTT


0 10 25 40 55 70
Time (minutes)


Figure 3-4. The reducing agent DTT increases NMDAR mediated synaptic responses to
a greater extent in aged than in the young animals. A) Time course of the
change in the normalized NMDAR-fEPSP slope in the aged (filled circles) (n =
16) and young animals (open circles) (n = 5) following application of DTT for
45 minutes. B) Quantification of the mean percent change in the NMDAR-
fEPSP slope following application of DTT in aged (filled bar) and young (open
bar) animals. C) Representative traces (average of 5 consecutive traces
under each condition) illustrating the change in NMDAR-fEPSP in the
presence of DTT in aged (top) and young animals (bottom). Calibration bars:
10 ms and 0.5 mV. D) Time course of the change in the normalized NMDAR-
fEPSP slope in aged animals (filled circles) (n = 5) upon application of DTT
for 45 minutes followed by a washout for 45 minutes.


85 100


^AA~


1











200-


S150





50-
0. 50-


I I


L-glutathione (0.7 mM)


'I I,


0 10
0 10


I I
25 40
Time (minutes)


0.5 mV


Figure 3-5. Extracellular application of reduced L-glutathione does not affect NMDAR
function. A) Time course of the normalized NMDAR-fEPSP slope in the aged
animals (n = 6) in response to extracellular application of the L-GSH. B)
Overlay of the means of 5 consecutive responses during the baseline (black
trace) and 45 minutes after application of L-GSH (gray trace). Calibration
bars: 20 ms (horizontal) and 0.5 mV (vertical).









A
L .400-
.W
W 300 I

S,,200- I

z0--- i
|S 100 '8^^--47.
0 Q L-GSH o Control
E E 0-
CU 0 5 10 15 20 25 30 35 40
Time (minutes)

B
L-GSH
(30min) Baseline


Baseline Bas

AP-5



Figure 3-6. Intracellular application of reduced L-glutathione enhances intracellular
NMDAR mediated synaptic potentials. A) Time course of the normalized
NMDAR mediated intracellular EPSP amplitude in the aged animals obtained
under control conditions (open circles, n = 3) or with L-GSH in the recording
pipette (filled circles, n = 6). B) Left: Overlay of means from 5 consecutive
responses obtained intracellularly during baseline (black trace) and 30
minutes after impalement (gray trace). Right: Overlay of means from 5
consecutive traces obtained intracellularly during baseline (black trace) and
25 minutes after application of AP-5 (gray trace). Calibration bars: 40 ms
(horizontal) and 2 mV (vertical).









A
a.
0.
a
uJ




L
0

<-
Qc


SA Nifedipine + L-GSH
0 5 10 15 20 25 30 35 40


Time (minutes)


Nifedipine+ L-GSH
(30 mn)



Baseline


Figure 3-7. Glutathione mediated recovery of NMDAR function in aged animals does not
involve L-type VGCC. A). Time course of normalized NMDAR mediated
intracellular EPSP amplitude obtained with nifedipine along with ACSF and L-
GSH in the recording pipette (filled triangles, n = 3). B) Overlay of means from
5 consecutive responses obtained intracellularly during baseline (black trace)
and 30 minutes after impalement (gray trace). Calibration bars: 40 ms
(horizontal) and 2 mV (vertical).









A 200. DTT (0.7 mM) B
DTNB (0.5 mM)

a 200

,, .*I =150
A 100 U.. .......... 1

S DTT DTNB
.-------.-,----. ,B -L
0 10 25 40 5S 70 s 100 Z DTT DTN
Time (minutes)
C

i 2)00 AP-5 (100 pM)
0o DTT (0.7 mM) D
(A 150
(0...160 --------.---- t----.a
CC;
10 "d r "






0 20 40 0 8i0
Time (min)


Figure 3-8. Redox modification of cysteine residues underlies NMDAR specific effect of
DTT. A) Time course of the change in the normalized NMDAR-fEPSP slope in
aged animals (n = 4) in response to the bath application of DTT followed by
DTNB. The increase in NMDAR mediated synaptic responses by DTT was
decreased by the oxidizing agent DTNB. Error bar in A is indicated for every
fourth point, for purposes of clarity. B) Quantification of the mean percent
change in NMDAR-fEPSP slope following application of DTT (filled bar) and
DTNB (open bar) in aged animals. C) AP-5 (100 pM) was applied on the
isolated NMDAR-fEPSP to block it, prior to DTT application. Time course of
the change in any negative slope measured in a 20 ms window (n = 8) upon
application of AP-5 followed by DTT. D) Representative traces (average of 5
consecutive traces) of the NMDAR-fEPSP recorded under control conditions
(solid black trace), and upon application of 100 pM AP-5 (dashed black trace)
for at least 30 min.








A B
150
DTT (0.7 mM)







20 35 50 65
Time (min)
C D
10- AP-5 (100 \ M)









0 DTT (0.7 mM)




0e ACSF + AP-5 (100 pMl)
C D










S 20 40 0 80
Time (min)


Figure 3-9. DTT does not affect the AMPAR function of aged animals. A) Time course of
the change in fEPSP upon application of DTT for 45 minutes in aged
hippocampal slices (n = 10). B) Representative traces (average of 5
consecutive traces) of the fEPSP recorded under control conditions (black
trace) and after application of DTT for 45 mi (gray trace). The downward
pointing arrows and cursors indicate the 15 ms time window, where the initial
descending phase of the fEPSP is predominantly mediated by AMPARs.
Calibration bars: 20 ms and 0.5 mV. C) Time course of the change in the
AMPAR mediated EPSP (isolated by the application of 100 pM AP-5 on the
fEPSP) upon application of 0.7 mM DTT in the aged animals (n = 5). D)
Representative traces (average of 5 consecutive traces) of the AMPAR
mediated EPSP obtained under control conditions (black trace) and after
application of DTT for 45 minutes (gray trace). Calibration bars: 20 ms, 0.5
mV.









Table 3-1. The NMDAR-fEPSPs from hippocampus of young and aged animals
PFV Bin Window Young NMDAR-fEPSP Aged NMDAR-fEPSP
(mV) (mean S.E.M) (mean S.E.M)
0 0.4 0.13 0.04 0.12 0.04
0.4 0.8 0.49 0.16 0.26 0.05
0.8 1.2 0.85 0.21 0.32 0.09
1.2 1.6 1.21 0.29 0.46 0.15
1.6 2.0 1.61 0.41 0.39 0.14
2.0 2.4 1.86 0.46 0.44 0.16
2.4 2.8 2.21 0.87 0.62 0.21
2.8 8.0 2.87 0.91 0.73 0.14
This table presents the NMDAR-fEPSP values recorded from the CA1 region of the
hippocampus from young and aged animals. The NMDAR-fEPSP has been grouped under bin
width of 0.4 mV of PFV amplitude.









CHAPTER 4
MOLECULAR MECHANISM UNDERLYING RECOVERY OF NMDAR FUNCTION AND
HIPPOCAMPAL SYNAPTIC PLASTICITY IN AGED ANIMALS

Introduction

Age-related decrease in the NMDAR function of the hippocampal CA1 pyramidal

neurons was reversed by the application of the reducing agent DTT or intracellular

application of L-GSH (results from chapter 3). The strong links to intracellular redox

state suggested a role for an intracellular signaling mechanism in causing the DTT-

mediated increase in NMDAR function.

Extensive empirical evidence suggests that the NMDAR function is regulated by

the phosphorylation state of the receptor. NMDAR function is increased upon

phosphorylation by several intracellular kinases (Ben-Ari et al., 1992; Westphal et al.,

1999; Li et al., 2001). Specifically the activation of tyrosine kinase (Wang and Salter,

1994; Heidinger et al., 2002), protein kinase C (PKC) (Ben-Ari et al., 1992; Chen and

Huang, 1992), and protein kinase A (Raman et al., 1996) increases NMDAR mediated

currents. In contrast, protein phosphatases, including calcineurin and protein

phosphatase 1, decrease NMDAR currents (Lieberman and Mody, 1994; Wang et al.,

1994; Raman et al., 1996). Moreover, phosphorylation state of NR2A and NR2B

subunits can rapidly regulate surface expression and localization of the NMDARs

(Gardoni et al., 2001; Chung et al., 2004; Hallett et al., 2006; Lin et al., 2006). For

example, phosphorylation of serine residues within the alternatively spliced cassettes of

the C-terminal tail of NR1 promotes receptor trafficking from the endoplasmic reticulum

and insertion into the postsynaptic membrane (Scott et al., 2001; Carroll and Zukin,

2002). On the other hand, increased phosphatase activity has been linked to the

internalization of NMDARs (Snyder et al., 2005). Hence, the kinases and phosphatases









act like molecular switches, which increase or decrease NMDAR function, respectively.

Interestingly, aging is associated with a shift in the balance of kinase/phosphatase

activity, favoring phosphatases (Norris et al., 1998b; Foster et al., 2001; Foster, 2007).

Thus, alterations in the kinase/phosphatase activity in the postsynaptic neuron could

underlie the decrease in the NMDAR function during aging. Moreover, the enzymatic

activity of these kinases can be regulated by the reduction and oxidation of the cysteine

residues located in their structure (Raynaud et al., 1997; Griendling et al., 2000; Knapp

and Klann, 2000).

We tested whether the DTT-mediated increase in the NMDAR function was

dependent on the activity of kinases and/or phosphatases that regulate the NMDAR

function. Indeed we demonstrate that the mechanism for the age-dependent redox

modulation of NMDARs involves CaMKII, but not PKC, PP1 or calcineurin/PP2B.

CaMKII activity assays established that DTT increased CaMKII activity in CA1 cytosolic

extracts in aged but not in young animals. Evidence is also provided to support the idea

that the reducing agent DTT increases LTP in CA1 region of aged but not young

hippocampal slices. The results presented in this chapter elucidate a molecular

mechanism for the age-related NMDAR hypofunction and links oxidative redox state to

impaired synaptic plasticity in aged CA1 pyrmaidal neurons.

Results

ROS Sensitive Dye Indicates Redox State of Live Hippocampal Neurons in in vitro
Slices

The ROS sensitive dye 5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein

diacetate (c-H2DCFDA) was used to detect ROS in live CA1 pyramidal neurons of the

hippocampus from aged (23 month old) F344 rat. As described in the methods section,









the hippocampal slices were incubated for 30 minutes with c-H2DCFDA. Images of the

aged hippocampal CA1 pyramidal neurons were obtained under bright field illumination

(Fig. 4-1A) and in the presence of a filter set to detect green fluorescence from the dye-

exposed slices (Fig. 4-1B). Dye-unexposed slices obtained from the same aged F344

rat served as the control (Fig. 4-1C); which were used to detect auto-fluorescence. The

green band pass filter (Excitation at 490 nm and Emission at 525 nm) was set to detect

green fluorescence at uniform exposure time of 100 ms, and uniform exposure intensity

set at 150%. The fluorescent signals from the dye-exposed slices were normalized to

the dye-unexposed slices as described in the methods section.

The fluorescent signals originating from the hippocampal neurons incubated with

c-H2DCFDA are used as a direct measure of the levels of ROS; thus mandating a

proper control for auto-fluorescent signals from the hippocampal tissue of aged animals.

For all experiments described here, auto-fluorescence was assessed from dye-

unexposed slices harvested from the same animal. No fluorescent signals were

detected in the dye-unexposed slices, from aged (23 month old) and young (7 month

old) hippocampi, when the imaging was performed with an exposure time of 100 ms

(Fig. 4-2, middle panels). However, when the imaging was performed with an exposure

time of 500 ms, clusters of fluorescent signals were detected in the aged but not in the

young hippocampal slices (Fig. 4-2, right panels). The auto-fluorescent signals in the

aged hippocampal slices could potentially arise from lipofuscin, an oxidized product

known to accumulate during aging. Hence for the purposes of this study the imaging

was performed at exposure times well below 500 ms, in order to eliminate auto-

fluorescent signals and detect the fluorescence predominantly from ROS-oxidized c-









H2DCFDA. For the results described in the following sections the imaging was

performed at an exposure time of 100 ms, which successfully eliminated the auto-

fluorescent signals.

Enhanced ROS Production in the CA1 Region of the Hippocampus of Aged
Animals

The ROS detection technique (standardized as described above) was used to

evaluate differential rates of ROS production in hippocampal slices from young and

aged animals. The slices were incubated with c-H2DCFDA for 30 minutes prior to

imaging (Fig. 4-3A). Dye-unexposed slices (aged control: 36.14 4.42%, n = 3 animals;

young control: 31.97 3.16%, n = 3 animals) showed no significant difference in

fluorescence across the age groups. Thus, fluorescence intensity from c-H2DCFDA -

unexposed slices was used to normalize the fluorescence intensity obtained from c-

H2DCFDA-exposed slices. Incubation of the hippocampal slices from young and aged

animals with c-H2DCFDA for 30 min resulted in significantly (unpaired t-test; p<0.05)

enhanced fluorescence in aged animals (242.19 20.96%, n = 3 animals) when

compared to young animals (141.61 11.78%, n = 3 animals) (Fig. 4-3B).

The dye was designed to detect ROS produced in the intracellular space of the

neurons. However, to further eliminate the possibility of signal contribution from ROS

outside the cells, aged hippocampal slices were incubated with superoxide dismutase

(SOD; 121 units/mL) and catalase (260 units/mL) along with the dye for 30 min prior to

imaging. There was no significant difference (p>0.05) in the ROS-oxidized c-H2DCFDA

fluorescence observed between the SOD/catalase exposed (292.388 28.04%, n = 3)

and unexposed aged hippocampal slices.









Broad Spectrum Ser/Thr Kinase Inhibitor Blocks DTT-Mediated Recovery of
NMDAR Function in Aged Hippocampal Neurons

To test if serine/threonine (Ser/Thr) kinases were involved in the DTT-mediated

increase of the NMDAR response, the broad-spectrum and membrane-permeable

Ser/Thr kinase inhibitor H-7 was bath applied prior to and during the application of DTT.

In the presence of H-7 (10 pM, 45 min), DTT application failed to produce the robust

increase (one-group t-test; p>0.05) in the NMDAR-fEPSP slope (111.86 6.92%, n = 7)

from the baseline levels (Fig. 4-4A).

In order to narrow down the identity of the Ser/Thr kinase that could potentially

underlie the DTT-mediated increase in NMDAR-fEPSP several specific inhibitors were

used. It was likely that Protein Kinase C (PKC) could underlie the DTT-mediated

increase in NMDAR-fEPSP, due to the fact that PKC increases NMDAR function

through phosphorylation mechanisms and PKC's activity could be regulated by redox

mechanisms. To test whether PKC was responsible for the DTT-mediated increase in

NMDAR-fEPSP, the membrane permeable PKC inhibitor, Bis-I (Knapp and Klann,

2002), was applied prior to, and during, the application of DTT on aged hippocampal

slices. Application of Bis-I (500 nM, 45 min) failed to block the DTT-mediated increase

in the NMDAR-fEPSP (142.58 13.06%, n = 6) (Fig. 4-4B). Finally, t-tests indicated no

effect of 0.01% DMSO (96.38 7.64, n = 5) or kinase inhibition per se (H-7: 103.01

4.21%, n = 7; Bis-l: 110.72 11.44%, n = 6) on the baseline NMDAR-fEPSP slope in

aged animals. Thus, DTT-mediated increase in NMDAR function was independent of

PKC activity.









CaM Kinase II specific Inhibitors Block DTT-Mediated Recovery of NMDAR
Function in Aged Hippocampal Neurons

One of the intracellular kinases that can enhance NMDAR function, and whose

activity is regulated by redox agents like DTT, is Ca2+/Calmodulin dependent Protein

Kinase II (CaMKII). To test if CaMKII underlies the DTT-mediated increase in NMDAR

function in aged neurons, the CaMK inhibitor, KN-62, was bath applied prior to and

during the application of DTT. In the presence of KN-62 (10 pM, 45 min) (Tokumitsu et

al., 1990), DTT application failed to produce the robust increase in NMDAR-fEPSP in

aged animals (97.9 7.98%, n = 5) (Fig. 4-5A). Furthermore, the specific peptide

inhibitor of CaMKII, myr-AIP (5 pM, 60 min), which was bath applied prior to and during

the application of DTT, effectively blocked (104.48 4.29%, n = 4) the DTT-mediated

increase in NMDAR-fEPSP in aged animals (Fig. 4-5B). Finally, t-tests indicated no

effect of 0.01% DMSO (96.38 7.64, n = 5) or CaM kinase inhibition per se (KN-62:

96.23 8.3%, n = 5; myr-AIP: 100.52 4.42%, n = 4) on the baseline NMDAR-fEPSP

slope in aged animals.

An ANOVA comparison of the effect of DTT in the presence and absence of the all

the pharmacological kinase inhibitors, indicated a significant effect of kinase inhibition

on the DTT effect in aged animals [F (4, 33) = 5.85, p<0.01]. Post hoc comparisons

(Fisher's PLSD) indicated that the DTT-mediated increase in the NMDAR response was

blocked by H-7, myr-AIP, and KN-62 but not Bis-I (Fig. 4-5C). This conclusively proves

that CaMKII underlies the DTT-mediated increase in the NMDAR-fEPSP slope in aged

CA1 hippocampal neurons.









DTT-Mediated Recovery of NMDAR Function in Aged Animals is Independent of
Neuronal Protein Phosphatases

It has been previously reported that neuronal kinases and phosphatases act in

tandem to regulate the function of the NMDARs. While kinases are known to increase

NMDAR function, phosphatases decrease NMDAR function. The activity of Ser/Thr

phosphatases like calcineurin (CaN) and protein phosphatase 1 (PP1), is thought to

contribute to altered synaptic plasticity during aging (Foster et al., 2001). Moreover,

increase in phosphatase activity decreases NMDAR function through dephosphorylation

of the cytosolic tails of the NMDARs (Lieberman and Mody, 1994; Wang et al., 1994).

To test if DTT effects were mediated by CaN, the CaN inhibitor FK-506 (10 pM) (Norris

et al., 2008) was bath applied for 45 minutes prior to and during the application of DTT

on aged hippocampal slices, while simultaneously recording the NMDAR-fEPSP.

Application of FK-506 per se did not affect the NMDAR-fEPSP slope (109.61 9.16%, n

= 5). Importantly, FK-506 failed to block the DTT-mediated increase in the NMDAR

response in aged animals such that the NMDAR-fEPSP slope increased to 148.61

16.42% (n = 5) (Fig. 4-6A).

To examine the role of PP1, in mediating the DTT effect on aged NMDAR function,

the PP1 inhibitor okadaic acid (OA) (1 pM, 30 min) (Schnabel et al., 2001) was

(Schnabel et al., 2001) applied prior to and during application of DTT on aged

hippocampal slices. Application of OA significantly increased the NMDAR-fEPSP slope

during the baseline recording period itself (121.46 9.19%, n = 5, p<0.05) (Fig. 4-6B).

Therefore, a new stable baseline was recorded prior to the application of DTT. Relative

to the new baseline, DTT increased the NMDAR-fEPSP slope (137.89 3.99%, n = 5)

in the presence of OA (Fig. 4-6C). An ANOVA comparing the effect of DTT in the









presence and absence of the phosphatase inhibitors, indicated no significant effect of

phosphatase inhibition on DTT's effect [F (2, 15) = 1.37, p>0.05]. A summary of the

results obtained with phosphatase inhibitors is presented in Fig. 4-6D.

Long-Term Potentiation is Enhanced in Aged Hippocampal Slices Exposed to
DTT

For studies on Long-Term Potentiation (LTP) and paired-pulse ratio, slices were

bathed in normal ACSF, in order to record the AMPAR and NMDAR component of the

synaptic response. LTP was induced by a single episode of high frequency stimulation

(HFS) of 100Hz secondnd. Hippocampal slices from aged animals were either

incubated in normal ACSF (Aged Control) or incubated in ACSF containing 0.7 mM DTT

(Aged DTT) for at least 45 min prior to the delivery of HFS. In addition, a second

pathway in the same slice that did not receive HFS (but received the baseline test

pulses at 0.033 Hz, at baseline stimulation intensity) was used as the control pathway.

The control pathway was used to monitor changes in slice health and to ensure stability

of recording. In each case the fEPSP was recorded for 20 min prior to, and 60 min after,

delivery of HFS. The magnitude of LTP was greater in aged hippocampal slices pre-

incubated with DTT for 45 min (136.53 2.77%; n = 10) (Fig. 4-7A), when compared to

the aged controls not exposed to DTT (118.15 4.63%; n = 9) (Fig. 4-7B). Although the

levels of LTP showed considerable variation (Fig. 4-7C), the LTP was significantly [F (1,

17) = 12.14, p<0.01] greater in Aged-DTT group than the Aged-Control group.

In order to evaluate the role of presynaptic transmitter release on the DTT

mediated enhancement in aged LTP, the paired pulse ratio was computed. Examination

of paired-pulses delivered at varying inter-pulse intervals (At = 50 ms, 100 ms, 150 ms,

200 ms) (Fig. 4-7D), under control conditions and 45 min after the bath application of









DTT, indicated no effect of treatment across the four inter-pulse intervals (Table. 4-1).

This suggests that the increase in aged LTP under DTT recruits postsynaptic

mechanisms, involving the NMDARs in the postsynaptic CA1 pyramidal neurons.

Reducing Agent does not Alter Long-Term Potentiation in Young Hippocampal
Slices

DTT had selectively enhanced NMDAR function in aged, but not in young,

hippocampal slices. In order to test whether DTT had any age-dependent effects in

enhancing LTP, young hippocampal slices were incubated with either normal ACSF

(Young Control) or ACSF containing 0.7 mM DTT (Young DTT), before delivering LTP

inducing HFS. In contrast to the effect observed in the aged animals, there was no

difference (p>0.05) in the levels of HFS-induced LTP between young controls (130.17 +

8.64%; n = 6) (Fig. 4-8A) and the young slices exposed to DTT (117.09 12.24%; n =

5) (Fig. 4-8B). Furthermore, the fEPSP observed in the HFS pathway was significantly

higher than the fEPSP in the control pathway (Fig. 4-8C).

CaMKII Activity is Enhanced in Aged Hippocampal CA1 Cytosolic Extracts
Treated with DTT

To determine whether DTT was directly influencing CaMKII activity, cytosolic

extracts from CA1 region of the hippocampus from aged animals were assayed for

CaMKII activity by examining the phosphorylation of the synthetic peptide, syntide-2, in

the presence and absence of DTT. Relative to baseline control levels, cytosolic CaMKII

activity was significantly enhanced (p<0.05) in the presence of 0.7 mM (113.11 3.47%,

n = 3) and 1.4 mM DTT (120.46 3.14%, n=3) in the aged CA1 cytosolic extracts (Fig.

4-9A). Higher levels of DTT (2.8 mM) resulted in a decrease in CaMKII activity,

presumably due to denaturation of the enzyme. In addition, the CaMKII activity was









significantly (p<0.05) blocked (Aged: 5.36 2.85%, n = 3) in the presence of Ca2+

chelator EGTA (2 mM) and CaMKII-specific peptide inhibitor myr-AIP (10 pM).

However DTT's effect on CaMKII activity from aged CA1 cytosolic extracts raised

the possibility that DTT was acting on the CaMKII activity regulator calmodulin (CaM),

and not exclusively on CaMKII. In this case the effect of DTT on CaMKII activity will be

reduced by the addition of exogenous and un-oxidized CaM. To test this idea the assay

was repeated in the absence of exogenously added CaM. The CaMKII activity from

aged CA1 cytosolic extracts, in the presence of 0.7 mM DTT, and in the absence of

exogenous CaM, (112.11 3.91%, n = 3) was not enhanced beyond that observed

following addition of exogenous CaM, suggesting that DTT effects were not mediated by

reducing effect on CaM (Fig. 4-9B).

DTT does not Alter CaMKII Activity in Young Hippocampal CA1 Cytosolic Extracts

It was possible that DTT's effect were specific to the oxidized CaMKII present in

the aged CA1 cytosolic extracts, and not the relatively un-oxidized CaMKII present in

the young CA1 cytosolic extracts. To test this idea, the CaMKII activity was measured

from the young CA1 cytosolic extracts, in the presence and absence of DTT. In contrast

to the effect observed in aged animals, DTT had either no effect or decreased CaMKII

activity in CA1 cytosolic extracts from young animals (Fig. 4-10A). However, as

observed in aged animals, the CaMKII activity was inhibited (p<0.05) by the addition of

Ca2+ chelator EGTA (2 mM) and CaMKII-specific peptide inhibitor myr-AIP (10 pM)

(Young: 19.99 9.01%, n = 3). Finally, addition of DTT to purified CaMKII (CycLex Co

Ltd) decreased CaMKII activity (p<0.05) (0.7 mM DTT: 86.62 6.04%, n = 3; 1.4 mM

DTT: 70.72 18.58%, n = 3) (Fig. 4-10B), indicating that the DTT effects were specific

for CaMKII present in the aged CA1 cytosolic extracts.









Discussion

The DTT-mediated enhancement of NMDAR responses was specific to CaMKII

activity because CaMKII inhibitors, myr-AIP and KN-62, blocked the DTT-mediated

increase in NMDAR-fEPSP in aged animals. The DTT effects were not blocked by

inhibition of PKC, PP1, or CaN/PP2B. The results point to CaMKII as a critical link

between the intracellular redox state and NMDAR hypofunction. The role of CaMKII was

further confirmed by enzyme activity assays which established that DTT increased

CaMKII activity only in CA1 cytosolic extracts from aged animals. In contrast, DTT did

not increase CaMKII activity in CA1 cytosolic extracts from young animals. In fact, DTT

decreased the CaMKII activity in a purified sample of CaMKII, presumably due to

enzyme denaturation by the reducing action of DTT.

Upon comparative analysis of all results, an interesting observation arises based

on one of our previous results in chapter 3. In Chapter 3, we demonstrated that the

DTT-mediated increase in NMDAR-fEPSP persisted for more than 45 min after aborting

DTT application (Fig. 3-4D). The results presented in this chapter complement the

previous findings and suggest that the lasting increase in NMDAR function was

sustained by DTT's effect on CaMKII. In fact DTT's effect on NMDARs expressed in

heterologous and non-neuronal systems has a quick onset and immediate washout

(Tang and Aizenman, 1993; Kohr et al., 1994; Choi et al., 2001). The persistent

increase in NMDAR function of aged neurons upon DTT application can be explained

only when we invoke the neuronal CaMKII signaling mechanism, as proved by the

results presented in here.

Oxidation of methionine residues on CaM has been reported to decrease the

ability of CaM to activate CaMKII (Robison et al., 2007). Our results are not likely due to









CaM methionine oxidation since DTT possesses higher selectivity to reduce cysteine

residues over methionine residues (Ciorba et al., 1997; Cai and Sesti, 2009; Long et al.,

2009). In addition, DTT had equivalent effects in activating CaMKII, regardless of

whether exogenous CaM was added to the CaMKII activity assay. The results indicate

that oxidation of CaMKII, rather than CaM, underlies the reduction in kinase activity and

are consistent with a recent report demonstrating that oxidative stress induced by

ischemia results in disulfide linkages on the cysteine residues of CaMKII which

decrease kinase activity (Shetty et al., 2008). While the data provide a link between

age-related changes in intracellular redox state, CaMKII activity, and NMDAR function,

the exact mechanism through which CaMKII regulates the NMDAR response remains to

be determined. In addition to regulating phosphorylation state of proteins, including

AMPARs, synaptic CaMKII participates in protein-protein interactions with several

proteins localized to the dendritic spine which could ultimately alter NMDAR location

and function (Lisman et al., 2002; Robison et al., 2005). In this context an independent

report suggests that reduced CaMKII activity is associated with a specific decrease in

synaptic NMDARs and decreased LTP (Gardoni et al., 2009).

In addition to a role for CaMKII, we observed that PP1 inhibition resulted in a

modest increase in the NMDAR-fEPSP in aged hippocampal neurons. Age differences

in the NMDAR response, which depend on kinase/phosphatase activity, are reminiscent

of the age-dependent effects of kinase and phosphatase inhibitors on the rapid

component of synaptic transmission mediated by AMPARs (Norris et al., 1998b; Hsu et

al., 2002; Foster, 2007) and suggest that PP1 activity contributes to a reduction in

AMPAR and NMDAR components of synaptic transmission (Foster et al., 2001;









Morishita et al., 2005), a characteristic specific to senescent CA1 synapses

(Rosenzweig and Barnes, 2003). Our results indicate that a shift in the intracellular

redox state towards oxidizing conditions during aging may cause or magnify the

imbalance in the kinase/phosphatase activity, favoring phosphatases (Foster, 2007).

Our results using the ROS-sensitive dye provided us a direct and real-time

indication of the intracellular redox state of the hippocampal neurons. Several points

indicate that the readout of our ROS detection experiment was an accurate indication of

the intracellular redox state. First the ROS sensitive dye c-H2DCFDA, is preferentially

cleaved by intracellular esterases to yield a non-fluorescent product; a pre-requisite for

subsequent oxidation by ROS. Second, upon oxidation by ROS, the dye is converted

into a fluorescent product which is membrane impermeable; thus the fluorescent

readout is primarily due to intracellular signals. Finally, extracellular application of SOD

and catalase did not affect c-H2DCFDA fluorescence, simply due to the fact that these

proteins are relatively membrane impermeable, and also because only intracellular

events gave rise to c-H2DCFDA fluorescence. While quantifying ROS-derived

fluorescence from aged neurons, a significantly large auto-fluorescent signal was

detected in dye-unexposed slices, mainly from lipofuscin. Lipofuscin, a breakdown

product of lipid oxidation, is reported to accumulate in the aged hippocampal neurons

(Landfield et al., 1981; Oenzil et al., 1994) and is capable of emitting auto-fluorescent

signals overlapping the upper end of green, and the lower end of the yellow emission

spectra (Haralampus-Grynaviski et al., 2003). Hence the imaging for detection and

quantification of c-H2DCFDA fluorescence was performed at 100 ms time-window in









order to eliminate the auto-fluorescent signals arising from lipofuscin accumulation in

aged neurons.

Finally, NMDAR function is critical to the induction of LTP and we observed that

DTT improved LTP in the CA3-CA1 synapses of aged animals. The interaction of

NMDARs with CaMKII has been proposed as a model of memory (Lisman et al., 2002)

and recent work indicates that disruption of the interaction between CaMKII and

NMDAR impairs the induction of LTP and spatial learning (Zhou et al., 2007). We have

provided evidence to indicate that a more oxidized redox state is a biological

mechanism that can progressively inhibit NMDAR function in the hippocampus during

senescence. Together, the results suggest that age-related changes in the redox state

contributes to a decline in CaMKII activity, which ultimately leads to a decline in the

NMDAR response. The outcome of such senescent mechanisms is an alteration in the

synaptic plasticity at the CA3-CA1 synapses which contributes to age-related cognitive

decline.














































Figure 4-1. Detection of ROS in live hippocampal slices. A) Bright field image of the
CA1 region of the hippocampus from an aged F344 rat. Indicated are the
various layers of hippocampal area CA1 stratum radiatum (s.rad), stratum
pyramidale (s.pyr) and stratum oriens (s.or). B) The same image as in (A)
was obtained with a green filter. White arrow heads indicate few of the many
CA1 pyramidal neurons that have oxidized the ROS detection dye into a
green fluorescent product. C) Image obtained from one of the dye-unexposed
slices from the same rat. Scale bars = 50 pm.









Bright Field
(100 ms)


Aged









Young


Green Emission
(100 ms)


Green Emission
(500 ms)


Figure 4-2. Detection of auto-fluorescence from dye-unexposed hippocampal slices.
Shown are the images of the CA1 region of the hippocampus from aged (top
panel) and young (bottom panel) animals. Dye-unexposed slices were used
to obtain the bright field image (left), and the images with a filter designed to
detect green fluorescence with exposure time set at 100 ms (middle) and 500
ms (right). Auto-fluorescent signals were detected, for a 500 ms exposure
time, in the stratum radiatum of aged hippocampal slices (white arrowheads)
but not in the young hippocampal slices. Scale Bar = 50 pm.










A Bright field (100ms) Green Emission (100ms) Green Emission (100ms)
Carboxy-HzDCFDA (30 min) Untreated Carboxy-H2DCFDA (30 min)


0






-I
<




B 400
D n.s
u 1 I I
CD --- I I


U.
S0

2-5


Iu
C. b


Aged
SOD
Catalase


Young Aged Aged
SOD+Catalase


Figure 4-3. Enhanced ROS production is observed in hippocampal tissue from aged
rats. A) Indicated above each image column are the imaging conditions
(bright field or green emission) and the exposure time (100 ms) for
hippocampal slices that were either untreated or treated (Carboxy-H2DCFDA)
with the dye. The rows are images of the CA1 region of young (top row) and
aged (second row) hippocampal slices. The lowermost image is a Carboxy-
H2DCFDA treated slice from an aged animal, which was incubated with SOD
+ catalase. The various layers of hippocampal area CA1- stratum radiatum
(s.rad), stratum pyramidale (s.pyr) and stratum oriens (s.or) are indicated in
the last set of images. Scale bars = 50 pm. B) Quantification of the mean
fluorescence intensity generated by the oxidation of c-H2DCFDA (c-H2DCFDA
Fluorescence) from young (n = 3) (open bar), aged (n = 3), and SOD +
catalase exposed aged hippocampal slices (n = 3) (gray bars) expressed as
percent of fluorescence in untreated (control) slices from the same animal. In
this and subsequent figures error bars represent standard error of mean
(S.E.M), asterisks indicate significant difference (p<0.05) between the groups
indicated, and n.s indicates no significant difference.


s.or


s. pyr


s.rad
s.or


s.pyr


s.rad

s.or


s.pyr


s.rad


.11









A
S 200
CL
0.









B

200


I 150
0.U

100

2


0 10 25 40 55 70 85 100
Time (minutes)
Bis-I (500 nM)


Time (minutes)


Figure 4-4. A Serine/Threonine (Ser/Thr) kinase, but not protein kinase C, mediates
DTT mediated increase in NMDAR function in aged hippocampus. A) Time
course of the change in the normalized NMDAR-fEPSP slope in the aged
animals that were incubated with the broad spectrum Ser/Thr kinase inhibitor
H-7 dihydrochloride (10 pM, n = 7) prior to and during DTT application. B)
Time course of the change in the normalized NMDAR-fEPSP slope in the
aged animals that were incubated with the PKC inhibitor Bis-I (500 nM, n = 6)
prior to and during DTT application.










A .200
0-
C. = 150
o-w
0:' 100-

Z
50-


KN-62 (10 pM) C n.s
DTT (0.7 mM)
^^^^^^^^^^^^^^a 200


& 200-
.-i #
6 l150-

, 100.


0 10 25 40 55 70 85 100 S |H
Time (minutes) 50
S 200-

B y^
200myr-AIP (5 pM)
0.
O DTT (0.7 mM)
I -150-


100-
Z
50-iI i
0 20 40 60 80 125
Time (minutes)



Figure 4-5. CaMKII involvement in the DTT mediated enhancement of NMDAR synaptic
responses in aged animals. A) Time course of the change in the normalized
NMDAR-fEPSP slope in the aged animals that were incubated with the CaMK
inhibitor KN-62 (10 pM, n = 5). B) Time course of the change in the
normalized NMDAR-fEPSP slope in the aged animals that were incubated
with the specific CaMKII inhibitor myr-AIP (5 pM, n = 4). C) Quantification of
the mean percent change in the NMDAR-fEPSP slope for aged animals under
DTT alone (filled bar), and DTT applied in the presence of H-7, KN-62, myr-
AlP and Bis-I (gray bars). Asterisk indicates a significant difference (p<0.05)
between the increases observed in presence of DTT alone relative to DTT
applied in the presence of H-7, KN-62, and myr-AIP; n.s indicates no
significant difference between the increases observed in presence of DTT
alone and DTT applied in presence of Bis-I. Pound sign indicates a significant
(p<0.05) increase in the response relative to baseline level of 100%, following
DTT application.


^I W










FK-506 (10 pM)
DTT (0.7 mM)



--- "
.. 7'


200-
2e-
NS 150-



Z


0 10 25 40 55 70 85 100
Time (minutes)

OA (1 pM)
D1 TT (0.7 mM)


--- --- '- -
OjA, ..^.*.VA1^^
.25;i' "C-'


SI i I
0 10 25 40 5
Time (minutes)


OA (1 pM)


0 10 20
Time (minutes)

D 250 *
n.s
n n-s
o 200. I n.s



S100.
z
,=

pt ll


<* # o //.^ 'r^


Figure 4-6. Calcineurin/PP2B and PP1 are not involved in the DTT mediated
enhancement of NMDAR synaptic responses in aged animals. A) Time
course of the increase in the NMDAR-fEPSP slope in slices from aged
animals that were incubated with FK-506 (10 pM), 45 minutes prior to and
during the application of DTT (n = 5). B) The NMDAR-fEPSP slope exhibited
a modest increase (121.46 9.19%) following a 30 minute incubation with OA
(1 pM) (n = 5). C) Following stabilization of the response in OA, the baseline
was recalculated. The figure illustrates the time course for the increase in the
re-normalized NMDAR-fEPSP slope following application of DTT (n = 5). D)
Quantification of the mean percent change in the NMDAR-fEPSP slope for
aged animals under DTT alone (filled bar), and in the presence of FK-506 +
DTT, OA + DTT and OA alone (gray bars).


a3
C.-- 150


z100


1 150-
a.

! 100.

z


30 40


sn ....-- r ---- i ---- i ----


au


~:tX;~"d~'jcJH~










A B
300- 300-
= 250- .
a Aged Control Aged DTT
A 200 .0200
S(ACSF) 200 (ACSF + DTT)
s 150- 150-
100 a0- m mi lN -P.ff -T] :r .i-i j'n-t .- 1 0 0
M HFS path HFSpath
S 100 Hz (Is) control pat 100 Hz (Is) o Control path
IL100 Hz (s) Control path I 0
LU 0 ________________________ i 0
0 20 40 M 80 o 20 40 60 80
Time (minutes) Time (minutes)

C D
1so Aged Control () DTT (e)
2,0
I 140- V V

S* 120 15-

.ioo .------- --
( Mean__
80 ACSF ACSF 50 100 150 200
+DTT Interpulse Interval (ms)



Figure 4-7. DTT enhances LTP in hippocampal area CA1 of aged animals. A) Time
course for the expression of LTP recorded in aged hippocampal slices bathed
in control ACSF (n = 9) for at least 45 minutes prior to HFS. Baseline
stimulation was applied to a control pathway (open circles) and to a second
pathway that received HFS (100 Hz, 1s) (filled circles). B) Time course for
the expression of LTP recorded in aged hippocampal slices bathed in ACSF
containing DTT (n = 10) for at least 45 minutes prior to HFS. Baseline
stimulation was applied to a control pathway (open circles) and to a second
pathway that received HFS (100 Hz, 1s) (filled circles). Arrows in A and B
denote HFS delivery. For purpose of clarity, each point represents the mean
of two consecutive responses. C) Distribution of the LTP magnitude for
individual slices from aged animals bathed in control ACSF and ACSF+DTT.
The rectangular boxes indicate the mean of each group. D) Quantification of
the mean percent change in the fEPSP slope recorded from the control (Cont)
and HFS (HFS) pathways from young slices bathed in ACSF or ACSF+DTT.
E) Plot of the paired-pulse ratio obtained under control conditions (black
circles) and after 45 minute bath application of DTT (gray circles) for four
inter-pulse intervals (50, 100, 150, 200 ms). Inset: Responses obtained upon
paired pulse stimulation (average of 5 consecutive traces; 50 ms inter-pulse
interval) under control conditions and under DTT application.










A
S300i
so
3 250-
S2oo- Young Control
o (ACSF)

S100- C Young
S0* HFS path 200
50 n.s
100 Hz (1s) Control path
LU 0 ,
0 20 40 60 80 S 150.
Time (minutes) 0 #
B
300- u
25s- .
S200- Young DTT so
r 4- (ACSF + DTT) Cof 4o01g anials
t ACSF ACSF
__+__O '"t DTT
S100- -p....
0. 50- t HFS path
C i 100 Hz (1s) o Control path
uJ 0- ----1
0 20 40 60 so80
Time (minutes)


Figure 4-8. DTT does not alter the LTP in hippocampal area CA1 of young animals. A)
Time course for the expression of LTP recorded in young hippocampal slices
bathed in control ACSF (n = 6) for at least 45 min prior to HFS. Baseline
stimulation was applied to a control pathway (open circles) and to a second
pathway that received HFS (100Hz, 1s) (filled circles). B) Time course for the
expression of LTP recorded in young hippocampal slices bathed in ACSF
containing DTT (n = 5) for at least 45 min prior to HFS. Arrows in A and B
denote HFS delivery. For purpose of clarity, each point represents the mean
of two consecutive responses. C) Quantification of the mean percent change
in the fEPSP slope recorded from the control (Cont) and HFS (HFS)
pathways of the young slices bathed in ACSF or ACSF+DTT.









B CaMKII+Ca2+


150

0
" 100
0

0 50
3 s


01

Aged CA1 Cytosolic extract
Calcium(2.5 mM)
Calmodulin(200 ng/mL)
Purified CaMKII(30 mU)
DTT(values in mM)
EGTA(2 mM) + Myr-AIP(10 pM)


(no
150


100


50


0


exogenous CaM)


+ + + + + + *1
+ + + + -+ *I
+ + + + 4


0 0.7 1.4 2.8 0
- *+


0 0.7


Figure 4-9. DTT enhances CaMKII activity in aged hippocampal CA1 cytosolic extracts.
A) CaMKII activity measured from the hippocampal CA1 cytosolic extracts of
aged F344 rats. CaMKII activity is represented as percent of control activity
(black bars) in the presence of exogenous calmodulin. CaMKII activity was
significantly enhanced in the presence of 0.7 mM and 1.4 mM DTT (gray
bars), and was blocked by the addition of EGTA (2 mM) + myr-AIP (10 pM)
(white bars). B) Removal of exogenous calmodulin did not further enhance
the DTT (0.7 mM) effect on CaMKII activity suggesting that DTT is not acting
through oxidized calmodulin in aged animals. Asterisk indicates a significant
difference (p<0.05) from respective controls. Plus and minus represent the
presence and absence (respectively) of the indicated component in the
reaction mix.












150 CaMKII+Ca2++CaM


150


100


Young CA1 Cytosolic extract
Calcium(2.5 mM)
Calmodulin(200 nglmL)
Purified CaMKII(15-30 mU)
DTT(values in mM)
EGTA(2 mM) + Myr-AIP(10 pM)


+ + + +
+ + + -
+ + + +


0 0.7 1.4 0
-+


+ +I. +
+ + +
+ + +
0 0.7 1.4


Figure 4-10. DTT does not enhance CaMKII activity in young hippocampal CA1
cytosolic extracts. A) CaMKII activity measured from the hippocampal CA1
cytosolic extracts of young F344 rats. CaMKII activity is represented as
percent of control activity (black bars) in the presence of exogenous
calmodulin. Addition of 0.7 mM and 1.4 mM DTT did not increase CaMKII
activity in hippocampal CA1 cytosolic extracts of young F344 rats. B) Addition
of 0.7 and 1.4 mM DTT decreased the activity of purified CaMKII. Asterisk
indicates a significant difference (p<0.05) from respective controls. Plus and
minus represent the presence and absence (respectively) of the indicated
component in the reaction mix.


. 100
0
U
so
0 50


0


Purified CaMKII









Table 4-1. Paired-pulse ratios from aged animals
Inter pulse Interval (ms) Paired pulse ratio Control Paired pulse ratio DTT
(mean S.E.M) (mean S.E.M)

50 1.51 0.12 1.38 0.11

100 1.34 0.08 1.21 0.08

150 1.24 0.05 1.15 0.07

200 1.38 0.09 1.28 0.06
The table represents the paired pulse ratio obtained from control, aged hippocampal slices and
aged hippocampal slices that were incubated with DTT for at least 45 minutes. The paired
pulses ratio is the ratio of the slopes of two consecutive fEPSPs elicited apart by a time interval
indicated by the inter pulse interval.









CHAPTER 5
REDOX MODULATION MEDIATES REDUCTION IN NEURONAL
AFTERHYPERPOLARIZATION OF AGED HIPPOCAMPAL NEURONS

Introduction

An age-related decline in hippocampus-dependent memory is thought to result

from dysregulation of Ca2+-dependent processes in CA1 pyramidal neurons including

synaptic plasticity and neuronal excitability (Foster, 1999, 2007; Kumar et al., 2009;

Burke and Barnes, 2010; Magnusson et al., 2010; Oh et al., 2010). The results

presented in chapters 3 and 4 dealt with NMDAR hypofunction, a significant biomarker

of aging in CA1 pyramidal neurons. One of the other well characterized markers of

aging in CA1 pyramidal neurons is an increase in the slow component of the Ca2+

activated, K- mediated afterhyperpolarization (sAHP) (Landfield and Pitler, 1984;

Moyer et al., 1992; Kumar and Foster, 2004; Thibault et al., 2007; Matthews et al.,

2009)

The exact mechanism that underlies the age-related increase in sAHP is unknown.

The increase in the sAHP may be due to altered Ca2+ regulation, including an increase

in L-type voltage gated Ca2+ channels (L-type VGCC) (Thibault and Landfield, 1996;

Veng and Browning, 2002) or increased release of Ca2+ from intracellular Ca2+ stores

(ICS) (Kumar and Foster, 2004; Gant et al., 2006) or an increase in the function or

density of K channels that mediate the sAHP (Power et al., 2001; Power et al., 2002).

Importantly, aging is associated with increased oxidative stress that could influence the

highly redox sensitive RyRs, which mediate Ca2+ release from ICS (Eager and

Dulhunty, 1998; Hidalgo et al., 2004; Bull et al., 2008; Huddleston et al., 2008).

Moreover, aged neurons are characterized by a decrease in their redox buffering

capacity (Parihar et al., 2008; Bodhinathan et al., 2010) and recent work from our lab


100









demonstrates that the shift in redox state contributes to altered Ca2+ regulation in CA1

neurons from aged animals (Bodhinathan et al., 2010). Based on these observations we

tested the hypothesis that the redox state of the aged neuron contributes to the increase

in sAHP (Foster, 2007; Kumar et al., 2009).

The results reveal that the sAHP is decreased by the reducing agent dithiothreitol

(DTT) in an age-dependent manner. Application of ryanodine, to block RyRs, prevented

the DTT-mediated decrease of sAHP in the aged neurons. Depletion of ICS by the

application of thapsigargin also blocked the DTT effect on aged-sAHP. The DTT-

mediated decrease in aged-sAHP was independent of the activity of L-type VGCC or

Ser/Thr kinase activity. Finally inhibition of the big conductance potassium (BK) channel

activity did not influence DTT-mediated decrease in aged-sAHP. The results point to an

ICS-dependent and RyR-mediated mechanism that links oxidative redox state during

aging and the enhanced sAHP in CA1 pyramidal neurons. Reversal of the redox state of

aged hippocampal CA1 pyramidal neurons is a potential therapeutic strategy to

ameliorate Ca2+ dysregulation, decrease sAHP and restore normal functionality in aged

neurons.

Results

Age Dependent Decrease in the sAHP Following DTT Application

To study the effects of oxidative redox state on the aged-sAHP, the reducing agent

DTT was applied to aged and young hippocampal CA1 pyramidal neurons while

continuously recording the sAHP. In confirmation of previous studies, the sAHP was

significantly (p<0.05) increased in aged (6.44 0.32 mV, n = 40) relative to young CA1

pyramidal neurons (4.23 0.17 mV, n = 12). The properties of the CA1 pyramidal

neurons recorded from the young and aged animals are indicated in Table. 5-1. In a


101









subset of these neurons, after a stable baseline recording for 10 min, DTT was applied

for 40 min. Application of DTT significantly (p<0.05) decreased the sAHP amplitude

form the baseline levels in the aged (48 14% of baseline, n = 5), but not in the young

animals (105 10%, n = 3) (Fig. 5-1A, 5-1B). The DTT-mediated reduction in sAHP of

aged neurons does not appear to be due to altered membrane properties, since the

holding current required to maintain the membrane potential at -63 mV did not differ

(p>0.05) between baseline and 45 minutes after DTT application. The DTT-mediated

decrease, specific to the aged-sAHP, suggests a link between oxidative redox state and

the increased sAHP amplitude in aged neurons.

DTT Mediated Decrease in Aged-sAHP Involves Intracellular Calcium Stores and
Ryanodine Receptors

To test the hypothesis that the DTT-mediated decrease in the aged-sAHP was due

to decrease Ca2+ mobilization from ICS, ICS were depleted by the application of

thapsigargin prior to and during the application of DTT to aged hippocampal slices.

Application of thapsigargin for 30 min significantly decreased (p<0.05) the amplitude of

aged-sAHP to 58 8% (n = 7) of the baseline levels (Fig. 5-2A). In a subset of these

cells (n = 4), a new baseline was established and DTT was applied. Application of DTT

for 50 min failed to decrease the aged-sAHP amplitude (104 23%) (Fig. 5-2B, 5-2C).

The results suggest that ICS provide a redox sensitive Ca2+ source that contributes to

the age-related increase in the sAHP.

RyRs mobilize Ca2+ from the ICS and are highly redox sensitive. To test whether

RyRs were involved in the DTT-mediated decrease in aged-sAHP, RyRs were block by

ryanodine prior to and during the application of DTT. Application of ryanodine for 40

min, significantly (p<0.05) decreased the aged-sAHP amplitude to 47 10% (n = 4).


102









Application of DTT for 50 min failed to further decrease the aged-sAHP amplitude such

that it was 54 7% (n = 4) of the original baseline (Fig. 5-3A, 5-3B).

Both DTT (Fig. 5-1) and ryanodine (Fig. 5-3) decreased the aged-sAHP to ~50%

of baseline. The similar magnitude effect raises the possibility of a "floor effect" of

ryanodine, which may have masked DTT influences on the sAHP. To address this

issue, the sAHP of aged neurons was enhanced by increasing the extracellular Ca2+

concentration from 2 mM to 4 mM. Increasing the extracellular Ca2+ to 4 mM increased

the sAHP almost two fold, from 6.71 0.79 mV (n= 1) to 11.06 1.09 mV (n = 6) (Fig.

5-4A, 5-4B). In five cells a baseline was recorded in 4 mM Ca2+, followed by application

of ryanodine for 40 min, which was then followed by the application of DTT for 50 min

(Fig. 5-4C). Application of ryanodine decreased (p<0.05) the aged-sAHP amplitude to

53 6% (5.31 0.86 mV) and application of DTT for 50 min failed to further decrease

the aged-sAHP amplitude (51 7%, 5.06 1.05 mV) of the original baseline (Fig. 5-4B,

5-4C, 5-4D). Thus, DTT failed to reduce the sAHP amplitude under high Ca2+ and

ryanodine application, despite the fact that sAHP amplitude was similar to the baseline

under normal 2 mM Ca2+ conditions (Fig. 5-4B). The results indicate that the ryanodine

blockade of the DTT-mediated decrease in the sAHP was not due to a floor effect of the

sAHP during ryanodine application. Rather, these data suggest that the DTT effect on

sAHP in aged animals is mediated by RyRs.

DTT Mediated Reduction in the Aged-sAHP is Independent of L-VGCC

L-type VGCCs are another major source of Ca2+ for the sAHP. To test the

hypothesis the DTT-mediated decrease in the aged-sAHP involves the L-type VGCC,

nifedipine was applied prior to and during the application of DTT to aged hippocampal

slices (Fig. 5-5A). Application of nifedipine for 20 min decreased the sAHP to 68 4%


103









(n = 5) of the baseline. Subsequent application of DTT for 30 min further decreased the

amplitude of aged-sAHP to 34 4% (n = 5) of the original baseline. The results suggest

that the effects of nifedipine and DTT may be independent. In fact, using the sAHP

responses recorded in nifedipine (bath application for at least 20 min) as the baseline,

application of DTT decreased the amplitude of the aged-sAHP to 48 6 % (p<0.05; n =

6) (Fig. 5-5B), a decrease comparable to that observed following DTT application in the

absence of nifedipine (Fig. 5-7B).

The activity of BK channels is sensitive to oxidation (DiChiara and Reinhart, 1997).

Moreover, an increase in BK channel activity can reduce the sAHP amplitude by

decreasing the action potential spike width (Giese et al., 1998; Shao et al., 1999;

Murphy et al., 2004). To test the hypothesis that the DTT-mediated decrease in aged-

sAHP involves the BK channels, paxilline first applied to inhibit BK channel activity

(Sanchez and McManus, 1996). Aged hippocampal slices were incubated in paxilline for

at least 60 min prior to recording the sAHP and applying DTT. Paxilline failed to block

the DTT-mediated decrease in aged-sAHP, such that DTT application was still able to

decrease the amplitude of the aged-sAHP to 28 11 % (n = 3) of the baseline (Fig. 5-

6A, 5-6B). Furthermore, the DTT-mediated decrease in the presence of paxilline was

not significantly (p>0.05) different from the decrease observed in the presence of DTT

alone.

Serine/threonine kinases provide another potential mechanism for regulating RyRs

and the K channels that mediate the sAHP. Protein kinase A increases the activity of

cardiac RyRs (RyR subtype 2) (Yoshida et al., 1992; Danila and Hamilton, 2004; Xiao et

al., 2007; Morimoto et al., 2009), and kinase activity inhibits the sAHP (Madison and


104









Nicoll, 1986; Malenka et al., 1986; Muller et al., 1992; Pedarzani and Storm, 1993;

Melyan et al., 2002). In order to test whether the changes in kinase activity underlies the

decrease in sAHP of aged neurons upon application of DTT, the broad spectrum

serine/threonine kinases inhibitor H-7 was applied prior to and during the application of

DTT. Aged hippocampal slices were incubated with H-7 for at least 60 minutes before

recording the sAHP. In the presence of H-7, application of DTT significantly (p<0.05)

decreased the aged-sAHP to 53 14 % (n = 3) of the baseline (Fig. 5-7A). The results

suggest that DTT is not altering the sAHP through modulation of kinase activity.

Fig. 5-7B summarizes the change in the sAHP amplitude following DTT application

under various conditions. In each case, the response was normalized to the pre-DTT

application baseline. In addition, the percent change for application of nifedipine-alone

or ryanodine-alone relative to the pre-drug baseline is illustrated for comparison.

Treatments that blocked Ca2+ release from ICS (thapsigargin, ryanodine) blocked the

DTT-mediated reduction in the sAHP. In all cases in which DTT reduced the sAHP;

including in the presence of nifedipine, the reduction was ~50%. A similar reduction was

observed following treatment with ryanodine-alone, consistent with previous reports in

aged animals (Kumar and Foster, 2004; Gant et al., 2006). Application of nifedipine-

alone decreased the sAHP by ~30%, consistent with previous reports in young and

aged animals (Power et al., 2002; Disterhoft et al., 2004).

Discussion

The results demonstrate a link between the age-related increase in the sAHP and

redox state, through the release of Ca2+ from ICS. A shift in Ca2+ regulation and altered

Ca2+ channel function is a characteristic of certain aging neurons (Foster, 1999, 2007;

Kumar et al., 2009; Burke and Barnes, 2010; Magnusson et al., 2010; Oh et al., 2010).


105









Recently we demonstrated that DTT could reverse an age-related decrease in NMDA

receptor function in region CA1 (Bodhinathan et al., 2010). In the current study, the

reducing agent, DTT, decreased the sAHP in aged, but not in young CA1 neurons. The

data are consistent with the weakened redox buffering in aged animals as a mechanism

contributing to Ca2+ dysregulation and electrophysiological changes observed in aged

neurons. Redox modulation has been observed for several ion channels including K

and Ca2+ channels (Ruppersberg et al., 1991; Chiamvimonvat et al., 1995; Stephens et

al., 1996; DiChiara and Reinhart, 1997; Hidalgo et al., 2004), which could contribute to

the sAHP. The identity of the K channel that underlies the sAHP is unknown (Furuichi

et al., 1994; Sah and Faber, 2002); however, the amplitude to the sAHP is reduced by

activation of Ser/Thr kinases, including PKA (Madison and Nicoll, 1986; Pedarzani and

Storm, 1993), CaMKII (Muller et al., 1992), and PKC (Malenka et al., 1986). In the

current study, the broad spectrum Ser/Thr kinase inhibitor H-7 had no influence on the

DTT-mediated decrease in aged-sAHP indicating that the reduction was not mediated

through kinase activity.

In the case of K channels, previous reports indicate cysteine specific oxidation

decreases BK channel activity (Tang et al., 2001; Tang et al., 2004), and that the

reducing agent DTT increases BK channel activity (DiChiara and Reinhart, 1997). The

BK channel is involved in repolarization of action potential, and an increase in BK

channel activity will reduce the width of the action potential (Shao et al., 1999).

Moreover, a decrease in the spike width can decrease the sAHP, by limiting the

duration of depolarization-induced Ca2+ entry through L-type VGCCs (Giese et al.,

1998; Murphy et al., 2004). Thus, DTT could be acting on the BK channels to decrease


106









L-type VGCC activity and the sAHP amplitude. However several pieces of evidence

suggest that this might not be the case. First, blockade of BK channels with paxilline did

not block the DTT-mediated decrease in aged-sAHP. Second, blockade of VGCC's with

nifedipine did not influence the DTT-mediated decrease in aged-sAHP. Finally, the fast

AHP, which is mediated by the BK channel, is not altered with age (Matthews et al.,

2009)

The amplitude of the sAHP is dependent on the level of cytosolic Ca2+. L-type

VGCCs play a role in determining the amplitude of the sAHP (Landfield and Pitler, 1984;

Moyer et al., 1992; Norris et al., 1998a) and contribute to the increase in the sAHP

during aging (Thibault and Landfield, 1996; Veng and Browning, 2002). However, it

does not appear that the DTT-mediated reduction in the aged-sAHP is acting through L-

channels. The DTT-mediated reduction in the sAHP was larger than that observed for L-

channel blockade, and was specific to aged animals. Previous research indicates that

the decrease in the AHP following blockade of L-channels is quantitatively larger in

aged animals; however, the percent decrease (~30%) is similar across ages, suggesting

other mechanisms contribute to the age-related increase in the AHP animals (Power et

al., 2002; Disterhoft et al., 2004). In the current study, blockade of the L-channel

reduced the aged-sAHP ~30%, consistent with previous reports (Power et al., 2002).

Regardless of L-channel function, DTT reduced the aged-sAHP by ~50% and the effect

of DTT was specific to aged animals. The results suggest that DTT is acting on

mechanisms other than the L-channel, which may mediate the age-related increase in

the sAHP.


107









Release of Ca2+ from ICS, through RyR activation, plays a role in determining the

sAHP amplitude (Sah and McLachlan, 1991; Usachev et al., 1993; Davies et al., 1996;

Borde et al., 2000; van de Vrede et al., 2007). Ca2+ from ICS contributes to altered

physiology during aging (Kumar and Foster, 2004, 2005; Gant et al., 2006). In addition,

the RyRs are highly redox sensitive (Bull et al., 2008), such that oxidation of the

cysteine residues increases the Ca2+ sensitivity and activity of RyR (Eager and

Dulhunty, 1998; Hidalgo et al., 2004; Huddleston et al., 2008). In the current study, the

DTT-mediated decrease in the sAHP was blocked upon depletion of ICS by

thapsigargin or blockade of RyRs by ryanodine indicating the involvement of ICS and

RyRs in the decreased Ca2+ mobilization by DTT application. Together the results

suggest that the increase in the sAHP in aged neurons is related to redox sensitive Ca2+

mobilization from the ICS through the RyRs. Interestingly, decreased sAHP is observed

in aged memory-unimpaired and young rats but not in aged memory-impaired rats

(Moyer et al., 2000; Tombaugh et al., 2005; Murphy et al., 2006; Matthews et al., 2009).

It is interesting to speculate that the decrease in the sAHP of memory unimpaired

animals may result from a shift in redox state associated with learning (Shvets-Teneta-

Gurii et al., 2007). Alternatively, treatments that modify intracellular redox state may

provide a novel therapeutic strategy to restore Ca2+ homeostasis in the aged neurons.


108









A 180 DTT
160



"E 100 la,-: .
20
u0,6 140

20 ;Young
0 Aged
0 10 20 30 40 50
B Time (minutes)




S Aged Young J
DTT
/ DTT

Control Control


Figure 5-1. Age-dependent reduction in the sAHP by DTT. (A) Time course of the
change in the normalized sAHP amplitude in the aged (filled circles) (n = 5)
and young (open triangles) animals (n=3), following application of DTT for 40
minutes. (B) Representative traces illustrating the change in the AHP of aged
(left) and young (right) animals under control conditions and at the end of a 40
minute application of DTT. The line beneath the traces indicates the onset
and offset of the step current used to elicit a train of 5 action potentials.
Calibration bars: 200 ms, 10 mV.


109










Thapsigargin


----,,-----'----'-'- '..


5 10 15 20
Time (min)


25 30 35


5 10 15 20 25 30
Time (min)


35 40 45 50


-63 mV


Thapsigargin
+DTTJ Thapsigargin


Control


Figure 5-2. Intracellular calcium stores underlie DTT-mediated decrease in aged-sAHP.
(A) Time course of the change in the normalized sAHP amplitude in the aged
animals that were incubated with thapsigargin (n = 7). (B) Time course of
change in the normalized sAHP amplitude in cells (n = 4) incubated with
thapsigargin prior to and during DTT application. (C) Representative traces
illustrating the AHP of aged animals under control condition (black trace), and
at the end of a 40 minute application of thapsigargin (gray trace) and at the
end of 50 min application of thapsigargin+DTT (gray trace). Calibration bars:
200 ms, 10 mV.


110


B 200
180
S 160
S140
120
E 100
S80
a_
1^ 60
S 40
20


U .g .. .. .. .. ....iN-


Thapsigargin
DTT




l I f











Ryanodine


DTT


a I. I ,.. ~ I


0I I I I0
70 80 90 100


Figure 5-3. RyR blockade inhibits DTT mediated decrease in aged-sAHP. (A) Time
course of the change in the normalized sAHP amplitude recorded from aged
animals that were incubated with the ryanodine receptor antagonist ryanodine
(n = 4) prior to and during application of DTT application. (B) Representative
traces illustrating the change in the AHP of aged animals under control
condition (black trace), at the end of a 40 minute application of ryanodine
(black trace), and at the end of 50 min application of ryanodine+DTT (gray
trace). Calibration bars: 200 ms, 20 mV. Inset: Magnified representation of
change in the aged AHP under control condition, ryanodine, and ryanodine +
DTT.


111


I I I I I I


10 20 30 40 50 60
Time (min)










A B


0 <
2mMCa2 4mMCa2+ E -2
-4
2 -6
-3nV -----.-- -------.--- -------------------.. -------------------------------- 4 8
S-10
F-1 0- -12
-6
< -14
C eo, Ryanodine D

S140 DTT I
S120,
W^ loo ---------------------------------
< 80 High Ca2(4 mM)
so Ryanodine
Ryanodine OTT
< 40 .... 1 111 '" ,'" -,63 mo -
cA 20 High Ca2'(4 mM)
0 I I I1I 1 1 Control
0 10 20 30 40 50 60 70 80 90 100
Time (minutes)

Figure 5-4. RyR blockade inhibits the DTT-mediated decrease in aged-sAHP when the
AHP is increased by increasing calcium in the recording medium. (A)
Representative traces illustrating the AHP from aged neurons recorded under
conditions of 2 mM or 4 mM Ca in the ACSF. Calibration bars: 200 ms, 10
mV. (B) Quantification of the mean sAHP amplitude in aged neurons recorded
under 2 mM Ca2+ (n = 11, open bar), under 4 mM Ca2+ (n = 6), under 4 mM
Ca2+ with ryanodine (n = 5), and under 4 mM Ca2+ with ryanodine + DTT (n =
5); all values under 4 mM Ca2+ are represented as filled bars. (C) Time course
of the change in the normalized sAHP amplitude recorded in 4 mM ACSF
from aged animals and incubated with ryanodine (n = 5) prior to and during
DTT application. (D) Representative traces illustrating the AHP of aged
animals under control condition, and at the end of a 40 minute application of
ryanodine and at the end of 50 min application of ryanodine + DTT.
Calibration bars: 200 ms, 20 mV.


112










A
A 160 Nifedipine
140 DTT
IS120
140
20





Time (min)

B son Nifedipine
S140 oDTT
rL 10 lO-w ^ f- -----------------------------






4 ^^", li
40
20






0
0 5 10 15 20 25 30 3 5 5 40
Time (min)



Nifedipine
140 DTT Nifedipine
r 120
E - -
<4 80
-63m 60
40
20
0
0 5 10 15 20 25 30 35 40
Time (min)





Nifedipine
-63mV + DTT Nifedipine

Control


Figure 5-5. DTT mediated decrease is independent of L-type calcium channel function.
A) Time course of the change in the normalized sAHP amplitude in the aged
animals (filled circles) that were incubated with nifedipine (n = 4). B) Time
course of the change in the normalized sAHP amplitude in the aged animals
(filled circles) that were incubated with nifedipine (n = 6) for at least 45 min
prior to the application of DTT for 30 min. Calibration bars: 200 ms, 10 mV. C)
Representative traces illustrating the change in the AHP of aged animals
under control condition (black trace), and at the end of a 20 min application of
Nifedipine (gray trace) and at the end of 30 min application of Nifedipine +
DTT (black trace).


113












0


Ecn
C .
u 0


0 5 10 15 20 25 30 35 40 45 50 55


Time (min)


-63mV


Paxilline


-J


Paxilline


Figure 5-6. DTT effects on aged-sAHP are independent of BK channel function (A) Time
course of the change in the normalized sAHP amplitude in the aged animals
(filled triangles) that were incubated with paxilline (n = 3) for at least 60 min
prior to DTT application. (B) Representative traces illustrating the change in
the AHP of aged animals under paxilline (black trace), and at the end of 45
min under paxilline+DTT (gray trace). Calibration bars: 200 ms, 10 mV.


114










A 160 H-7
o 140 DTT
BQ 140 I





20

0 5 10 15 20 25 30 35 40 45 50
Time (min)
B 140 4
120 5

100 ---------------------

E. 80 5 3
S60 4 I

S40




C o -, i I






response represents the percent change relative to the pre-DTT application
a.
>IV




Figure 5-7. SerlThr kinase activity does not mediate DTT effects on aged-sAHP. (A)
Time course of the change in the normalized sAHP amplitude in the aged
animals that were incubated with the broad spectrum Ser/Thr kinase inhibitor
H-7 (n = 3) for at least 60 min prior to DTT application. (B) Summary diagram
representing the mean percent change in the sAHP amplitude of aged
neurons following DTT application under various conditions. In each case, the
response represents the percent change relative to the pre-DTT application
baseline (dashed line). The effect of nifedipine or ryanodine (open bars) on
the pre-drug baseline is presented for comparison. The numbers above each
bar represents the number of neurons recorded in each condition


115









Table 5-1. Physiological properties of CA1 neurons from young and aged animals


IR (MO)


Young (n = 12)


Aged (n= 40)


37.0 2.5


37.8 1.1


RMP (mV)


-62.8 1.5


-61.9 0.6


Sp Amp (mV)


80.6 1.9


82.8 0.7


The values for input resistance (IR), resting membrane potential (RMP), and spike amplitude
(Sp Amp) are indicated as mean S.E.M. The values of holding current (HC) is presented as a
range. The number in parentheses indicates the number of cells from the young and aged
animals.


116









CHAPTER 6
CONCLUSION AND FUTURE DIRECTIONS

Conclusion

While numerous mechanisms could contribute to dysfunctional hippocampal

synaptic transmission during aging, we were primarily interested in the role of increased

oxidative stress and oxidative redox state in mediating NMDAR hypofunction, altered

synaptic plasticity, increased sAHP, and altered Ca2+ homeostasis. The results

presented in Chapter 3 describe the age-related changes in the baseline NMDAR

mediated synaptic transmission, and indicate how these changes are linked to the redox

state of the aged neurons. Briefly, measurement of the NMDAR-fEPSP amplitude in the

young and aged neurons, and comparison across 0.4 mV bins of PFV amplitude,

indicated a significant age-related decrease in the NMDAR function. The use of

oxidizing agent X/XO decreased NMDAR function in the young but not in the aged

neurons; and the use of reducing agent DTT, increased the NMDAR function in aged

but not in the young animals. The effect of X/XO was washed out in young animals,

even under higher concentrations of X/XO, indicating a transient effect on young

NMDAR function. This effect could possibly be due to a robust antioxidant capacity or

better redox buffering capacity in the young neurons. In contrast, the DTT effect in aged

animals could not be washed out even 45 minutes after switching-off DTT application.

This observation predicted secondary signaling mechanisms underlying the DTT-

mediated increase in NMDAR function. Quite remarkably, this prediction was accurate

and the data presented in Chapter 4 delineates the signaling mechanism underlying

DTT-mediated increase in NMDAR function of aged animals.


117









Results presented in Chapter 4 describe the age-related increase in the rate of

ROS production in the hippocampal slices. The use of dye-based ROS detection

technique in live hippocampal neurons suggested that the rate of oxyradical production

is higher in the aged neurons, when compared to young neurons. Moreover the use of

membrane impermeable antioxidant enzymes (SOD and catalase) in the extracellular

solution did not affect the dye-based fluorescence, suggesting that the source of

oxyradical production is intracellular. Furthermore, in Chapter 4, the use of kinase and

phosphatase inhibitors localized the specific effects of DTT to CaMKII, a Ca2+ sensitive

Ser/Thr kinase that participates in regulating the NMDAR function and is also redox

sensitive. First, physiological studies indicated that the DTT effect on aged NMDAR

function was blocked by CaMKII inhibitors KN-62 and myr-AIP. Second, biochemical

assays suggested that DTT increases CaMKII activity in aged but not in young CA1

cytosolic extracts of hippocampal neurons. In contrast no effect of phosphatase

inhibition was observed on the DTT-mediated increase in NMDAR function. Neither the

inhibition of CaN with FK-506 nor the inhibition of PP1 with OA had any effect on the

DTT-mediated increase in aged NMDAR function. Finally, DTT treatment increased LTP

in aged neurons following a single episode of 100 Hz stimulation. Taken together the

data presented in Chapter 4 is indicative of the link between oxidative redox state and

oxidized CaMKII signaling pathways in the aged neurons, which ultimately contributes

to the decreased NMDAR function observed in Chapter 3, and weakened hippocampal

function during aging. This complex relationship is summarized by the schematic in Fig.

6-1.


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NMDARs in aged neurons can be subject to numerous post-translational changes

that could mediate the observed deficit in activation. Results provided in Chapters 3 and

4 suggest a role for oxidative redox state. However numerous alternative mechanisms

could mediate deficit in NMDAR function. Altered mRNA and protein expression of

specific NMDAR subunits is proposed as a potential mechanism for the observed

decrease in NMDAR function (Magnusson, 2000). Significant decreases have been

observed in the expression of NR1 protein (Eckles-Smith et al., 2000; Mesches et al.,

2004; Liu et al., 2008) and NR1 mRNA (Adams et al., 2001) in aged hippocampus. In

contrast, other studies report no age-related decrease in NR1 protein expression in the

whole hippocampus (Sonntag et al., 2000; Zhao et al., 2009). These studies point to a

lack of congruent changes in hippocampal NR1 subunit expression. Some studies

indicate age-related changes in the modulatory NR2 subunits. A decrease in the NR2A

protein expression has been observed in the hippocampus (Sonntag et al., 2000; Liu et

al., 2008), which is not observed in the frontal cortex (Sonntag et al., 2000).

Furthermore, NR2A mRNA expression was reported to decline in the ventral

hippocampus (Adams et al., 2001). In contrast, other studies report no significant

change in the NR2A protein expression levels in the hippocampus and cortex (Sonntag

et al., 2000; Martinez Villayandre et al., 2004). Age-related changes have also been

reported for NR2B subunit of the NMDAR; in particular the expression of NR2B protein

(Mesches et al., 2004; Zhao et al., 2009) and NR2B mRNA (Adams et al., 2001;

Magnusson, 2001) declines in the aged hippocampus. This effect may be region

specific since a decline in NR2B protein is not observed in the frontal cortex (Sonntag et


119









al., 2000). In contrast, NR2B mRNA decreases in the frontal cortices of aging macaque

monkeys, but not in the hippocampus (Bai et al., 2004).

In conclusion, a lack of a clear model describing mRNA and protein changes of

various NMDAR subunits in aged hippocampus gives rise to another alternative

mechanism for reduced NMDAR activation in these neurons. The other possible

mechanism is that alterations in the NMDAR localization, through the insertion of

receptors into the membrane or recruitment of extra-synaptic receptors into the

synapse, may have important effects on NMDAR function during aging. It has been

suggested that NR2B containing receptors may be more prevalent at extra-synaptic

sites (Massey et al., 2004), before being internalized into the cytoplasm (Blanpied et al.,

2002; Lau and Zukin, 2007). A decrease in the NR2B protein expression in the synaptic

membrane fraction, but not in whole homogenates (Zhao et al., 2009) suggests an age-

related sequestration of NR2B in the extra-synaptic sites. Recent work indicates that

extra-synaptic NMDARs couple to different signaling cascades, and initiate mechanisms

that oppose synaptic potentiation, by shutting off the activity of cAMP response element

binding protein and decreasing expression of brain-derived neurotropic factor

(Hardingham et al., 2002; Vanhoutte and Bading, 2003). However, it remains to be

determined whether altered localization of the NMDARs (specifically extra-synaptic

localization) is the mechanism by which the NMDAR function declines during

senescence.

The other likely candidate mechanism for regulating NMDAR function during aging

is posttranslational modification of the receptor and/or its associated signaling cascades

(investigated in Chapters 3 and 4). NMDAR function can be altered by the


120









oxidation/reduction of sulfhydryl moieties on their structure. Previous research

demonstrates that oxidizing agents like 5,5'-dithiobis(2-nitrobenzoic acid) (Aizenman et

al., 1989), hydroxyl radicals generated by xanthine / xanthine oxidase (Aizenman, 1995)

and oxidized glutathione (Sucher and Lipton, 1991) decrease NMDAR function in the

neuronal cell cultures. The decrease in NMDAR function under oxidizing conditions is

thought to result from the formation of disulfide bonds on the sulfhydryl group containing

amino acid residues in NMDARs (Aizenman et al., 1990; Sullivan et al., 1994; Choi et

al., 2001); specifically the cysteine residues are more susceptible to oxidation over the

methionine residues (Shacter, 2000). The aging brain is associated with an increase in

the levels of oxidative stress and/or a decrease in redox buffering capacity, which

contributes to a shift in the redox state favoring an oxidative state (Foster, 2006; Poon

et al., 2006; Parihar et al., 2008). Thus conditions during aging should promote a

decrease in NMDAR function. Another candidate mechanism associated with

posttranslational modification is altered phosphorylation state of the receptor. In

particular, the function of the NMDAR is influenced by its phosphorylation state.

Activation of the tyrosine kinase (Wang et al., 1994; Heidinger et al., 2002), protein

kinase C (Ben-Ari et al., 1992; Chen and Huang, 1992) and protein kinase A (Raman et

al., 1996) increases NMDAR mediated currents. In contrast, protein phosphatases,

including calcineurin and protein phosphatase 1, decrease NMDAR currents (Lieberman

and Mody, 1994; Wang et al., 1994; Raman et al., 1996). Interestingly, Ser/Thr kinases

promote NMDAR trafficking from endoplasmic reticulum and insertion into the

postsynaptic membrane (Scott et al., 2001; Carroll and Zukin, 2002), while

phosphatases promote internalization of NMDARs into the cytoplasm (Snyder et al.,


121









2005). Thus, the kinases and phosphatases act like molecular switches which increase

or decrease NMDAR function, respectively. During aging there is a shift in the balance

of kinase/phosphatase activity, favoring an increase in the phosphatase activity (Norris

et al., 1998b; Foster et al., 2001; Foster, 2007). Thus alterations in the phosphorylation

state of the NMDARs could mediate the decreased NMDAR activation in aged neurons

(Coultrap et al., 2008).

In this study we have presented evidence that suggests that age-related increase

in oxidative stress or oxidative redox state contributes to the decrease in NMDAR

function. Alternatively it is also possible that increased nitrosative stress affects NMDAR

function. In the hippocampal neurons, nitric oxide is produced by neuronal nitric oxide

synthase (nNOS), which is activated upon stimulation of NMDARs. In memory impaired,

aged F344 rats there is no loss of NOS containing neurons, rather a decreased

production of NO (Meyer et al., 1998), probably due to decreased activation of NMDARs

themselves (Barnes et al., 1997; Billard and Rouaud, 2007; Bodhinathan et al., 2010).

Subsequently, nitric oxide reacts with superoxide to produce peroxynitrite, a potent

oxidant and nitrating agent (Squadrito and Pryor, 1998), which has been reported to

decrease NMDAR function (Lipton and Stamler, 1994; Lipton et al., 1998); and

proposed to inhibit NMDAR-dependent LTP (Wang et al., 2004). The nitrosative stress

mediated decrease in NMDAR function is thought to occur by S-nitrosylation of the

receptor (Lipton and Stamler, 1994; Choi et al., 2000; Takahashi et al., 2007). Thus,

excess amounts of superoxide plays an important role in mediating the effects of

nitrosative stress, by contributing to the production of the peroxynitrite, which leads to S-

nitrosylation of the NMDARs. Although we cannot completely discount the effect of


122









nitrosative stress on age-related decrease in NMDAR function, it is likely that the DTT

mediated increase in NMDAR function does not involve the removal of S-nitrosyl groups

on NMDARs.

The results presented in Chapter 5 describe the link between a more oxidized

redox state and increased sAHP during aging. Increased sAHP is a physiological

marker of aging, which makes it harder for the CA1 pyramidal neurons to reach the

threshold for action potential firing. Our results indicate that application of the reducing

agent DTT significantly decreases the sAHP in aged but not in young CA1 pyramidal

neurons. Moreover, the DTT-meditated decrease in sAHP amplitude involves

decreased Ca2+ mobilization from the ICS through a decrease in RyR function. Although

the L-type VGCC's and the BK channels contain redox sensitive cysteine residues, they

do not contribute to the DTT-mediated decrease in aged-sAHP. Furthermore, DTT's

effects are mediated by a direct reducing action on the cysteine residues of RyR, and

not due to redox modulation of Ser/Thr kinases which are known to regulate the function

of RyRs. In summary, our results suggest that during aging there is enhanced Ca2+

mobilization from the ICS through the RyRs, which leads to increased activation of the

Ca2+ dependent K current that underlies sAHP. The age-related increase in RyR

function does not appear to be due to increased RyR expression (Martini et al., 1994),

rather an oxidative stress induced shift in the intracellular redox state may enhance the

responsiveness of RyRs during aging (Hidalgo et al., 2004; Bull et al., 2007;

Gokulrangan et al., 2007). Hence posttranslational changes (primarily redox

modification) of the RyRs are thought to underlie age-related increase in sAHP.

Previous reports indicate that RyR contributes negatively to the induction of LTP and


123









interferes with normal spatial learning (Futatsugi et al., 1999). In fact blockade of RyRs

using ryanodine ameliorates hippocampal markers of aging (Gant et al., 2006). Thus

our model indicates that decreasing RyR function in aged hippocampal neurons, by

shifting the redox environment to a more reductive state would enhance LTP and

memory (as indicated in chapter 3 and 4), and decrease the sAHP amplitude (as

demonstrated in Chapter 5).

Therapeutic Potential of the Current Study

The fact that "functional lesion" of the hippocampus during normal aging can be

reversed to a certain extent, is an exciting starting point for the development of

therapeutic strategies aimed to treat memory loss and cognitive dysfunction. The

therapeutic potential of the current study is highlighted in the following points-

1). normal memory function could be restored in aging brains by reversing the subtle

physiological, biochemical and posttranslational changes to the neurons.

2). the ease of using antioxidant or pill-based therapeutics far outweighs the complexity

of the therapeutics based on cell replacement strategies. Therapeutics based on stem

cell would be more suited to treat neurodegenerative disorders that are characterized by

neuron loss; as opposed to treating memory dysfunction during normal aging that is

characterized by posttranslational changes like oxidation/reduction. In fact, antioxidants

(Socci et al., 1995; Cotman et al., 2002; Zhang et al., 2007; Li et al., 2009) and

antioxidant mimetics (Stoll et al., 1993; Liu et al., 2003) have been reported to

ameliorate age-related learning and memory deficits by reducing oxidative damage.

Antioxidants are also effective in improving spatial memory in rats following brain injury

(Long et al., 1996; Koda et al., 2008) pointing at a general protective role for

antioxidants in learning and memory.


124









3). the possibility of a non-genetic approach for the treatment of memory loss during

aging eschews the complexities of gene based therapeutics.

The results presented here indicate that a majority of neurons lose their function

during aging; however it is still possible that some neurons retain normal functionality in

the aged brain. If we assume that the normal functionality of these aged neurons

derives from proper functioning of certain mechanisms, then the identification of such

mechanisms is critical to treating cognitive impairment and memory dysfunction. The

strategy to prevent a normal neuron from becoming dysfunctional can be achieved by

creating suitable "therapeutic barriers" (Fig. 6-2). In this context, therapeutic barriers

could be strategies that keep intracellular Ca2+ concentrations within tolerable levels or

strategies that decrease oxidative damage to neurons like spin trap agents that

scavenge ROS, nutritional supplements, regular exercise, or caloric restriction. In the

provided conceptual framework (Fig. 6-2) integrated Ca2+ levels is considered as the

biomarker upon which a hypothetical therapeutic barrier could be applied. As an

extension of this idea, potentially any age-related biomarker, that marks the transition of

the neurons from a functional to a dysfunctional stage, could be subjected to a tailor

made therapeutic barrier. Based on the findings of the studies presented here, the

function of the NMDARs and/or the amplitude of sAHP of the CA1 pyramidal neurons

could be a biomarker for cognitive impairment and memory decline; and the therapeutic

barrier could be strategies that prevent an oxidative redox state or increased oxidative

stress.

Future Directions

Understanding the age-related changes in the Ca2+ handling mechanisms of the

neurons is critical to the development of therapeutics aimed to ameliorate cognitive


125









dysfunction and memory loss. Amongst the key players that maintain Ca2+ homeostasis

in the neurons (Fig. 6-3), NMDARs stand out for their significant role in synaptic

plasticity, learning and memory. The findings presented in this dissertation delineate the

biochemical and physiological changes to the NMDARs during aging. Debate

surrounding NMDAR function during aging and neurodegeneration are at the heart of

developing suitable therapeutics that reverse the biochemical and physiological

changes and reinstate normal function to dysfunctional aged neurons. One of the

important goals for future research is to distinguish between situations that demand an

increase in NMDAR function (like improving neuronal function) versus situations that

demand a decrease in NMDAR function (like preventing neuron death). The decrease in

the NMDAR function of aged neurons might represent a compensatory neuroprotective

mechanism associated with inappropriate receptor activity. It is well documented that

NMDAR associated Ca2+ influx triggers neurotoxicity and activates cell death programs

in neurons (Chen et al., 1992; Lei et al., 1992; Pivovarova et al., 2004). Thus aged

neurons could progressively down regulate neurotoxicity-associated NMDAR activation

for purposes of cell preservation (Foster, 1999). However one of the consequences of

down regulating NMDAR function is impaired NMDAR-dependent synaptic plasticity and

memory. Thus treatment strategies that deal with NMDARs in aged neurons have to

reconcile the opposing features of "functional rescue" by NMDAR activation and over

expression, with "neuroprotection" by NMDAR blockade and down regulation.

Interestingly, over expression of NR2B subunit improves synaptic plasticity and

memory in aged mice (Cao et al., 2007). However, NMDAR blockade by memantine

improves cognition and synaptic plasticity (Barnes et al., 1996; Norris and Foster, 1999;


126









Pieta Dias et al., 2007), possibly by blocking inappropriate NMDAR activation (Rosi et

al., 2006; Matute, 2007; Chang and Gold, 2008); thus indicating a "functional rescue". In

the case of neurodegenerative disease, decreased expression of NR1 mRNA has been

observed in brain regions that are most at risk for cell death, including Huntington's

disease, wherein a decrease in NR1 mRNA expression is observed in the neostriatum

(Arzberger et al., 1997). Furthermore, there is evidence for decreased NMDAR

expression in the hippocampus during the early stages of Alzheimer's disease

(Mishizen-Eberz et al., 2004; Jacob et al., 2007), hinting at "neuroprotection"

mechanisms employed by those neurons. Thus, it will be important for future research

to determine whether enhancing or inhibiting NMDAR function will be beneficial in

preserving hippocampus dependent learning and memory function during normal aging

and in the face of neurodegenerative disease. A sound strategy would be to find a

balance between the degree of functional rescue and the extent of neuroprotection

needed for successful aging and preserved neuronal function.


127










OXIDATIVE STRESS NMDAR FUNCTION
(DURING AGING) IN AGED NEURONS




REDOX MODULATION LTD
OF NEURONAL
KINASES/PHOSPHATASES
DECREASED
HIPPOCAMPAL FUNCTION &
SYNAPTIC TRANSMISSION



Figure 6-1. The biochemical model of brain aging and hippocampal dysfunction. The
proposed model linking increased oxidative stress and decreased NMDA
receptor activity during normal aging [either directly or indirectly through
kinases and phosphatases]. The outcome would be enhanced LTD and
impaired LTP for neural activity occurring at the modification threshold. If the
proposed model turns out to be true, it could explain the age related
weakening of synaptic connections in the hippocampus


128









NORMAL
NEURONS


AGED
NEURONS


NEURO-
DEGENERA'TON


THERAPEUTIC
BARRIER

INTEGRATED CA2 LEVELS INTRACELLULARR)


Figure 6-2. Conceptual framework for age-related neuronal dysfunction based on
intracellular calcium levels. Integrated Ca2+ levels within the neurons can be
used as an effective marker for differentiating the events associated with
normal aging and neurodegeneration. The integrated Ca2+ levels follow a
sigmoid pattern of increase as neurons transition from normal (light blue) to
aged category (dark blue). Apoptosis can be observed at these stages.
Sustained increase in integrated Ca2+ levels activates neurotoxic pathways
and leads to necrosis or neuron death observed in neurodegeneration (darker
shades of red). From a therapeutic standpoint, a "therapeutic barrier" could be
erected, in the form of nutritional or therapeutic intervention, which will
potentially prevent the normal neurons from exhibiting the intracellular Ca2+
profile of aged neurons. This crossover is proposed to precede neuronal
dysfunction and ultimately memory dysfunction.


129








































Figure 6-3. Integrative model of the impact of aging on the calcium handling
mechanisms and physiological processes. During aging there is an interaction
between increased oxidative stress and decreased neuron health with
mechanisms for Ca2+ regulation that includes the NMDA receptors (NMDAR),
voltage-dependent Ca2+ channels (VDCC), intracellular Ca2+ stores (ICS), and
Ca2+ buffering and extrusion mechanisms. An indication of regional specificity
(hippocampus, frontal cortex, cortex, basal forebrain) and the direction of
change (increase red arrow and decrease green arrow) for each
mechanism are also provided. The shift in Ca2+ homeostatic mechanisms
may represent neuroprotective mechanisms to decrease further rise in
intracellular Ca2+ by decreasing neuron activity. These changes impair the
function of the neuron (Adapted from Kumar A, Bodhinathan K, and Foster T
C, Front Ag Neurosci 2009)


130









APPENDIX A
DRUGS, SOLUTIONS, AND SUPPLIERS

AP-5 (DL-2-Amino-5-phosphonovaleric acid); NMDAR antagonist; Sigma, St. Louis, MO

ATP-Na lyophilized salt (Adenosine-5'-triphosphate); phosphate donor in kinase
reactions; CycLex Co Ltd, Nagano, Japan

Bis-I (bisindolylmaleimide-I); specific inhibitor of protein kinase C; Calbiochem, San
Diego, CA

c-H2DCFDA (5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein diacetate); fluorescent
dye used to detect reactive oxygen species; Molecular Probes Inc, Eugene, OR

CaM (Calmodulin, purified from bovine brain); Ca2+ binding protein and co activator of
CaM-Kinase II; CycLex Co Ltd, Nagano, Japan

CaMKII (Ca2+/calmodulin-dependent protein kinase II); Ser/Thr kinase; CycLex Co Ltd,
Nagano, Japan

Catalase (purified from human erythrocytes); antioxidant enzyme converts hydrogen
peroxide to water; Sigma, St. Louis, MO

DMSO (dimethyl sulfoxide); non-aqueous solvent for various drugs; Sigma, St. Louis,
MO

DNQX (6, 7-Dinitroquinoxaline-2, 3(1 H, 4H)-dione); AMPAR antagonist; Sigma, St.
Louis, MO

DTNB (5, 5'-Dithiobis (2-nitrobenzoic acid); oxidizing agent; Sigma, St. Louis, MO

DTT (Dithiothreitol); reducing agent; Sigma, St. Louis, MO

EGTA (Ethylene glycol-bis-(2-aminoethyl)-N, N, N', N'-tetraacetic acid); chelating agent;
CycLex Co Ltd, Nagano, Japan

Ethanol; non-aqueous solvent for various drugs; Fisher Scientific, Pittsburgh, PA

FK-506; Calcineurin/Protein Phosphates 2B inhibitor; LC Laboratories, Woburn, MA

H-7 (()-1-(5-Isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride); broad
spectrum Serine/Threonine kinase inhibitor; Tocris Bioscience, Ellisville, MO

HCI (Hydrochloric acid); general acid; Fisher Scientific, Pittsburgh, PA

HRP conjugated anti-phospho-Syntide-2 antibody; CycLex Co Ltd, Nagano, Japan


131









KN-62 (4-[(2S)-2-[(5-isoquinolinylsulfonyl) methylamino]-3-oxo- 3-(4-phenyl-1-
piperazinyl) propyl] phenyl Isoquinolinesulfonic acid ester); specific CaMKII
inhibitor; Tocris Bioscience, Ellisville, MO

L-Glutathione reduced form (L-GSH); biologically available reducing agent; Sigma, St.
Louis, MO

Myr-AIP (myristoylated autocamtide-2 related inhibitory peptide); specific peptide
inhibitor of CaMKII with the following sequence [Myr-N-Lys-Lys-Ala-Leu-Arg-Arg-
Gln-Glu-Ala-Val-Asp-Ala-Leu-OH]; Calbiochem, San Diego, CA

Nifedipine; L-type Voltage-gated Ca2+ Channel Antagonist; Tocris Bioscience, Ellisville,
MO

NaOH (Sodium Hydroxide); used to adjust the pH of solutions; Sigma, St. Louis, MO

OA (Okadaic acid); Protein Phosphatase 1 inhibitor; Tocris Bioscience, Ellisville, MO

Picrotoxin (PTX); GABAA receptor antagonist; Tocris Bioscience, Ellisville, MO

Ryanodine (RyR); Ryanodine receptor antagonist; Calbiochem, San Diego, CA

Superoxide Dismutase (SOD, from human erythrocytes); antioxidant enzyme converts
superoxide anion to hydrogen peroxide or oxygen; Sigma, St. Louis, MO

TMB (Tetra methyl-benzidine); chromogenic substrate for horseradish peroxidase;
CycLex Co Ltd, Nagano, Japan

Xanthine (X); substrate for Xanthine Oxidase which react together to produce
superoxide radicals; Calbiochem, San Diego, CA

Xanthine Oxidase (XO); an enzyme which reacts with Xanthine to produce superoxide
radicals; Roche Diagnostics, Indianapolis, IN


132









APPENDIX B
DRUG CONCENTRATIONS USED IN THE EXPERIMENTS

AP-5 (100 pM)

ATP-Na lyophilized salt (62.5 pM)

Bis-I (500 nM)

c-H2DCFDA (10 pM)

Ca2+/Calmodulin-dependent protein kinase II (15 to 30 mU)

Calmodulin (200 ng/mL)

Catalase (260 units/mL)

DMSO (final solvent concentration of < 0.01%)

DNQX (30 pM)

DTNB (500 pM)

DTT (700 pM)

EGTA (2 mM)

Ethanol (final solvent concentration of < 0.0001%)

FK-506 (10 pM)

H-7 (10 pM)

KN-62 (10 pM)

L-Glutathione reduced form (700 pM)

Myr-AIP (5 pM)

Nifedipine (10 pM)

Okadaic acid (1 pM)

Paxilline (10 pM)

Picrotoxin (10 pM)

Ryanodine (20 pM)

Superoxide Dismutase (121 units/mL)


133









Thapsigargin (1 pM)

Xanthine (20 pg/mL)

Xanthine Oxidase (0.25 to 1.00 units/mg of Xanthine)


134









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BIOGRAPHICAL SKETCH

Karthik was born in Madurai, India, to Rajeswari Bodhinathan and Bodhinathan

Sundarapandian. Karthik's primary schooling was at the Air Force School, Bangalore,

situated in the midst of India's scientific complex on Sir C. V. Raman road (named after

the late Nobel Laureate Sir C. V. Raman). Karthik graduated from T.V.S. Lakshmi

Matriculation Higher Secondary School in 2001. He then graduated "first-class honors"

in 2005 with a Bachelor of Technology (Major: Biotechnology) from P.S.G. College of

Technology in Coimbatore, affiliated with Anna University, one of India's eminent

engineering universities. His initial scientific pursuits were shaped by a Summer

Research Fellowship (2003 and 2004) awarded by The Jawaharlal Nehru Center for

Advanced Scientific Research in Bangalore, India. Consequently, the fellowship helped

him pursue his undergraduate research work in the lab of Dr. Saumitra Das at the

Indian Institute of Science in the spring of 2005. Karthik was recruited by the

Interdisciplinary Program for Biomedical Research at the University of Florida, in the fall

of 2005, with the Alumni Fellowship. The exciting and unanswered questions of brain

function and dysfunction led him, in the summer of 2006, to join the lab of Dr. Thomas

C. Foster, the McKnight Chair for Research on Aging and Memory in the Department of

Neuroscience at the University of Florida.


162





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ROLE OF REDOX STATE IN MEDI ATING AGE-RELATED CHANGES IN HIPPOCAMPAL SYNAPTIC TRANSMISSION, PLASTICITY AND NEURONAL EXCITABILITY By KARTHIK BODHINATHAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010 1

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2010 Karthik Bodhinathan 2

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To my family, my friends, and Ramana 3

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ACKNOWLEDGMENTS I thank my parents Rajeswari Bodhinathan and Bodhinathan Sundarapandian, for letting me chase my dreams. Special thanks to my little sister Soundharya Pradha, for her prayers. I thank my mentor Dr Thomas Foster for his cons tant encouragement and able guidance. I would also like to thank my committee members, Drs. Christiaan Leeuwenburgh, Harry Nick, and Charles Fr azier, and the Neuroscience program director Dr. Susan Semple-Rowland for their helpful comments and suggestions throughout my time at graduate school. My scientific pursuits would not have been possible without the guidance of past mentors, Drs. V. Ramamurthy and Saumitra Das, and the words of Ramana Maharishi and Dr. Abdu l Kalam. I also ex press my gratitude to the past and current members of Foster lab Dr. Ashok Kumar, Dr. Zane Zeier, Dr. Kristina Aenlle, Travis Jackson, Asha Rani, Wei-Hua Lee, Olga Tchigrinova, Michael Guidi, and Sylvia. This dissertation would not have been possible without the support of Sunitha Rangaraju, Emalick Njie, and numerous friends. Special thanks to Ms. Betty J. Streetman at the Neuroscienc e office, and Ms. Valerie Cloud-Driver at the IDP program. Finally, I would like to thank the Alumni Gr aduate Program for their four year fellowship that made a doctor and neurosci entist out of a kid who had the dreams but not the means. 4

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TABLE OF CONTENTS page ACKNOWLEDG MENTS .................................................................................................. 4LIST OF TABLES ............................................................................................................ 8LIST OF FI GURES .......................................................................................................... 9LIST OF ABBR EVIATION S ........................................................................................... 11ABSTRACT ................................................................................................................... 15 CHAPTER 1 INTRODUC TION .................................................................................................... 18Learning and Me mory ............................................................................................. 18Aging Effects on Hippocam pus ............................................................................... 19Learning and Memory Systems Dependent on Hippocampus ................................ 20Neuroanatomy of Hippocampus ............................................................................. 22Aging Effects on NMDAR Mediat ed Synaptic Trans mission ................................... 23Synaptic Transmission and Plasticity in CA1 Pyrami dal Neur ons ........................... 25Ionotropic Glutamat ergic Transmission ................................................................... 26NMDA Receptor Depe ndent Synaptic Plasti city: LTP and LTD .............................. 29Afterhyperpolarization in CA1 Pyramidal Neurons .................................................. 31Calcium Homeostasis in CA1 Ne urons ................................................................... 34Redox State and Agin g ........................................................................................... 36Summary ................................................................................................................ 392 MATERIALS A ND METHOD S ................................................................................ 41Drugs, Solutions and Supplie rs ............................................................................... 41Animal Proc edures ................................................................................................. 41Hippocampal Tissue Dissection for El ectrophysiological Experiments ................... 42Electrophysiological Recordings: Ex tracellular Field Potentia ls .............................. 42Extracellular Field Potent ials: Data Analysi s .................................................... 43Long-Term Potentiation and Paired-P ulse Ratio Recordings ........................... 44Isolation of NMDAR Mediated Extracellular Synaptic Potentia ls ...................... 45Electrophysiological Recordings: Intracellular Sharp Microelectrode Recording .... 45Intracellular Synaptic Potentials: Data Analysis ................................................ 46Intracellular Afterhyperpolar ization: Data Analysis ........................................... 47Measurement of ROS in Hippocampal S lices ......................................................... 48CaMKII Activity Assay ............................................................................................. 49Statistical Methods for Analysis of Data .................................................................. 49 5

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3 REDOX STATE DEPENDENT CHANGES IN NMDA RECEPTOR MEDIATED SYNAPTIC TRANSMISSION IN AGED HIPPOCAMPUS ....................................... 53Introducti on ............................................................................................................. 53Results .................................................................................................................... 54NMDA Receptor Function Decreases in the Hippocampus of Aged Animals at Various Levels of Pre-synapt ic Fiber Volley Amplitud e ............................. 54Oxidizing Agents Decrease NMDAR Func tion in Young, but not in Aged, Hippocampal Slices ....................................................................................... 55NMDAR Function in Young Animals Re covers From Exposure to Higher Concentrations of Ox idizing Ag ents .............................................................. 56Reducing Agents Increase NMDAR Function Selectively in Aged Hippocamp us ................................................................................................ 57Intracellular Location of Redox Sensit ive Cysteines Revealed by Differential Application of Biologically Availabl e Reducing Agent LGlutathione ............. 58Reducing Agent Mediated Recovery of NMDAR Function is Reversed by Oxidizing Agent, and Spec ific to NM DARs .................................................... 59Discussio n .............................................................................................................. 614 MOLECULAR MECHANISM UNDERLYING RECOVERY OF NMDAR FUNCTION AND HIPPOCAMPAL SYNAPTIC PLASTICITY IN AGED ANIMALS 75Introducti on ............................................................................................................. 75Results .................................................................................................................... 76ROS Sensitive Dye Indicates Redox Stat e of Live Hippocampal Neurons in in vitro Slices ................................................................................................. 76Enhanced ROS Production in the CA1 Re gion of the Hippocampus of Aged Animals ......................................................................................................... 78Broad Spectrum Ser/Thr Kinase Inhibito r Blocks DTT-Mediated Recovery of NMDAR Function in Aged Hippocampal Ne urons ......................................... 79CaM Kinase II specific Inhibitors Bl ock DTT-Mediated Recovery of NMDAR Function in Aged Hippo campal N eurons ....................................................... 80DTT-Mediated Recovery of NMDAR Function in Aged Animals is Independent of Neuronal Pr otein Phosph atases ........................................... 81Long-Term Potentiation is Enhanced in Aged Hippocampal Slices Exposed to D TT ........................................................................................................... 82Reducing Agent does not Alter L ong-Term Potentiation in Young Hippocampal Slices ....................................................................................... 83CaMKII Activity is Enhanced in Aged Hippocampal CA1 Cytosolic Extracts Treated with DTT .......................................................................................... 83DTT does not Alter CaMKII Activity in Young Hippocampal CA1 Cytosolic Extracts ......................................................................................................... 84Discussio n .............................................................................................................. 855 REDOX MODULATION MEDIATES REDUCTION IN NEURONAL AFTERHYPERPOLARIZATION OF AGED HIPPOCAMPAL NEURONS ............. 100 6

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Introducti on ........................................................................................................... 100Results .................................................................................................................. 101Age Dependent Decrease in the sAHP Following DTT A pplication ................ 101DTT Mediated Decrease in Aged-sAHP Involves Intracellular Calcium Stores and Ryanodine Receptor s ............................................................... 102DTT Mediated Reduction in the Aged-sAHP is Independent of L-VGCC ....... 103Discussio n ............................................................................................................ 1056 CONCLUSION AND FUTU RE DIRECT IONS ....................................................... 117Conclusion ............................................................................................................ 117Therapeutic Potential of the Current Study ........................................................... 124Future Direc tions .................................................................................................. 125APPENDIX A DRUGS, SOLUTUIONS, AND SUPPLIE RS ......................................................... 131B DRUG CONCENTRATIONS USED IN THE EXPERI MENTS .............................. 133LIST OF REFE RENCES ............................................................................................. 135BIOGRAPHICAL SK ETCH .......................................................................................... 162 7

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LIST OF TABLES Table page 3-1 The NMDAR-fEPSPs from hippocampus of young and a ged animals ............... 744-1 Paired-pulse ratios from aged ani mals ............................................................... 995-1 Physiological properties of CA1 neur ons from young and aged animals .......... 116 8

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LIST OF FIGURES Figure page 1-1 Calcium homeostasis in the neuron .................................................................... 402-1 Hippocampal dissection and setup for electrophysiological recordings .............. 512-2 Analysis of electrophysiologic al signals from hippocampal slices ....................... 523-1 NMDAR mediated synaptic potentials (NMDAR-fEPSP) are reduced in area CA1 of the hippocampus during aging ................................................................ 653-2 The oxidizing agent X/XO decreases NMDAR mediated synapt ic potentials in young animals but not in aged anima ls .............................................................. 663-3 Effect of maximal concentrations of X/XO on NMDA R mediated synaptic potentials in y oung animal s ................................................................................ 673-4 The reducing agent DTT increases NM DAR mediated synaptic responses to a greater extent in aged t han in the young animals ............................................ 683-5 Extracellular applicati on of reduced L-glutathione does not affect NMDAR functi on ............................................................................................................... 693-6 Intracellular application of reduc ed L-glutathione enhances intracellular NMDAR mediated synapt ic potentia ls ................................................................ 703-7 Glutathione mediated recovery of NMDAR function in aged animals does not involve L-ty pe VGCC .......................................................................................... 713-8 Redox modification of cysteine residues underlies NMDAR specific effect of DTT .................................................................................................................... 723-9 DTT does not affect the AMPAR function of aged animals ................................. 734-1 Detection of ROS in live hippocampal slices ...................................................... 894-2 Detection of auto-fluorescence from dye-unexposed hippocampal slices .......... 904-3 Enhanced ROS production is observed in hippocampal tissue from aged rats .. 914-4 A Serine/Threonine (Ser/Thr) kinase, but not protein kinase C, mediates DTT mediated increase in NMDAR fu nction in aged hippocampus ............................ 924-5 CaMKII involvement in the DTT m ediated enhancement of NMDAR synaptic responses in aged animals ................................................................................. 93 9

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4-6 Calcineurin/PP2B and PP1 are not involved in the DTT mediated enhancement of NMDAR synaptic re sponses in ag ed animals .......................... 944-7 DTT enhances LTP in hippocampal area CA1 of aged animals ......................... 954-8 DTT does not alter the LTP in hi ppocampal area CA1 of young animals ........... 964-9 DTT enhances CaMKII activity in aged hi ppocampal CA1 cytosolic extracts ..... 974-10 DTT does not enhance CaMKII activity in young hippocampal CA1 cytosolic extracts ............................................................................................................... 985-1 Age-dependent reduction in the sAHP by DTT ................................................. 1095-2 Intracellular calcium stores underlie DTT-mediated decrease in aged-sAHP ... 1105-3 RyR blockade inhibits DTT m ediated decrease in aged-sAHP ......................... 1115-4 RyR blockade inhibits the DTT-m ediated decrease in aged-sAHP when the AHP is increased by increasing ca lcium in the recording medium .................... 1125-5 DTT mediated decrease is independent of L-type calcium channel function .... 1135-6 DTT effects on aged-sAHP are indepen dent of BK channel function ............... 1145-7 Ser/Thr kinase activity does not mediate DTT effect s on aged-sAHP .............. 1156-1 The biochemical model of brai n aging and hippocampal dysfuncti on ............... 1286-2 Conceptual framework for agerelated neuronal dysfunction based on intracellular calc ium levels ................................................................................ 1296-3 Integrative model of the impact of aging on the calcium handling mechanisms and physiological pr ocesses ............................................................................. 130 10

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LIST OF ABBREVIATIONS C Degree Celsius (unit for expressing temperature) x g g-force (unit for expressing relative centrifugal force) + plus-minus sign (symbol fo r variability around a value) AC Alternating current ACSF Artificial Cerebro Spinal Fluid ADP Adenosine diphosphate ad lib ad libitum AHP Afterhyperpolarization ANOVA Analysis of Variance AMPAR Alpha-amin o-3-hydroxy-5-Methyl-4-isoxazole Propionic Acid Receptor AP-5 2-Amino-5-Phos phonovaleric acid APamp Action Potential Amplitude (expressed in milli volts) ATP Adenosine-5'-triphosphate BCA assay Bicinchoninic acid assay (a method to determine total protein levels in a sample) Ca2+ Calcium (ionic form) [Cai] Intracellular Calcium concentration; usually expressed in nanomoles to micromoles CA1 Cornu Ammonis Area 1 CA3 Cornu Ammonis Area 3 CaMKII Ca2+/Calmodulin-Dependent Protein Kinase II CaN Calcineurin 11

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Cat Catalase (Enzyme) DC Direct current DG Dentate Gyrus DNA Deoxyribonucleic acid (genet ic material of living cells) DTNB 5, 5-dithiobis (2-nitrobenzoic acid) DTT Dithiothreitol EC Entorhinal Cortex EPSP Excitatory Post Synaptic Potential F(x, y) F-test statis tic value of F-distribution F344 Fischer 344 (strain of rat commonly used in aging studies) fEPSP Field Excitatory Post Synaptic Potential Fishers PLSD Fishers Protec ted Least Significant Difference GABA Gama Amino butyric acid GSSG Glutathione Disulfide (a di mer of two glutathione molecules) H2O2 Hydrogen peroxide HFS High Frequency Stimulation Hz Hertz (unit for represen ting frequency of periodic events) ICS Intracellular Calcium Stores kHz Kilo Hertz (1000 Hertz) L-GSH L-Glutathione L-type VGCC L-type Vo ltage gated calcium channel LTD Long-Term Depression LTP Long-Term Potentiation 12

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m Micro Meter (1/1000000 of a meter; unit of length) M Micro Molar (unit for repr esenting concentration of solutions) s Microsecond (1/1000000 of a second; unit of time) M Molarity (unit for repres enting concentration of solutions) M Mega Ohms (1000000 Ohms; unit of electrical resistance) mg Milli Grams (1/1000 of a gram; unit of mass) Mg2+ Magnesium (ionic form) min Minutes (unit of time) mL Milli Liters (1/1000 of a liter; unit of volume) mm Milli Meter (1/1000 of a meter; unit of length) mM Milli Molar (1/1000 of a Mol; unit for representing concentration) mo Months Old (Ex. 24 mo means 24 months old) mV Milli Volt (1/1000 of a Volt; uni t of representing potential difference) mU Milli Units (1/1000 of a Unit of enzymatic activity) myr-AIP Myristoylated Autocamti de-2 Related Inhibitory Peptide N Normality (unit for representing concentration of solute in solution) nA Nano Ampere (1/1000000000 of an Ampere; unit for current) Na+ Sodium (ionic form) nm Nano meters (1/1000000000 of a meter; unit of length) NMDAR N-methyl D-aspartate Receptor NMDAR-fEPSP NMDAR Mediat ed Field Excitatory Post Synaptic Potential O2 Superoxide anion OA Okadaic Acid 13

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OHHydroxyl Radical PFV Pre synaptic Fiber Volley pH Measure of acidity or basicity of solutions PKC Protein Kinase C post hoc Post Hoc Ergo Propter Hoc (Latin for after this) PP1 Protein Phosphatase Type 1 PP2B Protein Phosphatase Type 2B Rin Input Resistance RNA Ribonucleic acid ROS Reactive Oxygen Species RyR Ryanodine Receptor S.E.M Standard E rror of the Mean s.or Stratum Oriens s.pyr Stratum Pyramidale s.rad Stratum Radiatum SC Schaffer collateral sec Seconds (unit of time) Ser/Thr Serine/Threonine SOD Superoxide Dismutase (Enzyme) Vm Resting Membrane Potentia l (expressed in milli volts) X/XO Xanthine/Xanthine Oxidase 14

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Abstract of Dissertation Pr esented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Doctor of Philosophy ROLE OF REDOX STATE IN MEDI ATING AGE-RELATED CHANGES IN HIPPOCAMPAL SYNAPTIC TRANSMISSION, PLASTICITY AND NEURONAL EXCITABILITY By Karthik Bodhinathan August 2010 Chair: Thomas C Foster Major: Medical Sciences Neuroscience The mechanisms that disrupt normal neur onal function during aging are poorly understood. Due to this fact we do not yet possess a reliable therapeutic strategy to treat age-related memory loss and cognitive d ysfunction. Given the central role played by hippocampal CA1 pyramidal neurons in learning and memory, understanding the senescent changes to the biochemical and physiological properties of these neurons has become a necessary first-step in devel oping effective therapeutic strategies. The hypothesis that forms the basis for this disse rtation is that increas ed oxidative stress or a more oxidative redox state mediates an age-related shift in Ca2+ homeostasis The experiments presented in this dissertation were designed to delineate the age-related changes to the N-methyl D-aspartate rec eptor (NMDAR) function of CA1 pyramidal neurons. We tested the hypothesis that the age-related decline in NMDAR function was linked to a more oxidative redox state of the neuron. We confi rmed that the NMDAR function declines in the CA1 region of aged hippocampus. The results indicate that the intracellular redox state of the aged neurons shifts to a mo re oxidative environment. The oxidizing agent xanthine/xanthine oxi dase (X/XO) decreased the NMDAR mediated 15

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synaptic responses at hippocampal CA3-CA1 synapses, in slices from young (3-8 mo), but not aged (20-25 mo) F344 rats. Conversely, the reducing agent dithiothreitol (DTT) selectively enhanced the NMDAR mediated synaptic response in aged but not in young hippocampal slices. The age-dependent sensitiv ity of the NMDAR function to DTT was associated with facilitated induction of long term potentiation (LTP) in aged but not young animals. Moreover, experiments usi ng membrane impermeable reducing agent L-glutathione (L-GSH) indi cated that the NMDAR response was dependent on the intracellular redox state. The effect of DTT was not observed for the alpha-amino-3hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (AMPAR). The intracellular redox state dependent effects of DTT on NMDAR function indicated a role for various intracellular si gnaling cascades. We tested the hypothesis that the DTT-mediated increase in NMDAR function involved in tracellular kinases and/or phosphatases. The blockade of DTT effect by H-7 indicated the involvement of Ser/Thr kinase(s) in mediating the increase in NM DAR function. The DTT-mediated increase in NMDAR function was not blocked by Bis-I (a protein kinase C inhibitor), but was blocked by the Ca2+/calmodulin-dependent pr otein kinase II (CaMKII) inhibitor myristoylated autocamtide-2 related inhibitory peptide (myr-AIP), and the general CaM kinase inhibitor KN-62. Furthermore, the i nhibition of the ac tivity of protein phosphatasesPP1 and calcineurin had no effe ct on the DTT-mediated increase in NMDAR function. These results suggest a ro le for the CaMKII signaling cascade. Our results with CaMKII activity assays establish ed that DTT increases CaMKII activity in CA1 cytosolic extracts from aged but not from young animals. The findings provide a link between intracellular redox state and Ca MKII activity during aging, which causes 16

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17 the decline in the NMDAR function, and subsequent ly impairs synaptic plasticity in the aged hippocampal neurons. Taken together t he results provide a link between a hypothesized mechanism of aging (increased oxidative stress) and mechanisms of impaired memory (decreased NMDAR func tion and impaired synaptic plasticity). We further tested the hypot hesis that increased oxid ative stress or a more oxidative redox state decreas es neuronal excitability of aged neurons by increasing the post burst afterhyperpolarization (AHP). Application of DTT decreased the slow component of afterhyperpolarization (sAHP) in CA1 pyramidal neurons of aged but not young animals. The DTT-mediated decreas e in aged-sAHP was blocked by the depletion of intracellular Ca2+ stores (ICS) using thapsigargin or blockade of ryanodine receptor (RyR) by ryanodine. Neither the inhibition of L-type voltage gated calcium channels (L-type VGCC) nor the inhibition of Ser/Thr kinases by H-7 had any effect on the DTT-mediated decrease in aged-sAHP. The results sugges t that a more oxidative redox state during aging contributes to RyR oxidation, increases Ca2+ mobilization from the ICS, and increases the sAHP. The results presented in this disserta tion link oxidative redox state of aged neurons to decrease in the NMDAR function, and increase in sAHP. These two processes are very potent and functionally significant biomarkers of aging in the hippocampus. Hence the results of this study will have significant impact on the development of therapeutics t hat can offset senescent changes to the biochemical and physiological properties of hippocampal neurons and provide fundamental insights into the mechanisms that mediat e age-related memory lo ss and neuronal dysfunction.

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CHAPTER 1 INTRODUCTION Learning and Memory The mammalian brain is endowed with the amazing capacity for learning new information that can be stored as memories. It is fascinating that the brain possesses the unique capacity for learning and memory, in addition to being the seat of a wide variety of human faculties (Crick, 1995). Cert ain regions of the brain are designed to take part in distinct forms of learni ng and memory, for exam ple, the hippocampus located in the medial temporal lobe of the brain is involved in the formation and retrieval of declarative/explicit memory. The two main classes of cells in the hippocampus that possess unique properties (Kupfermann et al., 2000), and enable the learning and memory function of the brain are the neur ons and the glial cells. The neurons are the predominant type of signaling cells, which communicate through chemical neurotransmitters and receptor s. The glial cells are the supporting cells, which provide nutrition and recycle neurotransmitters releas ed by the neurons (Kande l, 1991, 2000a; Alberts et al., 2002). The learning and me mory function of the hippocampus is accomplished by the utilization of a comp lex array of molecules and signaling mechanisms present in these cells. The studies presented in this dissertati on were designed to test the hypothesis that an increased oxidative stress or a more oxidative redox state mediates age-related shift in Ca2+ homeostasis and contributes to neuronal dysfunction. Neuronal function is defined by the neurons synaptic transmission, pl asticity and excitability (described in detail in the following sections). Our experim ents were designed to test the effects of redox state modulators on impor tant markers of neuronal function, namely the synaptic 18

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transmission mediated by the N-methyl D-aspartate receptors (NMDARs), synaptic plasticity and neuronal excitability. Before describing the results in the following chapters, an overview of the key component s of the hypothesis is provided in the following sections of this introduction. Aging Effects on Hippocampus The hippocampal function is particularl y vulnerable to dysfunction during aging. The National Institute on Agin g (NIA) has identified cognitive impairment due to memory dysfunction as a normal part of aging. In par ticular, the hippocampal function is impaired in aged animals such that they learn slow er and forget easily (Barnes, 1979; Foster, 1999). In this context it is worthwhile to differentiate the effects of normal aging on hippocampus-dependent memory functi on and the effects arising from neurodegenerative processes. The pattern of changes to hippocampus during normal aging is different from that observed in individuals suffering from neurodegenerative disorders like Alzheimers disease (AD). The primary difference seems to be the absence of major neuron loss in people wi th normal aging. Al though initial data suggested neuronal loss during aging (Ball, 1977; Brizzee et al., 1980; Coleman and Flood, 1987), subsequent studies have conclusi vely proved that no significant neuron loss is observed (West et al., 1994; Rapp and Gallagher, 1996; Rasmussen et al., 1996; Gazzaley et al., 1997; Morrison and Ho f, 1997; Pakkenberg and Gundersen, 1997; Merrill et al., 2001). Furthermore, the loss of memory function, due to neuron loss, is associated with neurodegenerat ive disorders but not normal aging (Rapp and Gallagher, 1996; Rasmussen et al., 1996). In other words, the basic elements required for hippocampus dependent learning and memory seems to be intact during normal aging; however these system s are progressively weakened in their function. 19

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The absence of neuron loss indicates t hat the hippocampus does not exhibit an anatomical lesion during aging. Neve rtheless, the memory systems dependent on hippocampus become dysfunctional during aging, such that aged animals learn slower and forget rapidly, as noted above (Barnes, 1 979; Dunnett et al., 1990; Mabry et al., 1996; Oler and Markus, 1998; Foster, 1999; Norris and Foster, 1999). Based on the evidence presented in the followi ng chapters, it is becoming in creasingly clear that the neurobiological correlates of memory loss during normal aging are subtle physiological, biochemical and posttranslational changes ac cumulated in the hippocampal neurons. These lines of evidence support the idea of a functional lesion of hippocampus during aging characterized neuronal dysfunction. Learning and Memory Systems Dependent on Hippocampus In chapters 3, 4 and 5 we have present ed results that suggest that increased oxidative stress or more ox idative redox state contribut es to the physiological and biochemical changes in the hippocampal CA1 pyramidal neurons during aging. In order to better understand the functional significance of these results, a br ief overview of the memory systems in the brain and a detailed overview of the role of CA1 pyramidal neurons in hippocampus dependent learning and me mory are provided in the following section. Although several memory systems are t hought to utilize the neural networks of hippocampus (Riedel et al., 1999), the declarative /explicit memory system is particularly dependent on the intact functioning of the hippocampus (Eichenbaum, 1997; Mingaud et al., 2007). Declarative memory includes semantic and episodic memory (Squire and Zola, 1998). Semantic memory is the capacity to store general knowledge and recollect factual information (Squire and Zola, 1998; Kapur and Brooks, 1999; Holdstock et al., 20

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2002; Manns et al., 2003) and episodic memory is the capacity to store and recollect information about time, places and their contex t in a temporal order (Vargha-Khadem et al., 1997; Tulving and Markowitsch, 1998). Model s of memory impairment in nonhuman primates (Mishkin, 1982; Squire et al., 2004), combined with extensive behavioral characterization (Zola-Morgan et al., 1994) has indicated that hippocampal pyramidal neurons are necessary for the acquisition an d consolidation of declarative memory; while long-term storage occurs in the neocorti cal regions of the br ain (McClelland et al., 1995; McClelland and Goddard, 1996; Eichenbaum, 2000; Fell et al., 2001; Kali and Dayan, 2004). The hippocampal CA1 pyramidal neurons mediate memory consolidation (Shimizu et al., 2000; Remondes and Schum an, 2004; Frankland and Bontempi, 2005; Ji and Wilson, 2007; Takehara-Nishiuchi and McNaughton, 2008), and the retrieval or recollection of recently form ed memories (Gabrieli et al ., 1997; Roozendaal et al., 2001; Smith and Squire, 2009). All these observations support the idea that the CA1 pyramidal neurons of the hippocampus play a cr ucial role in the formation, consolidation and retrieval of declarative/explicit memories in the mammalian brain. The hippocampal formation is also important for the acquisi tion of spatial memory, which denotes the capacity to store and retrieve information regarding the spatial location and relative orientation of objects (O'K eefe, 1993; O'Keefe and Burgess, 1996; Nakazawa et al., 2004; McNaughton et al., 2006). Spatial informa tion is represented in the hippocampus through alterations in the firing properties of the CA1 pyramidal neurons in the hippocampus (O'Keefe and Speakman, 1987; Foster et al., 1989). The intricate anatomy of the hippocam pus enables its learning and memory function. One of the ideas used in constructing the hypotheses in chapters 3, 4, and 5 21

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has been that age-related changes to t he learning and memory function of the hippocampus arises, in part, from the ph ysiological and biochemical changes to the CA1 pyramidal neurons. Since all studies presented in this dissertation concern the CA1 pyramidal neurons, a brief overview of the hippocampal neuroanatomy and the organization of the CA1 pyramidal neurons wi thin the hippocampal CA1 subfield are provided below. Neuroanatomy of Hippocampus The hippocampus is a sea horse shaped stru cture located in the medial temporal lobe of the brain. It is part of the hippocampal formation in the medial temporal lobe, which includes the entorhinal cortex (EC), the subiculum, the presubiculum and the parasubiculum. The hippocampus is divided into three major subfields: the CA1 region, the CA3 region, and the dentate gyrus (D G). The abbreviation CA stands for cornu ammonis due to its semblance to a rams horn. The experiments described in the following chapters were all designed to study the synaptic transmission and plasticity in the CA3-CA1 synaptic contacts, which are part of the tri-synaptic pathway. In the trisynaptic pathway, the first set of synaptic contacts occur between the axonal afferents from EC onto the DG pyramidal neurons, the second set of synaptic contacts between the afferents from DG pyramidal neurons on to the CA3 pyramidal neurons, and the third set of synaptic contacts between the afferent s from the CA3 pyrami dal neurons onto the CA1 pyramidal neurons (Kandel, 2000b; Amaral and Lavenex, 2007). In addition to the tri-synaptic connection between t he principal pyramidal cells of the hippocampus, there are many recurrent and interneuronal connecti ons in all the major subfields of the hippocampus thus providing a massive, yet organized, network of neurons. 22

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The subfields of hippocampus are further di stinguished into various layers which reflect the underlying laminar organization, or ientation and location of the principal pyramidal cells. The CA1 subfield is distinguished into stratum lacunosum molecular (s.l.m) stratum radiatum (s.r) stratum pyramidale (s.p or the pyramidal cell layer), and stratum oriens (s.o) (Amaral and Lavenex, 2007). The dendrites of the CA1 pyramidal neurons are located in s.l.m and s.r ; the cell body is located in s.p ; and axon passes through s.o In addition, the DG subfie ld is distinguished into the molecular layer, the granule cell layer and the polymorphic cell laye r, and the CA3 subfield is distinguished into stratum radiatum stratum lucidum stratum pyramidale (or pyramidal cell layer) and stratum oriens The function of the CA1 pyramidal neurons is defined by the neurons synaptic transmission, plasticity and exci tability. In chapters 3, 4, and 5 we tested the hypothesis that a shift in the redox st ate to a more oxidative environment contributes to neuronal dysfunction by altering synaptic transmission, plasticity and excitability. In order to clarify the key components of this hypothesis, a brief description of the above mentioned parameters of CA1 pyramidal neurons is provided in the following sections. Aging Effects on NMDAR Mediat ed Synaptic Transmission The decline in NMDAR function is thought to be one of the critic al biomarkers of aging in CA1 pyramidal neurons (Foster, 2006) which is also supported by previous reports (Barnes et al., 1997; Billard and Ro uaud, 2007). In chapter 3, we tested the hypothesis that age-related dec line in NMDAR function is caused by increased oxidative stress or a more oxidative r edox state. In order to bette r understand the hypothesis and results, a brief description of the properties and functi on of NMDARs in the CA1 pyramidal neurons and their role in synaptic transmission and plasticity is provided in 23

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the following sections. But first, an eval uation of the age-relat ed changes to NMDAR function is discussed. The NMDAR mediated synaptic transmission is critical for acquisition and consolidation of hippocam pus dependent spatial learning and memory (Bannerman et al., 1995). As noted above, there is considerabl e evidence to indicate that aging is associated with a decline in NMDAR function within regions involved in processing and performing higher brain functi on including learning and memo ry (Gonzales et al., 1991; Pittaluga et al., 1993; Barnes et al., 1997; M agnusson, 1998; Eckles-Smith et al., 2000; Gore et al., 2002; Liu et al., 2008; Zhao et al., 2009). Perhaps the strongest evidence for a reduction in NMDAR function comes from physiological studies which indicate that the NMDAR mediated excitatory post synaptic potentials in the CA3-CA1 synapses of the hippocampus are reduced by approximately 50% in aged animals (Barnes et al., 1997; Eckles-Smith et al., 2000; Bodhinat han et al., 2010). However, age-related changes in the amplitude of NMDA-evoked res ponses were not observed in dissociated cortical neurons suggesting t he possibility of regional specif icity in the loss of NMDAR function (Kuehl-Kovarik et al., 2003). Several st udies indicate a decrease in the level of NMDAR protein expression in the hippocampus during aging (Bonhaus et al., 1990; Kito et al., 1990; Miyoshi et al., 1991; Tamaru et al., 1991; Wenk et al., 1991; Magnusson, 1995; Magnusson et al., 2006; Billard and Rouaud, 2007; Das and Magnusson, 2008; Liu et al., 2008; Zhao et al., 2009). Moreover the decrease has been localized to area CA1 of the hippocampus (Magnusson and Cotman, 1993; Gazzaley et al., 1996; Magnusson, 1998; Wenk and Barnes, 2000); w herein the studies report reduced binding of [3H] glutamate (agonist site), [3H] glycine (NR1 site), [3H] CPP (a competitive 24

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antagonist to the L-glutamate binding site), and [3H] MK-801 (an open channel blocker) in the hippocampus of aged rats. However, others have reported no age-related change in antagonist binding (Kito et al., 1990; Miyosh i et al., 1991; Araki et al., 1997; Shimada et al., 1997), or an increased MK-801 binding in animals with learning and retention deficits (Ingram et al., 1992; Topi c et al., 2007). It is important to note that MK-801 binds to the hydrophobic channel domain of NMDAR, exclusively labeling open channels. Thus, an apparent increase in NMDAR channel open time may act as a compensatory mechanism for the decrease in receptor num ber (Serra et al., 1994). However, the majority of reports, including our recent findi ngs, indicate that t he net function of the NMDARs decreases at CA3-CA1 hippocam pal synaptic contacts during aging (Bodhinathan et al., 2010). Thus, our working hypothesis is based on the idea that the age-related changes in the NMDAR functi on are predominantly posttranslational, probably involving oxidation/ reduction and/or changes in phosphorylation. Before describing the results which indicate a link between oxidative redo x state and decrease in NMDAR function during aging, a brief overview of the general properties of synaptic transmission and plasticity involving the CA 1 pyramidal neurons is provided in the following section. Synaptic Transmission and Plasticity in CA1 Pyramidal Neurons Information is transmitted between neurons through the synapses (a narrow cleft between two neurons). Neurotransmitters re leased by presynaptic neurons bind to and activate the receptors at the postsynaptic sites. This action constitutes synaptic transmission. The ability to modify the st rength of synaptic transmission between the neurons is thought to underlie t he learning and memory function of the brain, including hippocampal-dependent learning and memory (Bliss and Collingridge, 1993; Foster et 25

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al., 1996; Malenka and Nicoll, 1999; Bailey et al., 2000; Martin et al., 2000; Dragoi et al., 2003). In particular, the neurons possess a vast array of signaling molecules that are responsive to various aspects of learning and memory (Lisman, 1994; Tsien et al., 1996; Benson et al., 2000; Abel and Lattal, 2001; Genoux et al., 2002; Koekkoek et al., 2003). Unfortunately the learning and memory functions of the hippocampus are weakened during aging and disease. The role of hippocampus in learning and memory, described previously, depends on the complex synaptic properties of its neurons. The hypothesis presented in chapter 3 is that age-related increase in oxidative stress or oxidative redox state decreases the NMDAR mediated synaptic transmission. The focus of this hypot hesis has been the CA3-CA1 synaptic contacts located in the CA1 subfield of the hippocampus. A part of the CA3 axons constitute the Schaffer colla teral pathway that release L-glutamate, which binds to ionotropic and metabotropic glutamate receptors on the CA1 pyramidal neurons. Synaptic transmission is completed upon the activation of the receptors, leading to inflow of Na+ and Ca2+ ions into the neuron, and outflow of K+ ions from the neurons. The ability to modify the strength of synaptic transmission between neurons is termed synaptic plasticity. In this study we have investigat ed the influence of redox state on synaptic transmission and plasticity during agi ng. Specifically we tested whether the redox agents can modulate NMDAR mediated synaptic transmission and plasticity in an age-dependent manner. Ionotropic Glutamatergic Transmission The experiments described in chapters 3 and 4 were designed to analyze the effects of redox agents on NMDARs, and we al so tested if they had any effect on AMPARs. The NMDARs and AMPARs are ionotropic glutamate receptors (iGluRs), 26

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which are activated by the amino acid neurot ransmitter L-glutamate (Collingridge et al., 1983) released from the axonal terminals of CA3 pyramidal neurons. The L-glutamate can also activate metabotropic glutamate receptors (mGluRs) on the CA1 pyramidal neurons; however all the studies presented in this dissertation involved the iGluRs. The iGluRs mediate fast sy naptic transmission and are cla ssified into three major subtypes, named after the synthetic agonists that activate them. They are the alphaamino-3-hydroxy-5-methyl-4-isoxazole pr opionic acid receptor (abbreviated as AMPAR), the kainate receptor and the N-meth yl D-aspartate receptor (abbreviated as NMDAR). The AMPARs and kainate receptors are also categorized as non-NMDARs. All the iGluRs are non-selective cationic channels that are permeable to both Na+ and K+ (Aidley, 1989; Johnston and Wu, 1995). However the NMDARs are relatively more permeable to Ca2+ ions (Garaschuk et al., 1996). The ionic fluxes mediated by the iGluRs in the CA1 pyramidal neurons are measured as the excitatory post synaptic potential (EPSP). The non-NMDA Rs generate the early phase of the EPSP (~ 0 to15 ms from stimulation) and the NMDARs contri bute to the late phase of the EPSP (greater than 15 ms time window from stimulation). In this study we have analyzed both the AMPAR-mediated and NMDAR-medi ated synaptic transmission. Decreased NMDAR function is an important biomarke r of aged CA1 pyramidal neurons, and is also the central aspect of hypotheses presented in chapters 3 and 4. We tested the hypothesis that increase in ox idative stress or oxi dative redox state of aged neurons contributes to decrease in t he NMDAR mediated synaptic transmission and plasticity. In order to better understand the resu lts in support of this hypothesis, it is critical to understand the properties and function of NMDAR mediated synaptic 27

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transmission and plasticity. The following section provides a brief overview of the ionic currents and synaptic mechanism that are associated with NMDAR function in neurons. The NMDARs are hetero-tetrameric protein complexes composed of two classes of subunits, the ubiquitously expressed and essential subunit NR1; and a modulatory subunit NR2A/2B/2C/2D (Moriyoshi et al., 1991; Kutsuwada et al., 1992; Meguro et al., 1992; Monyer et al., 1992; Cull-Candy et al ., 2001). The activation of NMDAR requires binding of a ligand (glutamate), me mbrane depolarization (to remove the Mg2+ block on the channel), and binding of a co-agonist (glycine). These requirements for NMDAR activation makes it the ideal coincidence detector to integrate presynaptic and postsynaptic activity. Since NMDAR is a nonselective cation channel, its activation and opening leads to simultaneous conductance of Na+, K+, and Ca2+ ions (Chen et al., 2005). However the NMDARs are at l east 19 times more permeable to Ca2+ ions than the AMPARs, which are the other major subtype of iGluR in the CA1 pyramidal neurons (Garaschuk et al., 1996), primarily because a ll the NMDAR subunits carry the polar but neutral residue arginine in the M2 region of their pore domain (Zarei and Dani, 1994; Ferrer-Montiel et al., 1996; Premkumar and A uerbach, 1996). The NMDARs also play a critical role in influencing the properti es of AMPARs. For example the AMPARs are rapidly inserted into recently activated synapses (Hayashi et al., 2000; Zhu et al., 2000; Song and Huganir, 2002) where NMDARs have been activated (Petralia et al., 1999; Shi et al., 1999). This feature serves as the molecular basis for the induction and expression of synaptic plasticity at CA3CA1 synapses (described in detail in the following section). Although AMPAR subunits carry an arginine, instead of glutamine in their pore domain, can generate significant Ca2+ influxes, akin to the NMDARs (Jonas et 28

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al., 1994; Lomeli et al., 1994); the NMDARs ar e the primary source of iGluR derived Ca2+ in the CA1 pyramidal neurons. NMDA Receptor Dependent Synaptic Plasticity: LTP and LTD As described in more detail in the pr evious section, the NMDA receptor component of synaptic transmission is decreas ed in aged animals. This is important because, the influx of Ca2+ through the NMDARs is critical to the activation of signaling cascades in close proximity to the sy napses, such that those synapses undergo a change in the strength of synaptic transmi ssion. A change in the synaptic strength between the hippocampal neurons constitutes hippocampal synaptic plasticity which is thought to underlie the formation of memories in the mamma lian brain (Morris et al., 2003). Agerelated decline in memory functi on is associated with altered hippocampal synaptic plasticity (Foster, 1999). In chapt er 4 we tested the hypothesis that the agerelated increase in oxidative stress and decr ease in NMDAR function contributes to the alteration in synaptic plasticity. In order to better understand the results, a brief overview of the relationship between NMDAR function, and synaptic plasticity is provided below. One aspect of synaptic plasticity is long term potentiation (LTP), first reported in 1973 (Bliss and Lomo, 1973), as a long lasti ng enhancement in synaptic transmission between two neurons following brie f high-frequency electrical stimulation. In contrast, long term depression (LTD) is a long lasti ng decrease in the strength of synaptic transmission between two neurons, which is observed after prolonged low-frequency stimulation. LTD was firs t reported in 1977 (Lynch et al., 1977), and an integrative model of the interplay between LTP and LTD has emerged since then. Although LTP and LTD are processes of synaptic plasticity with opposite outcomes, they are governed by changes in the Ca2+ dynamics of the postsynaptic neuron (Cummings et al., 1996; 29

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Shouval et al., 2002), involving calcium influx predominantly through the NMDARs. In this dissertation we have investigated t he effects of redox modulators on NMDAR function. Experiments have shown that LTP is initiated upon a quick and large amplitude rise in the intracellular Ca2+ concentration in the postsynaptic neuron, while LTD induction requires a moderate rise in intracellular Ca2+ concentration over longer periods of time (Cho et al., 2001; Cormier et al., 2001). Other studi es have shown that elevations in the cytosolic Ca2+ levels in the postsynaptic neuron is sufficient, by itself, to cause bidirectional changes in synaptic st rength without presynaptic activity (Neveu and Zucker, 1996a, b; Yang et al., 1999). All these studies support the idea that Ca2+ influx into the postsynaptic neuron through the NMDARs is necessary for initiating LTP and LTD (Bliss and Collingri dge, 1993; Bear and Male nka, 1994). The NMDARmediated Ca2+ influx into the neuron activates numerous Ca2+ sensitive signaling cascades that are involved in the induction a nd expression of LTP and LTD. The role of NMDARs in synaptic plasticity, learning and memory is supported by evidence showing that NMDAR antagonists (used at concentrations that block LTP in vitro ) block acquisition of hippocampus dependent memory (Bolhuis and Reid, 1992; Davis et al., 1992). In addition, genetic models carrying a CA1 region specific knockout of the NMDAR subunit NR1 exhibit impaired hip pocampal synaptic plasticity, and poor memory skills (McHugh et al., 1996; Tsien et al., 1996). Subsequent models connecting synaptic plasticity, learning and memory ha ve proposed a sliding threshold for the direction of synaptic modification dependent on the frequency of neural activity (Shouval et al., 2002; Kumar an d Foster, 2007). 30

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One of the contribut ing factors for the NMDAR mediated Ca2+ influx during the induction of LTP and LTD is postsynaptic me mbrane depolarization. In the context of the current studies, neuronal excitability is closely linked to the membrane depolarization and determines synaptic transmi ssion and plasticity. The depolarization of the postsynaptic membrane potential is necessary to remove the Mg2+ block on the NMDARs. Normally K+ channels in the postsynaptic neu rons gate the efflux of K+, through the afterhyperpolarization (AHP) cu rrent that maintains the neurons in a hyperpolarized state. The synapt ic inputs from the presynapt ic neurons counteracts the effect of K+ channels and shifts the postsynaptic membrane potential to more depolarized potentials, which ultimately aids in the removal of the Mg2+ block on the NMDARs. Thus a more hyperpolarized state makes it difficult for the neurons to depolarize and activate the NMDARs. The co mplex relationship that exists between processes that regulate membrane potent ial and processes that modify synaptic strength ultimately determines the expr ession of LTP and LTD. Increased AHP amplitude is a biomarker of aged CA1 pyramidal neurons, and is also the focus of a separate set of studies presented in this disse rtation. We tested t he hypothesis that the oxidative redox state of aged neurons contributes to increase in the amplitude of the slow component of AHP or sAHP In order to better understand the results in support of this hypothesis, it is critical to under stand the properties and f unction of AHP. The following section provides a brief overview of the ion channels, currents and signaling mechanisms that mediat e the AHP in neurons. Afterhyperpolarization in CA1 Pyramidal Neurons The AHP is a post burst hyperpolarizati on in the membrane potential of the neurons which lasts over 1-2 seconds after the offset of the depolarizing pulse. The 31

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AHP is divided into three broad phases based in time kinetics into the fast component (fAHP), the medium component (mAHP), and the slow component (sAHP) (Sah and Faber, 2002). Each component of AHP is mediat ed by distinct classes of ion channels that differ in their pharmacology and time kinet ics. The fAHP, which lasts several tens of milliseconds, is mediated by the BK c hannels. The BK channels generate large K+ currents with single channel conductance r eaching 400 pS (Marty, 1981). The BK channels are dependent on Ca2+ binding and membrane depolarization for their activation (McManus, 1991; Cui et al ., 1997). In the context of Ca2+ signaling in the CA1 pyramidal neurons, the BK channels contain a Ca2+ detection site on their intracellular domain (Wei et al., 1994; Schreiber and Salk off, 1997). Although activated by cytosolic Ca2+, the Ca2+sensitivity of BK channels is highly dependent on the membrane potential, which enables it to generate the hyperpolarizing K+ currents within few milliseconds of the onset of the depolarizing pulse. The mAHP, which lasts a few hundred milliseconds, is generated by the SK channels. The SK channels have small K+ conductance ranging from 2-20 pS (Blatz and Magleby, 1986). In contrast to the BK channels, the SK channe ls are voltage insensitive (Hirschberg et al., 1998); however, their activation is dependent on rises in cytosolic Ca2+. In the context of Ca2+ signaling, the SK channels possess unique Ca2+ binding properties. The SK channels do not directly bind to Ca2+ like the BK channels; however, reports indicate that they can bind to Ca2+ binding proteins like calmodulin (Xia et al., 1998; Keen et al., 1999; Schumacher et al., 2001), which leads to activation of the SK channel through a conformational change. All these properties of SK channels ideally suit them to generate the relatively late onset K + current which underlie the mAHP. 32

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The sAHP is the primary focus of a separ ate set of studies presented in this dissertation. Unfortunately, the molecular identity of the ion channel underlying the sAHP is unknown. Nevertheless, numerous observations regarding the current that underlies the sAHP have given rise to in teresting predictions. The current underlying sAHP has been observed to be voltage independent, but Ca2+ dependent (Sah, 1996), which is modulated by a range of neurotransmitters including glutamate, acetylcholine and serotonin (Nicoll, 1988). Although a clas s of SK channels could possibly mediate the sAHP current (Marrion and Tavalin, 1998; Bowden et al., 2001), experiments using clotrimazole analogs, which are highly selectiv e synthetic inhibitors of sAHP current, have eliminated this possibility (Shah et al., 2001). Recent advances in understanding the molecular mechanisms underlying the sA HP current suggest that a diffusible molecule or second messenger system coul d be operating at the interface between cytosolic Ca2+ and the channels that mediate the sA HP current. For example, recent studies indicate that the neuronal Ca2+ sensing protein hippocalcin has been reported to activate channels that mediate the sA HP current in its membrane bound form (Tzingounis et al., 2007; Tzingounis and Nicoll, 2008). Another study suggests that the ionotropic kainate receptors could decrease sAHP currents through a unique metabotropic action involving protein kinase C (Melyan et al., 2002). A separate set of studies point to a phosphorylation mechanism for mediating the sAHP involving kinases like cAMP-dependent protein kinase A (Madison and Nicoll, 1986; Pedarzani and Storm, 1993), Ca2+/Calmodulin dependent protein kinas e II (Muller et al., 1992), and protein kinase C (Malenka et al., 1986) In summary, numerous slow-activating secondary systems have been implicated in mediating and modul ating the sAHP 33

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current; however the identity of the ion channel that actually conducts the K+ ions underlying the sAHP current remains to be discovered. Nevertheless, the dependence of this unknown ion channel on cytosolic Ca2+ is a veritable starting point for investigations into the mec hanisms that modulate sAHP, es pecially in the context of aging, because sAHP is reported to incr ease in aged hippocampal neurons (Landfield and Pitler, 1984; Moyer et al., 1992; Kuma r and Foster, 2004; Matthews et al., 2009). The cytosolic Ca2+ level, which determines the activation of K+ channels underlying the sAHP current, is the outcome of processes involved in maintaining neuronal Ca2+ homeostasis. The Ca2+ homeostasis is maintained by the complex interaction between mechanisms that allow Ca2+ entry into the neuron and those mechanisms that remove Ca2+ from the cytosol and/or buffer them in internal stores. In addition to the sAHP, the intracellular Ca2+ concentration also modulates synaptic plasticity. Due to the fundamental role of intracellular Ca2+ homeostasis in the development of several hypotheses presented in this dissertation a brief discussion of the key components and regulators of Ca2+ homeostasis in CA1 pyramidal neurons is provided below. Calcium Homeostasis in CA1 Neurons There are three major sources for Ca2+ mobilization in neurons the NMDARs, the L-VGCCs, and the intracellular Ca2+ stores (ICS). Cytosolic elevation in Ca2+ concentration, from these sources, activa tes signaling cascades involved in LTP/LTD, and mediates physiological processes like t he AHP. Excessive levels of cytosolic Ca2+ can lead to excitotoxicity and neuronal death, which is offset by pumps and buffering mechanisms that remove Ca2+. The interplay between the processes that control the elevation and decrease in cytosolic Ca2+ levels maintains the Ca2+ homeostasis in the 34

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neurons (Fig. 1-1). Age-related neuronal dysfuncti on is thought to originate, in part, from the perturbation of Ca2+ homeostatic mechanisms. In aged neurons there is a shift in the relative contribution of Ca2+ by various sources; such that the NMDARs contribute less Ca2+, and the L-VGCCs and ICS contribute more Ca2+ (Thibault and Landfield, 1996; Norris et al., 1998a; Thibault et al ., 2001; Foster and Kumar, 2002; Kumar and Foster, 2004; Gant et al., 2006; Foster, 2007; Thibault et al., 2007; Kumar et al., 2009) for the maintenance of Ca2+ homeostasis. Poor regulat ion of intracellular Ca2+ concentration contributes to improper activa tion of the signaling cascades involved in synaptic plasticity, thus impairing LTP and LTD (Foster, 1999; Burke and Barnes, 2006, 2010). Age-related changes to Ca2+ homeostasis is highlighted in the calcium hypothesis of brain aging which states that t he disruption of normal Ca2+ homeostasis underlies neuronal dysfunction during aging (Landfield and Pitler, 1984; Gibson and Peterson, 1987; Khachaturian, 1989, 1994). Howe ver, the mechanisms that cause the disruption in normal Ca2+ homeostasis are, as yet, poo rly understood. The hypothesis that forms the basis for this dissertation is that increased oxidat ive stress or a more oxidative redox state mediates the age-related shift in Ca2+ homeostasis, and contributes to neuronal dysfunction. The re sults presented in chapter 3, 4, and 5 delineate the link between oxidative redox state and age-related changes to neuronal synaptic transmission, plasticity, and sAHP. In order to better understand the hypotheses and results, it is important to understand the caus e and consequence of increased oxidative stress or an oxidative redox state. Hence an overview of the neuronal redox state, in the c ontext of aging, is provided in the following section. 35

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Redox State and Aging Biological aging is thought to be the out come of accumulation of changes in an organism over time (Hayflick, 1985, 2007). O ne school of thought considers aging as a biological process controlled by the expressi on pattern of genes (Kennedy et al., 1995; Martin et al., 1996; Kirkwood and Austad, 2000; Martin, 2007; Budovskaya et al., 2008) and/or alterations in the stru cture of chromosomes, specif ically the events associated with the length and state of t he ends of chromosomes called telomeres (Harley et al., 1990; Bodnar et al., 1998; Wr ight and Shay, 2002; Stewart et al., 2003; Blasco, 2005). However our current understanding is that accu mulation of changes to the biological molecules (lipids, proteins, DNA, RNA etc) contributes to the process of aging by altering the structure and f unction of the biological mole cules (Rattan et al., 1992; Butterfield et al., 1998; Finkel and Holbrook, 2000). In particular, protein oxidation is a commonly observed age-related change which alters the structure and function of proteins (Stadtman, 1988, 1992; Yin and Chen 2005; Widmer et al., 2006; Riemer et al., 2009), including several neuronal proteins (Smith et al., 1992) Protein oxidation results in the formation of disulfide bonds on the thiol moieties of cysteine and/or methionine residues (Shacter, 2000; Davies 2005). The formation of disulfide bonds modifies NMDAR function too, which is one of the major focus of this study (Choi and Lipton, 2000; Choi et al., 2001). Protein ox idation during aging arises from increased oxyradical production and/or weakened antioxidant capacity of the neurons (Foster, 2006; Poon et al., 2006), which shifts the re dox state to a more oxidative environment and weakens the redox buffering capacity (Par ihar et al., 2008). Thus progressive accumulation of oxidative damage due to increased oxidative stress and a more oxidative redox state leads to protein dysfunction and contributes, in part, to the age36

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related alteration in the struct ure and function of prot eins. One of the central tenets of all the hypotheses presented in the following chapt ers is that aging is associated with an increase in oxidative stress or an oxidative re dox state. In order to better understand the results based on this idea, it is very critic al to understand the production and removal of the free radicals that prom ote protein ox idation. One of the widely accepted t heories of aging is the Free Radical theory of aging proposed by Denham Harman in 1956. The theory hypothesized that biological aging is the consequence of free radical damage to the biological molecules (Harman, 1956, 1972). The free radicals commonly encountered in biological systems are the reactive oxygen species (ROS) and the reactive ni trogen species (RNS). Among its various functional roles, the free radicals partici pate in cellular signaling, immunological response, neurotransmission, and oxidative metabolism. However, due their toxic nature, cells have evolved an el aborate detoxificati on system to remove and neutralize them. Thus a balance exists between the pr oduction and removal free radicals. During aging there is an excessive production and impr oper removal of free radicals that leads to an accumulation of abnormal levels of ROS, and RNS, which leads to oxidative and nitrosative stress respectively. Although there is insufficient evidence to support the idea that free radicals determine lif e-span; there is nev ertheless a consensus that increased oxidative stress has a significant role to play in age-related disorders (Beckman and Ames, 1998; Migliaccio et al., 1999; Finkel and Holbrook, 2000; Melov, 2000; Schriner et al., 2005; Muller et al., 2007). Excessive levels of superoxide are one of the hallmarks of increased oxidative stress. The increase in the accumulation of fr ee radicals shifts t he redox environment of 37

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the neurons to a more oxidat ive state, and contributes to the free-radical induced damage proposed by Harman. Normally at the end of the electron transport chain, the free electrons are absorbed by oxygen, which is reduced to water. However incomplete reduction of oxygen yields superoxide (denoted by O2 -) that contains one free electron. The enzyme superoxide dismutase (SOD) catalyzes the dismutation of superoxide into hydrogen peroxide (H2O2). However H2O2 is a potent oxidizing agent, which can still cause oxidative damage to cellular component s. The enzyme catalase then converts H2O2 into oxygen and water. In addition, ce lls utilize the enzyme glutathione peroxidase to convert H2O2 into water by simultaneous oxidati on of reduced glutathione to oxidized glutathione. The age-related increase in the production of ROS overwhelms these antioxidant systems and ultimately leads to excessive accumulation of ROS. The increase in oxidative stress creates an oxidative redox state and weakens the redox buffering capacity of the aged neurons which is al so indicated by lower resting levels of reduced nicotinamide adenine di nucleotide (phosphate), and r educed L-glutathione (LGSH) (Parihar et al., 2008). The results pres ented in chapters 3, 4, and 5 indicate that oxidative redox state is one of the critical factors that paves the way for neuronal dysfunction during aging. A key component of all the hypothes es presented so far has been neuronal dysfunction. Our experiments were designed to detect abnormal changes in neuronal function and understand the mechanisms that cause such dysfunction. As described in the previous sections, the neuronal dysfuncti on is closely linked to processes that regulate intracellular Ca2+ homeostasis. Increased oxidative stress or a more oxidative redox state during aging can modulat e processes that maintain Ca2+ homeostasis by 38

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decreasing NMDAR function (Bodhinathan et al., 2010), increasing the activity of L-type VGCC (Lu et al., 2002; Akaishi et al., 2004), and increasing Ca2+ mobilization from ICS (Hidalgo et al., 2004; Kumar and Foster, 2004) Redox state of the aged neuron can also affect synaptic plasticity in hippocampal slices (Serrano and Klann, 2004; Bodhinathan et al., 2010). T he results presented in the following chapters enhance our understanding of the link between these diverse processes. Summary The overview presented above highlight s the complex interaction between mechanisms that mediate normal functioning of hippocampal CA1 pyramidal neurons, and mechanism during aging (more oxidative redox state) that contribute to its dysfunction. At the outset normal aging has significant impact on NMDAR function, NMDAR-dependent synaptic plasticity, and neuronal excitability. One of the central themes highlighted in the following chapters is that the CA1 pyra midal neurons express a broad profile of changes in intracellular Ca2+ homeostasis during aging. This phenomenon is central to many mechanisms inve stigated in this dissertation including changes in synaptic transmission, plasticity, and neuronal excitability. Notably, the ideas presented in the preceding sections take a reductionist approach (in the context of aging) to describe age-related changes in learning and memory dependent on hippocampus, the oxidative redox state arising from increased oxidative stress, and the subtle age-related biochemical and physiol ogical changes in the CA1 pyramidal neurons. The following chapters have built upon these ideas and provide novel results that enhance our understanding of neur onal dysfunction during aging. 39

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40 Figure 1-1. Calcium homeostasis in the neuron. Model depicting various Ca2+ sources, sequestrating, buffering mechanisms, and Ca2+ signaling events in a healthy neuron. Indicated are the voltage dependent Ca2+ channels (VDCC), nmethyl-d-aspartate receptor (NMDAR), -amino-3-hydroxy-5-methyl-4isoxazolepropionate receptor (AMPAR ), and g-protein coupled receptor (GPCR) involved in Ca2+ (red balls ) influx into the cytosol ( blue dashed arrows ). The release of Ca2+ into the cytoplasm also occurs from the intracellular Ca2+ stores (ICS) through inositol (1, 4, 5)-triphosphate receptor (IP3R) and ryanodine receptors (RyRs) Organelles, including the endoplasmic reticulum (ER), mitoc hondria, and lysosomes act as Ca2+ buffering systems, releasing and sequestering Ca2+. Further, the model depicts Ca2+ buffering and extrusion pathways ( red dashed arrows ), involving Na+/Ca2+ exchanger (NCX) and plasma membrane Ca2+ATPase (PMCA), sarcoplasmic reticulum Ca2+ ATPases (SERCAs), nicotinic acid adenine dinucleotide phosphate (NAADP), various Ca2+ binding proteins (CBPs). Mitochondrial permeability transition pore (mPTP) and mitochondrial sodiumCa2+ exchanger (mNCX) and secretory pathway Ca2+-ATPases (SPCA) contribute to Ca2+ regulation (Adapted from Ku mar A, Bodhinathan K, and Foster T C, Front Ag Neurosci 2009). .

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CHAPTER 2 MATERIALS AND METHODS Drugs, Solutions and Suppliers All drugs were prepared according to the manufacturers specifications and ultimately dissolved in ACSF prior to bath ap plication on the slices. Appendix A provides a comprehensive list of all the drugs, soluti ons and their suppliers, used in this study. Drugs that need either DMSO or ethanol as the solvent were initially dissolved in DMSO or ethanol respectively and diluted in ACSF to a final DMSO concentration of less than 0.01% and final ethanol concentration of le ss than 0.0001%. Appendix B provides a list of the all the concentrations of various drugs used in this study. Commonly used laboratory chemicals were acqui red from either Sigma-Aldric h (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Animal Procedures Procedures involving animals hav e been reviewed and approved by the Institutional Animal Care and Use Committee at the University of Florida. All procedures were in accordance with the guidelines establ ished by the U.S. Public Health Service Policy on Human Care and Use of Laboratory Animals. Male Fischer 344 rats, young (38 mo) and aged (20 mo), were obtained from National Institute on Aging colony at Harlan Sprague Dawley Inc (Indianapolis, IA). All animals were group housed (2 per cage), and maintained on a 12:12 hr light schedule, and provided ad libitum access to food and water. Animal health was regularly m onitored with the help of the Animal Care Services at the University of Florida. 41

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Hippocampal Tissue Dissection for El ectrophysiological Experiments The protocol to prepare live hippocam pal slices for electrophysiological experiments are derived from initial reports by Li and McIlwain (Li and McIlwain, 1957), which has been suitably modified and standar dized in our lab (Kumar and Foster, 2004). Animals were deeply anaesthetized using isoflurane (Webster, Sterling, MA) and decapitated with a guillotine (MyNeurolab, St Louis, MO). The layer of skin covering the skull was pared open and the skull was removed using bone snips. The brains were rapidly removed and transferred to a beaker containing ice-cold artificial cerebrospinal fluid (ACSF) which was calcium free. The hi ppocampi were dissected out carefully for slicing. Hippocampal slices (~ 400 m) were cut parallel to the alvear fibers using a tissue chopper (Mickle Laborator y Engineering Co, Surrey, UK). The slices were incubated in a holding chamber (at room te mperature) with ACSF containing (in mM): 124 sodium chloride (NaCl), 2 potassium chloride (KCl), 1.25 potassium phosphate monobasic (KH2PO4), 2 magnesium sulfate (MgSO4), 2 calcium chloride dihydrate (CaCl2), 26 sodium bicarbonate (NaHCO3), and 10 D-glucose. At least 30 min before recording, slices were transferred to a standard interface recording chamber (Warner Instrument, Hamden, CT). The chamber was continuously per fused with oxygenated ACSF (95%-O2 and 5%-CO2) at the rate of 2 mL/min. T he pH was maintained at 7.4 initially adjusted using 10N hydrochloric acid or 10M sodium hydroxide. The temperature was maintained at 30 0.5C us ing the automatic te mperature controller TC-324B (Warner Instrument Hamden, CT) (Fig. 2-1). Electrophysiological Recordings: Ex tracellular Field Potentials The extracellular f ield e xcitatory p ostsynaptic p otentials (fEPSP) represent the net influx of Na+ and other positive ions like Ca2+ into the postsynaptic neuron. The net 42

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movement of positive ions from the recording electr ode is measured as a negative deflection on the oscilloscope. This negativ e deflection is called the fEPSP (Aidley, 1989; Johnston and Wu, 1995; Kandel, 2000b) and is indicated as total fEPSP ( black trace ) in Fig. 2-2A. The fEPSPs for st udies described here were generated by stimulating the CA3 afferent fibers onto CA1 pyramidal neurons, known as the Schaffercollateral pathway or the CA3-CA1 pathway. The fEPSPs were recorded using glass micropipette electrodes filled with artificial ce rebrospinal fluid as the recording medium. The glass micropipette electrode resistances ranged from 4-6 M The glass micropipettes were pulled from thin-wall ed borosilicate capillary glass using a Flaming/Brown horizontal micropipette puller (S utter Instruments, San Rafael, CA). The borosilicate capillary glass had an outer diameter of 1 mm, an inner diameter of 0.75 mm and was about 4 inches in length. Two c oncentric bipolar stimulating electrodes (FHC, Bowdoinham, ME) were localized to the middle of the stratum radiatum on either side of the recording electrode in order to stimulate the afferent s onto CA1 pyramidal neurons. The outer pole of the bipolar electrode was made of stainless steel and was 200 m in diameter. The inner pole was made of platinum/iridium alloy and was about 25 m in diameter. Diphasic stimulus pulses of 100 s duration were delivered by a stimulator (SD9 Stimulator ; Grass Instrument, West Wa rwick, RI) and alternated between the two pathways such that eac h pathway was activated at 0.033 Hz. Extracellular Field Potentials: Data Analysis The signals corresponding to the extracellular field potentials were sampled at a frequency of 20-kHz; filtered and amplified between 1 Hz and 1 kHz using Axoclamp-2A (Molecular Devices, Sunnyvale, CA) and a di fferential AC amplifier (A-M Systems, Sequim, WA) and stored on a computer disk (De ll Inc, Texas) for off-line analysis. A 43

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separate output from the differential AC amplifier was fed into an oscilloscope (Tektronix 2214, Tektronix Inc, Beaver ton, OR) and audio monitor (A M8, Grass Technologies, West Warwick, RI) for real ti me visualization of the signals. Two cursors were placed to cover the initial descending phase of the waveform and the maxi mum negative slope (mV/ms) of the fEPSP was determined by a Sciworks computer algorithm (Datawave Technologies, Berthoud, CO) which determined the maximum change across a set of 20 consecutively recorded points between the cu rsors. To measure the amplitude of the fEPSP, the cursors were placed to co ver the entire waveform of the fEPSP. Subsequently a Sciworks com puter algorithm was used to compute the maximum amplitude (mV) of the f EPSP at the peak of the waveform (Fig. 2-2B). Long-Term Potentiation and Paired-Pulse Ratio Recordings For induction of LTP, the stim ulation intensity was set to elicit 50% of the maximal fEPSP obtained by stimulating the CA3 affe rents on CA1 pyramidal neurons. After stable baseline recording at 0.033 Hz for at least 20 min, high frequency stimulation (HFS) was delivered to the pathway at 100 Hz for 1 sec (100 pulses) at the baseline stimulation intensity, and recorded for at least 60 min post-HFS. A simultaneously recorded control (non-HFS) pathway received t he test stimulation but not the HFS. In some cases the fEPSP was monitored in the presence of drugs to account for changes on baseline synaptic transmission, before delivering the HFS. The average fEPSP slope corresponding to the last 5 minutes from each pathway was used to compare changes in synaptic strength relative to the baseline. For measuring the paired-pulse ratio, paired pulses were delivered through a single stimulating electrode at varying inter pulse intervals. The first pulse was set to elicit 50% of the maximal fEPSP. The vari ous inter pulse intervals between successive 44

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pulses were 50 ms, 100 ms, 150 ms, and 200 ms. The ratio of the maximum negative slope of the second pulse to the maximu m negative slope of the first pulse was computed as the paired-pulse ratio. Isolation of NMDAR Medi ated Extracellular Synaptic Potentials To obtain the NMDAR-mediated field exci tatory postsynaptic potential (NMDARfEPSP) at the CA3-CA1 synapses, the slices were incubated in ACSF containing low extracellular Mg2+ (0.5 mM), 6, 7-dinitroquinoxalin e-2, 3-dione (DNQX, 30 M), and picrotoxin (PTX, 10 M). Low extracellular Mg2+ was used to facilitate the removal of the Mg2+ block on the NMDAR; DNQX (AMPAR ant agonist) was used to block the AMPAR component of the fEPSP; and PTX (GABAA antagonist) was used to minimize the GABA-ergic inhibition on the CA1 neurons. In each case the baseline response was collected for at least 10 min before experim ental manipulations (drug application). The NMDAR-fEPSP is indicat ed in Fig. 2-2A ( blue trace). Successful pharmacological isolation of the NMDAR-f EPSP was demonstrated by the application of the NMDAR antagonist AP-5 (100 m), which is indicated by the red trace in Fig. 2-2A. Changes in the levels of synaptic transmission, induced by drug application, were calculated as percentage change from the averaged baseline responses. Electrophysiological Recordings: Intracellu lar Sharp Microelectrode Recording Intracellular excitatory post synaptic potentials were recorded from the CA1 pyramidal neurons using sharp microelectr odes. Sharp microelectrodes were pulled from thin walled borosilicat e capillary glass (1 mm out er diameter; 0.75 mm inner diameter; 4 inches in length) using the Flaming/Brown hori zontal micropipette puller (Sutter Instruments, San Ra fael, CA). All microelectrode tips were filled with 3M potassium acetate and in some cases were filled with potassium acetate solution 45

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containing the drug (for ex ample L-GSH). The microelec trode resistances typically ranged from 39-55 M Microelectrodes were visually positioned in the CA1 pyramidal cell layer using a dissecting microscope (SZH10, Optical Elements Corp, Washington, DC) and a bipolar stimulating electrode was pos itioned to stimulat e the CA3 afferents onto CA1 pyramidal neurons. On cell entry, pos itive or negative current was applied to clamp the neuronal membrane potential at -65 mV. Only neurons with a resting membrane potential (Vm) more hyperpolarized than -57 mV, and having an input resistance (Rin) >20 M and an action potential amplitude (APamp) rising 70 mV from the point of spike initiation were incl uded in the analysis. The resting membrane potential and holding current was monitored through the entire course of the experiment. Variations in the resting membrane potential, holding current, input resistance, action potential amplitude or the microelectrode resistance was also monitored for those cases in which drugs were included in the pipette and eliminated accordingly. Diphasic stimulus pulses of 100 s duration were delivered at 0.033 Hz and the stimulation intensity was adjusted to elicit an intrace llular synaptic response, which was below the spike threshold. Baseline response recording began within 3 minutes after cell entry. To obtain the NMDAR-mediat ed intracellular synaptic potentials from CA1 pyramidal neurons, slices were incubat ed in ACSF containing low extracellular Mg2+, DNQX and PTX as described above. An example of the NMDAR mediated intracellular synaptic potential is indicated in Fig. 2-2C. Intracellular Synaptic Potentials: Data Analysis The signals corresponding to the intracellula r synaptic potentials were sampled at a frequency of 20 kHz; filtered and amplified between 1 Hz and 1 kHz using Axoclamp2A (Molecular Devices, Sunnyvale, CA) and a differential AC amplifier (A-M Systems, 46

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Sequim, WA) and stored on a computer disk (De ll Inc, Texas) for off-line analysis. A separate output from the differential AC amplifier was fed into an oscilloscope (Tektronix 2214, Tektronix Inc, Beaver ton, OR) and audio monitor (A M8, Grass Technologies, West Warwick, RI) for real ti me visualization of the signals. Two cursors were placed to cover the entire waveform of the intracellula r EPSP; from the pre-stimulus baseline to > 100 ms of the waveform. A Sciworks computer algorithm was used to compute the maximum amplitude (mV) of the intracellu lar EPSP at the peak of the waveform. Intracellular Afterhyperpolarization: Data Analysis The signals corresponding to the intracellular AHP were sampled at 20 kHz; filtered and amplified between 1 Hz and 1 kH z and processed as described previously for the intracellular synaptic potentials. T he AHP was recorded from the neurons in the following manner. Depolarizing current pulses ( duration: 100 ms; amplitude: 0.3 to 1.2 nA) were delivered every 20 seconds through the intracellular electrode in order to elicit a train of action potentials with 5 spikes Since the AHP amplitude varies with the number of spikes in a train of action potent ials, a train of 5 action potentials were maintained throughout the recording to study the effect of various treatment conditions on the AHP amplitude. The AHP recorded du ring the baseline and under the application of various drugs were elicited at the sa me membrane potential (-63 mV), which was achieved by manually clamping the membrane potential with DC current injection (-1 to +1 nA). The sAHP amplitude was measur ed as the difference between the average membrane potential during the 100 ms period immediately preceding the onset of the depolarizing current and the average membrane potential 400 to 500 ms after the offset of the depolarizing current. The sAHP am plitudes were computed in mV and changes under experimental conditions are assesse d as percent change from the average 47

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baseline value over 5 to 10 min period. A repr esentative trace of the intracellular AHP is indicated in Fig. 2-2D. Measurement of ROS in Hippocampal Slices Hippocampal slices were incubated for 30 minutes in ACSF containing 10 M of the ROS detection reagent 5-(and-6)-carboxy-2 7 -dichlorodihydrofluorescein diacetate (c-H2DCFDA; Molecular Probes Inc, Eugene, OR). Slices incubated for 30 minutes in absence of c-H2DCFDA were used to detect background or auto fluorescence. Slices that were incubated with c-H2DCFDA were imaged to quantify the levels of ROS. Fluorescent images were obtained with an Ax iovert 40 CFL fluorescent microscope and Axiocam digital CCD camera (Carl Zeiss, Thornwood, NY). Fluorescence intensity was quantified as follows: fluorescent microscope was used to obtain images under uniform exposure time (100 ms) and intensity (150%). The images were then converted to grayscale images in Adobe Photoshop 5.5; t he resulting images were quantified by densitometry analysis using Image J software (h ttp://rsbweb.nih.gov/ ij). An area of about 225 m along the medial-lateral axis and 187.5 m along the anterior-posterior axis, centered on the CA1 pyramidal neurons was selected for analysis of fluorescence intensity. The mean gray value intens ities obtained from the aged and young animals are represented as the mean fluorescence int ensities. The mean fluorescence intensity from the dye-exposed slices is normalized to the mean fluorescence intensity obtained from dye-unexposed slices (harvested from the same animal) using the following relationship: Mean c-H2DCFDA Fluorescence (% of control) = [(Fe-Fu)/Fu] x 100 Where Fe and Fu were the mean fluorescence intensities obtained from dyeexposed and dye-unexposed slices respectively. 48

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CaMKII Activity Assay Hippocampi were isolated from aged F344 rats as described above. CA1 region was separated from the rest of the hippoc ampus, collected in an eppendorf tube, flash frozen in liquid nitrogen and stored at -80C. The froz en CA1 tissue samples were placed in a dounce homogenizer containi ng 1 mL of the homogenization buffer (sucrose, 1M Tris pH 7.5, 1M KCl, protease inhibitor, protein phosphatase 1 inhibitor, protein phosphatase 2 inhibitor, 100 mg/m L sodium butyrate, 0.1 M phenyl methyl sulfonyl fluoride; all prepared in distilled H2O) and homogenized us ing at least six strokes of the pestle. Homogenates were cent rifuged at 7700 x g for 10 minutes at 4C. The supernatant (containing the cytosolic frac tion) was carefully isolated and stored at 80C. Protein concentrations of the cytosol ic fractions were determined using the BCA assay method (Pierce, Rockford, IL). CaMKII activity in the cytosolic fraction was measured using the CaMKII assay kit (CycL ex Co., Ltd, Nagano, Japan). Briefly, uniform amount of cytosolic ex tracts (protein concentration = 2.0 g per well) were loaded onto micro titer wells coated with a specific peptide substrate for CaMKII Syntide-2, along with kinase r eaction buffer with or without Ca2+/calmodulin. Purified CaMKII (30 milli units per reaction; CycLex Co Ltd) was used as positive control and cytosolic extracts incubated with EGTA + myr-AIP (CaMKII specific peptide inhibitor) was used as negative control to obtain a measure of comparison for the CaMKII activity in the DTT treated and untreated cytosolic extracts. CaMKII ac tivity is expressed as spectral absorbance units at 450 nm, normalized to the control. Statistical Methods for Analysis of Data Statistical analyses for purposes of hypot hesis testing were performed using Stat View 5.0 (SAS Institute Inc, NC) and Exce l 2003 (Microsoft Corp., Seattle, WA). 49

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Students t -tests (paired or unpaired as applicable) were used to examine for differences between data sets. The statistical level of significance was set at p<0.05. For tests involving more than one factor, analysis of variance (ANOVA) was employed. In general, ANOVA was followed by Fishers protected least significant difference (PLSD) post hoc analysis in order to localiz e individual differences among various data sets. Unless otherwise stated, the effects of pharmacological treat ments on the synaptic transmission are represented as percent of baseline (Mean Standard Error of Mean). Repeated measures ANOVA was used for statistical analysis of data sets obtained sequentially from the same experimental setup over time. For example, repeated measures ANOVA was used to compare t he NMDAR-fEPSP obtained for consecutively higher stimulus intensities fr om a single hippocampal slic e and recording setup. Where stated, n represents th e number of slices used in each experiment, an indication of the statistical power of the analysis. 50

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Figure 2-1. Hippocampal disse ction and setup for electrophysiological recordings. A). Depiction of the dissecting plane in the hippocampus. The dissecting plane is parallel to the alvear fibers. B). Depict ion of the hippocampal slice indicating the position of the recording (R) and st imulating (S) electrodes. The gray outline indicates the CA1 pyramidal ce ll layer. C). The fEPSP recorded from one such arrangement. Calibration Bars: 10 ms (horizontal) and 0.5 mV (vertical). 51

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52 Figure 2-2. Analysis of electrophysiolog ical signals from hippocampal slices. A) Representative traces demonstrating the total field potential (Total fEPSP, black trace), the pharmacologically isolated NMDA receptor mediated synaptic response (NMDAR-fEPSP, blue trace) and the NMDA R-fEPSP in the presence of the NMDAR antagonist AP-5 (NMDAR-fEPSP (+AP-5), red trace). B) Representation of the measurement of t he fEPSP amplitude (blue) and the fEPSP slope (red) from a representative fEPSP (black trace). Calibration bars in A and B: 20 ms; 0.5 mV. C) Representation of the measurement of the amp litude (blue) of the intr acellular NMDAR mediated synaptic potentials obt ained from single CA1 py ramidal neurons in the hippocampus of aged animals. Calibration bars: 40 ms, 2 mV. D) Representative trace of t he intracellular post-burst afterhyperpolarization (red trace), indicating the fast (fAHP) medium (mAHP), and slow (sAHP) components. The step current used to elic it a train of 5 action potentials is indicated beneath the AHP trace. Ca libration bars: 100 ms, 10 mV.

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CHAPTER 3 REDOX STATE DEPENDENT CHANGES IN NMDA RECEPTOR MEDIATED SYNAPTIC TRANSMISSION IN AGED HIPPOCAMPUS Introduction The NMDAR is a major source of Ca2+ influx into the postsynaptic neurons during the induction of LTP at hippocampal CA3-CA1 synapses (Bliss and Collingridge, 1993). The CA1 region specific knockout of the NR1 subunit of the NMDAR abolishes LTP and impairs spatial learning and memory (Tsi en et al., 1996). Similar deficits in LTP and spatial memory are observed in aged, memo ry impaired animals. Most importantly, preliminary studies have indicated that the NMDAR component of the synaptic transmission at the CA3-CA1 synapses is de creased in aged animals (Barnes et al., 1997; Billard and Rouaud, 2007). These resu lts have given rise to the NMDAR hypofunction hypothesis in the hippocampu s during aging. The NMDAR hypofunction hypothesis suggests that age-related LTP and me mory deficits are due to a decrease in the NMDAR mediated component of synaptic transmission (Foster, 1999; Rosenzweig and Barnes, 2003; Foster, 2007). This idea is further supported by reports indicating that the NMDARs contribute less Ca2+ to the induction of LTP in the CA1 region of aged hippocampus, when compared to the young hippoc ampus (Norris et al., 1998a; Shankar et al., 1998; Boric et al., 2008). However, it is still unclear what age-related mechanism underlies the NMDAR hypofunction. Age-related alterations that may contribut e to the NMDAR deficits include altered subunit expression, composition, and splice forms (Magnusson et al., 2005; Magnusson et al., 2006). However there is a debate concerning whether the NMDAR subunit expression decreases at the CA3-CA1 synaptic sites (Fos ter and Kumar, 2002; Kumar et al., 2009). It is thus possibl e that NMDAR hypofunction is related to posttranslational 53

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modifications associated with oxidation/r eduction and/or phosphoryl ation state rather than number and/or type of receptor subunits. In terestingly previous research examining the ability of reducing and oxidizing (redox) agents to modulate NMDAR activity in cell cultures and in tissue from neonates sugges ts that redox stat e is an important determinant of NMDAR function (Aizenman et al., 1989; Aizenm an et al., 1990; Bernard et al., 1997; Choi and Lipton, 2000; Choi et al., 2001). In this study we tested the hypothesis that the age-related NMDAR hypofunction is due to increased oxidative stress or a more oxidative redox st ate of the aged neuron. The current studies confirm that the NMDAR mediated synaptic potentials are decreased at CA3-CA1 synaps es of the aged hippocampus. The NMDAR responses were modified by redox agents in an age-dep endent manner; such that oxidizing agents decreased NMDAR responses to a greater extent in young than in aged animals, and reducing agents increased NMDAR responses to a greater extent in aged than in young animals. However, using a combination of extr acellular and intracellular recordings with the relatively membrane impermeable reduci ng agent L-GSH, we found that intracellular redox state mediates that age-dependent shift in NMDAR responses. Moreover the intracellular redox state depende nt increase in NMDAR function was independent of Ltype VGCC activity. Thus, the results prov ide a link between oxidative redox state and decrease in NMDAR mediat ed synaptic transmission. Results NMDA Receptor Function Decreases in the Hippocampus of Aged Animals at Various Levels of Pre-synapt ic Fiber Volley Amplitude One of the potential mechani sms that might explain the loss of hippocampus dependent learning and memory function is decrease in the NMDAR function of the 54

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CA1 pyramidal neurons. To study the alte rations in the NMDAR function during aging, the NMDAR mediated field excitatory postsynaptic potentials (NMDAR-fEPSPs) were pharmacologically isolated (Kumar and Fost er, 2004), recorded an d analyzed from the hippocampus of young and aged F344 rats (F ig. 3-1A). The stimulation evoked presynaptic fiber volley (PFV) served as an indi cator of the level of axon activation that gave rise to the NMDAR-fEPSP; thus enabling the compar ison of NMDAR-fEPSP amplitudes across the age groups at increasingly higher PF V amplitudes (Table. 3-1). To examine the relationship between PFV and NMDAR-fEPSP across the two age groups, the PFV amplitude was separated into 0.4 mV bins and plotted against the corresponding NMDAR-fEPSP amplitude obtained from the aged and the young animals (Fig. 3-1B). An ANOVA revealed that the amplitude of the NMDAR-fEPSP was reduced in the aged animals (n = 6 animals) when compared to young animals (n = 5 animals) [F (1, 47) = 27.47, p<0.0001]. In fact, the maximum am plitude of the NMDARfEPSP was 0.73 0.14 mV and 2.87 0.9 mV in aged and young animals, respectively, approximately exhibiting a fourfold decrease during aging (Table 3-1). Oxidizing Agents Decrease NMDAR Function in Young, but not in Aged, Hippocampal Slices To test the hypothesis that the decrease in the NMDAR response was related to oxidizing conditions X/XO, an enzyme substrat e combination which produces two types of ROS superoxide anion and hydrogen peroxide, was applied to hippocampal slices of young and aged animals. X/XO has been pr eviously used in an independent study that evaluated the effects of ROS on hippocampal synaptic transmission and plasticity (Knapp and Klann, 2002). 55

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The stimulation intensity was set to evoke a response which was 30% to 50% of the maximal NMDAR-fEPSP attainable in t hat pathway, and the slope of the NMDARfEPSP was measured before and after pharmacological mani pulations. Paired Students t test revealed that application of X/XO (20 g/mL/ (0.25 units/mg of xanthine)) for 60 min, significantly decreased the slope of the NMDAR-fEPSP from the baseline levels in the young (p<0.01) but not in the aged ani mals. Furthermore, application of X/XO significantly [F (1, 10) = 15. 49, p<0.01] decreased the NMDA R-fEPSP slope to a greater extent in the young animals (66.37 7. 04%, n = 5), when compared to the aged animals (96.41 6.14%, n = 7) (Fig. 3-2). Paired t -test on the percent change in the PFV amplitudes (corresponding to the last 5 mi n of the 60 min record ing) from the X/XO treated files indicated no effect (p > 0.05) of X/XO on the level of presynaptic axonal activation across the age groups (young: 95.16 + 8.29%; aged: 108.23 + 19.44%). NMDAR Function in Young Animals R ecovers From Exposure to Higher Concentrations of Oxidizing Agents The effect of oxidizing agents on the NMDAR function in young animals was reversible, such that even in the presence of a higher concentration of xanthine oxidase (X/XO: (20 g/mL) / (1 unit/mg of xanthine)), the NM DAR mediated synaptic response decreased to 54.21 5.79%; however the res ponse recovered to 88.11 5.41% of the baseline (n = 5) following a 50 minute washou t (Fig. 3-3). One possi bility is that the redox state of the young hi ppocampal neurons is comparatively more reduced than oxidized, thus enabling a quick recovery of the NMDAR-fEPSP in the young animals upon washout of the oxidizing agent. Furthermore, the recovery of the NMDAR-fEPSP following washout indicates that young anima ls possess sufficient antioxidant and/or redox buffering capacity to absorb the excess oxyradicals due to application of X/XO. 56

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Reducing Agents Increase NMDAR Function Selectively in Aged Hippocampus The age-dependent sensitivity of NMDAR-fEPSP to oxidizing conditions suggests that the components of NMDAR signaling system are initially oxidized to a greater extent in aged animals. To test whether the decline in the NMDAR response in aged animals might be due to the age-dependent increase in the formation of disulfide linkages on the cysteine residues, the reduci ng agent DTT was applied to hippocampal slices from young and aged animals, and its effe ct on the NMDAR function in both the age groups was studied. DTT can reduce the disulfide bonds on cysteine residues of proteins into free thiols (Ciorba et al., 1997; Cai and Sesti, 2009; Long et al., 2009), thus partially reversing the effect of incr eased oxidative stress during aging. Paired t test revealed that DTT significantly enhanced the slope of the NMDAR-fEPSP from the baseline levels in both the aged (p<0.0001) and young (p<0.05) anim als. However, bath application of DTT (0.7 mM, 45 min) significantly [F (1, 19) = 5.49, p<0.05] increased the slope of the NMDAR-fEPS P to a greater extent in the aged animals (171.38 13.26%, n = 16) when compared to young animals (114.55 4.41%, n = 5) (Fig. 3-4A, 3-4B, 3-4C). Paired t -test on the PFV amplitude before and after application of DTT confirmed no change (p > 0.05) in the PFV amplitude for aged (102.81 + 3.84) and young (97.08 + 5.41) animals indicating that the effect of DTT on the NMDAR mediated response was not due to changes in the number of axons activated. In a subset of these files, DTT was allo wed to washout of the recording chamber, while its effect on the aged NM DAR-fEPSP was continuously mo nitored. Interestingly, the enhancement of the NMDAR-fEPSP in the aged animals was maintained (167.32 20.91%, n = 5) following a 45 mi n washout of DTT (Fig. 3-4D). This finding indicates a persistent change associated with the NMDAR function upon applicatio n of the reducing 57

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agent DTT. It raises the possibility that intr acellular signaling cascades that are known to regulate the NMDAR function could underlie the DTT-mediated increase observed in aged animals. Intracellular Location of Redox Sensiti ve Cysteines Revealed by Differential Application of Biologically Available Reducing Agent L-Glutathione The results from the experiments using the membrane permeable reducing agent, DTT (Susankova et al., 2006), left open the ques tion of the exact loca tion of the redox sensitive cysteine residues. Sequence comparis on with the structurally similar bacterial periplasmic binding protein predicts that the NMDARs mu st possess at least 7 cysteine residues on their extracellular surface (Choi and Lipton, 2000; Choi et al., 2001), which are redox sensitive and controls the func tion of NMDARs. In or der to delineate the location of the cysteines that are responsive to the application of DTT, the biologically available and membrane impermeable redu cing agent L-GSH was used to study its effect on the aged NMDAR-fEPSP. L-GSH is relatively membrane impermeable such that exogenous application of L-GSH is not effe ctive in increasing intracellular free thiols when compared to DTT (Mazor et al., 1996; Zou et al., 2001; Susankova et al., 2006). Moreover previous findings suggest that L-GSH protects hippocampal neurons against damage due to oxidative stress (Shin et al., 2005; Shih et al., 2006; Yoneyama et al., 2008). Surprisingly, extracellular application of the reduced form of L-GSH (0.7 mM, 45 min) did not alter NMDAR-fEPSP in the aged animals (104.83 8.39%, n = 6) (Fig. 3-5). Since extracellular applic ation of the membrane impermeable L-GSH failed to enhance NMDAR function, L-GSH was delivered in to the intracellular compartment of the aged neurons using sharp microelectro des. The NMDAR mediated intracellular EPSP was simultaneously recorded. Inclusion of the reduced form of L-GSH (0.7 mM to 58

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1.4 mM) in the intracellular recording pipe tte significantly [F(1,6) = 6.87, p<0.05] enhanced the amplitude of the NMDAR mediat ed synaptic potentials (203.90 31.38%, n = 5) in single hippocampal CA1 pyrami dal neurons from aged hippocampus when compared to age matched control cells (91. 55 22.94%, n = 3), for which L-GSH was not included in the intracellular recording pi pette (Fig. 3-6A). Application of 100 M of NMDAR antagonist AP-5 abolished the intracellular EPSP (Fig. 3-6B, right), suggesting that the recorded component wa s specifically due to the activation of the NMDARs in the aged hippocampal neurons. The fa ct that intracellular deliv ery, but not extracellular application, of L-GSH enhanced the NMDAR response provides strong evidence for intracellular redox status as a mechanism for the age-associated modulation of NMDAR function. One possibility was that intracellular de livery of the reduced form of L-GSH was increasing the activity of the Ltype VGCCs To test whether L-channel activity was modified, intracellular NMDAR EPSP was reco rded in the presence of L-GSH and the Ltype VGCC antagonist nifedipine. Intracellula r delivery of L-GSH in the presence of nifedipine (10 M) continued to enhance th e amplitude of the NMDAR mediated intracellular synaptic potentials (223.54 46. 56%, n = 3) suggesting that the DTT effect is not due to differences in L-channel activity (Fig. 3-7A, 3-7B). Reducing Agent Mediated Recovery of NMDAR Function is Reversed by Oxidizing Agent, and Specific to NMDARs To test whether the NMDAR-fEPSP in aged animals was indeed affected by the redox environment, the oxidizi ng agent 5, 5-dithiobis (2-n itrobenzoic acid) (DTNB) was applied subsequent to the DTT-mediated en hancement of the NMDAR response in aged animals. Bath application of DTNB (0 .5 mM, 45 min) significantly decreased 59

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(p<0.05) the DTT-mediated increase in NM DAR-fEPSP in aged animals, such that the NMDAR-fEPSP slope was 86.49 5. 54% (n = 4) of the baseli ne (Fig. 3-8A). A repeated measures ANOVA comparing the mean NMDAR-fEPSP slope corresponding to the last 5 min under each condition indicated a signifi cant [F(2,9) = 6.24, p<0.05] difference (Fig. 3-8 B). Post-hoc analysis revealed t hat the NMDAR-fEPSP slope was significantly (p<0.05) increased under DTT compared to baseline or DTNB (p<0.05), and no significant difference was observed between the NMDAR-fEPSP slopes in the baseline and under DTNB. In order to delineate the specificity of the DTT effect on the NMDARs, the NMDAR-fEPSP was isolated and 100 M AP-5 was applied prior to and during the application of DTT. With NMDA Rs blocked, there was no effect of DTT on any residual component of the field potentia l, such that the percent change in the residual fEPSP was 109.43 7.15% (n = 6) of the baseline (Fig. 3-8C). Finally, the NMDAR-fEPSP was abolished by application of AP-5 (Fig. 3-8D) i ndicating that the response was generated by the activation of NMDARs. To further examine the spec ificity of the DTT effect s, the AMPAR component of synaptic transmission was studied before and after application of DTT in aged hippocampal slices. Application of DTT did not affect (p>0.05) the AMPAR component of the synaptic response such that respons es were 101.63 2.89% (n = 10) of the baseline after application of DTT for 45 minutes (Fig. 3-9A). In this case, the AMPAR component was recorded as the initial descending phase of the synaptic response (covering a 15 ms to 20 ms window) indi cated in Fig. 3-9B. Finally, the AMPAR component of the fEPSP was isolated in the presence of 100 M AP-5, and DTT was 60

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applied to the non-NMDAR or predominant ly AMPAR component of the synaptic transmission. Application of 0.7 mM DTT under these conditions did not significantly alter (p>0.05) the AMPAR-fEPS P from the baseline levels ( 94.27 4.42%, n = 10) (Fig. 3-9C, Fig. 3-9D). Together, the results indicate that oxi dation of sulfhydryl groups can rapidly regulate responsiveness of NMDARs, and t hat an age-related reduction in the NMDAR response is linked to an oxidativ e redox state. Moreover this effect was exclusive to the NMDARs and had no discernable effect on the AMPAR component of the synaptic transmission. Discussion In this study we have used extracellular and intracellular recording techniques to understand the alterations in NMDAR respon ses during aging. The results presented in this chapter confirmed an age-related decr ease in the NMDAR response (or NMDAR hypofunction) and demonstrate age-dependent effects of redox modulators on the NMDAR response. First, the oxidizing agent X/XO selectively decreased NMDARfEPSP in young but not aged hippocampal neur ons. The age dependent sensitivity of NMDAR function to oxidizing condition sugg ests that the NMDAR signaling system is oxidized to a greater extent in aged ani mals. Moreover, the recovery of NMDAR response under higher concentrations of X/ XO in young neurons indicates sufficient redox buffering capacity in the young animals. Second, application of the reducing agent DTT increased NMDAR-fEPSP select ively in the aged but not in young hippocampal slices, suggesting that NMDAR signaling components are relatively less oxidized in young animals. 61

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The redox state of the ex tracellular cysteine residues of the NMDARs have been implicated in regulating t he NMDAR function in cell cultures and neonatal animals (Aizenman et al., 1989; Aizenman et al., 1990; Bernard et al., 1997). However extracellular application of the membrane impermeable bi ological reducing agent LGSH failed to increase NMDAR-fEPSP. In contrast, intracellular application of L-GSH increased NMDAR mediated synapt ic responses in single aged neurons. The use of NMDAR antagonist AP-5 confi rmed that the intracellular response was mediated solely by NMDARs. The L-GSH mediated increase in intracellular NMDAR response was independent of L-type VGCC activity. Taken together, these results indicate that the age-related decrease in NMDAR function is due to a shift in the intracellular redox state favoring an oxidative state. Th is particular result is consistent with recent work in hippocampal cell cultures indicating a decreas e in the intracellular redox ratio during aging, due in part to a deficit in the reduced fo rm of GSH inside neurons (Parihar et al., 2008). Furthermore, in accordance with previous reports (Gozlan et al., 1995; Abele et al., 1998), the reducing agent DTT had no e ffect on the AMPAR function of aged neurons. We have demonstrated this by st udying DTTs effect on total fEPSP and AMPAR-mediated fEPSP, which was isolated by the application of NMDAR antagonist AP-5. With NMDARs blocked, there was no e ffect of DTT on any residual component of the field potential. One of the consequences of NMDAR hypofunction is that aged neurons could engage a compensation mechanism in order to maintain Ca2+ homeostasis. Thus during aging the decreased Ca2+ contribution from the NMDARs is offset by the increased Ca2+ 62

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contribution from the L-type VGCCs and ICS, as discussed previously. This situation poses a problem from a functional standpoin t. The NMDARs are located at synaptic sites that are in close proximity to the Ca2+ sensitive kinases (Ex. CaMKII) and the postsynaptic density, which c ontain numerous signaling molecu les that are critical for the induction and expression of synaptic pl asticity. NMDAR hypofunction during aging would decrease the activation of these signaling cascades. Furthermore, Ltype VGCCs are mostly clustered at the base of dendrites (Westenbroek et al., 1990), thus limiting their ability to activate the Ca2+ sensitive signaling ca scades at the distant synaptic sites. Our results suggest that age-related NMDAR hypofunction is the consequence of a shift in the intracellular redox state to a more oxidative state. The various alternative explanations for NMDAR hypofunction are discussed below. Excessive NMDAR activation is thought to contribute to ex citotoxicity and neuronal death (Waxman and Lynch, 2005). Thus NMDAR hypofunction could be regarded as a cellular response to prevent excitotoxicity, and impaired NMDA R-dependent synaptic plasticity and memory decline may be the consequence of processes t hat mediate cell preservation (Foster, 1999). Alternatively it is possible that altera tions in the NMDAR localization, through the insertion of receptors into the membrane or recruitment of extra-sy naptic receptors into the synapse, may have important effects on NMDAR function du ring aging. However, it remains to be determined whether altered lo calization of the NM DARs (specifically extra-synaptic localization) is the mechani sm by which the NMDAR function declines during senescence. Preliminary reports indica te that the NR2B subunit levels, but not the obligatory NR1 subunit levels of NMDARs decrease in the synaptic sites (Zhao et 63

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al., 2009). However these findings raise the possibility that the loss in NR2B at the synaptic sites could possibly be compensat ed by NR2A/2C/2D subunits that can combine with the unaltered NR1 subunits an d form a normally functioning receptor. Another likely candidate mechanism extends the idea of posttranslational changes investigated in this chapter. The NMDAR fu nction is regulated by phosphorylation state of its cytosolic tail, which is determined by the activity of neuronal kinases and phosphatases. In the next chapter we have dem onstrated the role of one such kinase (CaMKII) in the DTT-mediated increase in the NMDAR function of aged animals. In conclusion, the results presented in this chapter demonstrate a link between age-related decline in NMDAR function and increased oxidative stress or a more oxidative redox state of the aged neurons. Most importantly we have demonstrated that the NMDAR function can be recovered in the aged CA1 pyramidal neurons upon application of DTT. This is a veritabl e starting point for treatment of NMDAR hypofunction. 64

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Figure 3-1. NMDAR mediated syn aptic potentials (NMDAR-fEPS P) are reduced in area CA1 of the hippocampus during aging. A) Representative traces of NMDARfEPSPs obtained at consecutively higher stimulus intensities from the young ( left) and aged animals ( right ). Open and filled arrows indicate the PFV and NMDAR-fEPSP, respectively. As observ ed in the traces, the aged animals exhibit a markedly reduced NMDAR mediated synaptic potential. Calibration bars: 10 ms, 1 mV. B) Plot of the mean NMDARfEPSP amplitude versus the PFV amplitude (at 0.4 mV binning width). The aged animals ( filled circles ) (n = 6) exhibited reduced NMDAR-fEPSP when compared to the young animals ( open circles ) (n = 5). In this and subsequent figures error bars represent S.E.M. 65

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Figure 3-2. The oxidizing agent X/XO decreases NMDAR mediated synaptic potentials in young animals but not in aged animals. A) Time course of the change in the normalized NMDAR-f EPSP slope in the aged ( filled circles ) (n = 7) and in the young animals ( open circles ) (n = 5) following app lication of X/XO (20 g/mL/ (0.25 unit/mg of xanthine)) for 60 minutes. B) Representative traces (average of 5 traces under each condi tion) illustrating the change in the NMDAR-fEPSP in the young ( top ) and aged animals ( bottom ) under control conditions and at the end of a 60 minute application of X/XO Calibration bars: 20 ms, 0.5 mV. C) Quant ification of mean percent change in the NMDARfEPSP slope from baseline ( dashed line ), corresponding to the last 5 minutes of a 60 minute application of X/XO in aged ( filled bar ) and young ( open bar ) animals. 66

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Figure 3-3. Effect of maximal concentra tions of X/XO on NMDAR mediated synaptic potentials in young animals. A) Time course of the change in the normalized NMDAR-fEPSP slope in the young animals (n = 5) following application of X/XO (20 g/mL/ (1 uni t/mg of xanthine)) for 30 minutes and followed by a washout for 50 minutes. B) Representativ e traces (average of 5 consecutive traces) obtained during the indicated time points: baseline (1), under X/XO (2), and upon washout (3). Calibration bars: 20 ms, 0.5 mV. C) Quantification of the mean percent change in the NM DAR-fEPSP slope in young animals following the application of X/XO for 30 minutes (filled bar) and after washout for 50 minutes (open bar). In this and subsequent figures dashed lines represent the baseline level of 100%; pound signs indicate significant difference (p<0.05) from baseline level of 100%; asterisks indicate significant difference (p<0.05) betw een the indicated groups. 67

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Figure 3-4. The reducing agent DTT increases NMDAR mediated synaptic responses to a greater extent in aged than in the young animals. A) Time course of the change in the normalized NMDAR-fEPSP slope in the aged ( filled circles ) (n = 16) and young animals ( open circles ) (n = 5) following application of DTT for 45 minutes. B) Quantification of t he mean percent change in the NMDARfEPSP slope following applic ation of DTT in aged ( filled bar ) and young ( open bar ) animals. C) Representative traces (aver age of 5 consecutive traces under each condition) illustrating the change in NMDAR-fEPSP in the presence of DTT in aged ( top ) and young animals ( bottom ). Calibration bars: 10 ms and 0.5 mV. D) Time course of the change in the normalized NMDARfEPSP slope in aged animals ( filled circles ) (n = 5) upon application of DTT for 45 minutes followed by a washout for 45 minutes. 68

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Figure 3-5. Extracellular app lication of reduced L-glutathi one does not affect NMDAR function. A) Time course of the no rmalized NMDAR-fEPSP slope in the aged animals (n = 6) in response to extracellular application of the L-GSH. B) Overlay of the means of 5 consecutive responses during the baseline (black trace) and 45 minutes after application of L-GSH (gray trace). Calibration bars: 20 ms (horizontal) and 0.5 mV (vertical). 69

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Figure 3-6. Intracellular application of reduced L-glut athione enhances intracellular NMDAR mediated synaptic pot entials. A) Time course of the normalized NMDAR mediated intracellular EPSP am plitude in the aged animals obtained under control conditions (open circles, n = 3) or with L-GSH in the recording pipette (filled circles, n = 6). B) Left: Overlay of means from 5 consecutive responses obtained intracellularly dur ing baseline (black trace) and 30 minutes after impalement (gray trace) Right: Overlay of means from 5 consecutive traces obtained intracellu larly during baseline (black trace) and 25 minutes after application of AP-5 (g ray trace). Calibration bars: 40 ms (horizontal) and 2 mV (vertical). 70

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Figure 3-7. Glutathione mediat ed recovery of NMDAR function in aged animals does not involve L-type VGCC. A). Time co urse of normalized NMDAR mediated intracellular EPSP amplitude obtained with nifedipine along with ACSF and LGSH in the recording pipette (filled triangl es, n = 3). B) Overlay of means from 5 consecutive responses obtained intracellularly during baseline ( black trace) and 30 minutes after impalement ( gray trace ). Calibration bars: 40 ms (horizontal) and 2 mV (vertical). 71

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Figure 3-8. Redox modification of cysteine residues underlies NMDAR specific effect of DTT. A) Time course of the change in the normalized NMDAR-fEPSP slope in aged animals (n = 4) in response to t he bath application of DTT followed by DTNB. The increase in NMDAR mediat ed synaptic responses by DTT was decreased by the oxidizing agent DTNB. E rror bar in A is indicated for every fourth point, for purposes of clarity. B) Quantification of the mean percent change in NMDAR-fEPSP slope following a pplication of DTT (filled bar) and DTNB (open bar) in aged animals. C) AP-5 (100 M) was applied on the isolated NMDAR-fEPSP to block it, prior to DTT application. Time course of the change in any negative slope measur ed in a 20 ms window (n = 8) upon application of AP-5 followed by DTT. D) Representativ e traces (average of 5 consecutive traces) of the NMDAR-f EPSP recorded under control conditions ( solid black trace), and upon application of 100 M AP-5 ( dashed black trace ) for at least 30 min. 72

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Figure 3-9. DTT does not affect the AMPAR f unction of aged animals. A) Time course of the change in fEPSP upon applicati on of DTT for 45 minutes in aged hippocampal slices (n = 10). B) Representative traces (average of 5 consecutive traces) of the fEPSP re corded under control conditions (black trace) and after application of DTT fo r 45 min (gray trace). The downward pointing arrows and cursors indicate t he 15 ms time window, where the initial descending phase of the fEPSP is pr edominantly mediated by AMPARs. Calibration bars: 20 ms and 0.5 mV. C) Time course of the change in the AMPAR mediated EPSP (isolated by the application of 100 M AP-5 on the fEPSP) upon application of 0.7 mM DTT in the aged animals (n = 5). D) Representative traces (average of 5 consecutive traces) of the AMPAR mediated EPSP obtained under control conditions ( black trace) and after application of DTT for 45 minutes ( gray trace ). Calibration bars: 20 ms, 0.5 mV. 73

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Table 3-1. The NMDAR-fEPSPs from hippocampus of young and aged animals PFV Bin Window (mV) Young NMDAR-fEPSP (mean S.E.M) Aged NMDAR-fEPSP (mean S.E.M) 0 0.4 0.13 0.04 0.12 0.04 0.4 0.8 0.49 0.16 0.26 0.05 0.8 1.2 0.85 0.21 0.32 0.09 1.2 1.6 1.21 0.29 0.46 0.15 1.6 2.0 1.61 0.41 0.39 0.14 2.0 2.4 1.86 .46 0.44 0.16 2.4 2.8 2.21 0.87 0.62 0.21 2.8 8.0 2.87 0.91 0.73 0.14 This table presents the NMDAR-fEPSP values recorded from the CA1 region of the hippocampus from young and aged animals. The NMDAR-fEPSP has been grouped under bin width of 0.4 mV of PFV amplitude. 74

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CHAPTER 4 MOLECULAR MECHANISM UNDERLYING RE COVERY OF NMDAR FUNCTION AND HIPPOCAMPAL SYNAPTIC PLASTICITY IN AGED ANIMALS Introduction Age-related decrease in the NMDAR function of the hippocampal CA1 pyramidal neurons was reversed by the application of the reducing agent DTT or intracellular application of L-GSH (results from chapter 3). The strong links to intracellular redox state suggested a role for an intracellular signaling mechanism in causing the DTTmediated increase in NMDAR function. Extensive empirical evidence suggests that the NMDAR function is regulated by the phosphorylation state of the receptor. NMDAR f unction is increased upon phosphorylation by several intracellular kina ses (Ben-Ari et al., 1992; Westphal et al., 1999; Li et al., 2001). Specific ally the activation of tyrosine kinase (Wang and Salter, 1994; Heidinger et al., 2002), protein kinase C (PKC) (Ben-Ari et al., 1992; Chen and Huang, 1992), and protein kinase A (Raman et al., 1996) increases NMDAR mediated currents. In contrast, pr otein phosphatases, including calcineurin and protein phosphatase 1, decrease NMDAR currents (L ieberman and Mody, 1994; Wang et al., 1994; Raman et al., 1996). Moreover, phos phorylation state of NR2A and NR2B subunits can rapidly regulate surface ex pression and localization of the NMDARs (Gardoni et al., 2001; Chung et al., 2004; Halle tt et al., 2006; Lin et al., 2006). For example, phosphorylation of serine residues wit hin the alternatively spliced cassettes of the C-terminal tail of NR1 promotes receptor trafficking from the endoplasmic reticulum and insertion into the postsynaptic membrane (Scott et al., 2001; Carroll and Zukin, 2002). On the other hand, increased phosphat ase activity has been linked to the internalization of NMDARs (Snyder et al., 2005). Hence, the kinases and phosphatases 75

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act like molecular switches, which increase or decrease NMDAR function, respectively. Interestingly, aging is associated with a sh ift in the balance of kinase/phosphatase activity, favoring phosphatases (Norris et al ., 1998b; Foster et al ., 2001; Foster, 2007). Thus, alterations in the kinase/phosphatase ac tivity in the postsynaptic neuron could underlie the decrease in the NMDAR function during aging. Moreover, the enzymatic activity of these kinases can be regulated by the reduction and oxidation of the cysteine residues located in their structure (Raynaud et al., 1997; Griendling et al., 2000; Knapp and Klann, 2000). We tested whether the DTT-mediated increase in the NMDAR function was dependent on the activity of kinases and/or phosphatases that regulate the NMDAR function. Indeed we demonstr ate that the mechanism for the age-dependent redox modulation of NMDARs involves CaMKII, but not PKC, PP1 or calcineurin/PP2B. CaMKII activity assays established that DTT increased CaMKII activity in CA1 cytosolic extracts in aged but not in young animals. Evi dence is also provided to support the idea that the reducing agent DTT increases LTP in CA1 region of aged but not young hippocampal slices. The results presented in this chapter elucidate a molecular mechanism for the age-related NMDAR hypofunction and links oxidative redox state to impaired synaptic plasticity in aged CA1 pyrmaidal neurons. Results ROS Sensitive Dye Indicates Redox Stat e of Live Hippocampal Neurons in in vitro Slices The ROS sensitive dye 5-(and-6)-carboxy-2 7 -dichlorodihydrofluorescein diacetate (c-H2DCFDA) was used to detect ROS in live CA1 pyramidal neurons of the hippocampus from aged (23 month old) F344 rat. As described in the methods section, 76

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the hippocampal slices were incubated for 30 minutes with c-H2DCFDA. Images of the aged hippocampal CA1 pyramidal neurons were obtained under bright field illumination (Fig. 4-1A) and in the presence of a filter set to detect gr een fluorescence from the dyeexposed slices (Fig. 4-1B). Dye-unexposed slices obtained from the same aged F344 rat served as the control (Fig. 4-1C); which were used to detect auto-fluorescence. The green band pass filter (Excitation at 490 nm and Emission at 525 nm) was set to detect green fluorescence at uniform exposure time of 100 ms, and uniform exposure intensity set at 150%. The fluorescent signals from t he dye-exposed slices were normalized to the dye-unexposed slices as descr ibed in the methods section. The fluorescent signals originating from the hippocampal neurons incubated with c-H2DCFDA are used as a direct measure of the levels of ROS; thus mandating a proper control for auto-fluorescent signals from the hippocampal tissue of aged animals. For all experiments described here, aut o-fluorescence was assessed from dyeunexposed slices harvested from the same animal. No fluorescent signals were detected in the dye-unexposed slices, from aged (23 month old) and young (7 month old) hippocampi, when the imaging was performed with an exposure time of 100 ms (Fig. 4-2, middle panels). However, when th e imaging was performed with an exposure time of 500 ms, clusters of fluorescent si gnals were detected in the aged but not in the young hippocampal slices (Fig. 4-2, right panels). The auto-fluorescent signals in the aged hippocampal slices could potentially arise from lipofuscin, an oxidized product known to accumulate during aging. Hence fo r the purposes of this study the imaging was performed at exposure times well belo w 500 ms, in order to eliminate autofluorescent signals and detect the fluoresc ence predominantly from ROS-oxidized c77

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H2DCFDA. For the results described in t he following sections the imaging was performed at an exposure time of 100 ms, which successfully eliminated the autofluorescent signals. Enhanced ROS Production in the CA1 Region of the Hippocampus of Aged Animals The ROS detection technique (standardiz ed as described above) was used to evaluate differential rates of ROS production in hippocam pal slices from young and aged animals. The slices were incubated with c-H2DCFDA for 30 minutes prior to imaging (Fig. 4-3A). Dye-unexposed slices (aged control: 36.14 4.42%, n = 3 animals; young control: 31.97 3.16%, n = 3 animals ) showed no significant difference in fluorescence across the age groups. Thus, fluorescence intensity from c-H2DCFDA unexposed slices was used to normalize the fluorescence intensity obtained from cH2DCFDA-exposed slices. Incubation of the hippocampal slices from young and aged animals with c-H2DCFDA for 30 min resulted in significantly (unpaired t -test; p<0.05) enhanced fluorescence in aged animals (242 .19 20.96%, n = 3 animals) when compared to young animals (141.61 11.78% n = 3 animals) (Fig. 4-3B). The dye was designed to detect ROS produc ed in the intracellular space of the neurons. However, to further eliminate the possibility of signal contribution from ROS outside the cells, aged hippocampal slices were incubated with superoxide dismutase (SOD; 121 units/mL) and catala se (260 units/mL) along with t he dye for 30 min prior to imaging. There was no significant difference (p>0.05) in the ROS-oxidized c-H2DCFDA fluorescence observed between the SOD/catalase exposed (292.388 28.04%, n = 3) and unexposed aged hippocampal slices. 78

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Broad Spectrum Ser/Thr Kinase Inhibi tor Blocks DTT-Mediated Recovery of NMDAR Function in Aged Hippocampal Neurons To test if serine/threonine (Ser/Thr) kinases were involved in the DTT-mediated increase of the NMDAR response, the broad-spectrum and membrane-permeable Ser/Thr kinase inhibitor H-7 was bath applied prior to and during t he application of DTT. In the presence of H-7 (10 M, 45 min), DT T application failed to produce the robust increase (one-group t -test; p>0.05) in the NMDAR-fE PSP slope (111.86 6.92%, n = 7) from the baseline levels (Fig. 4-4A). In order to narrow down the identity of the Ser/Thr kinase that could potentially underlie the DTT-mediated increase in NMDARfEPSP several specific inhibitors were used. It was likely that Protein Kinase C (PKC) could underlie the DTT-mediated increase in NMDAR-fEPSP, due to the fact that PKC increases NMDAR function through phosphorylation mechanisms and PKCs activity could be regulated by redox mechanisms. To test whether PKC was res ponsible for the DTT-mediated increase in NMDAR-fEPSP, the membrane permeable PKC inhibitor, Bis-I (Knapp and Klann, 2002), was applied prior to, and during, the application of DTT on aged hippocampal slices. Application of Bis-I (500 nM, 45 min) failed to blo ck the DTT-mediated increase in the NMDAR-fEPSP (142.58 13.06%, n = 6) (Fig. 4-4B). Finally, t -tests indicated no effect of 0.01% DMSO (96.38 7.64, n = 5) or kinase inhi bition per se (H-7: 103.01 4.21%, n = 7; Bis-I: 110.72 11.44%, n = 6) on the base line NMDAR-fEPSP slope in aged animals. Thus, DTT-mediated increase in NMDAR function was independent of PKC activity. 79

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CaM Kinase II specific Inhibitors Bl ock DTT-Mediated Recovery of NMDAR Function in Aged Hippocampal Neurons One of the intracellular kinases t hat can enhance NMDAR function, and whose activity is regulated by redox agents like D TT, is Ca2+/Calmodulin dependent Protein Kinase II (CaMKII). To test if CaMKII under lies the DTT-mediated increase in NMDAR function in aged neurons, the CaMK inhibitor, KN-62, was bath applied prior to and during the application of DTT. In the presence of KN-62 (10 M, 45 min) (Tokumitsu et al., 1990), DTT application failed to produce the robust increase in NMDAR-fEPSP in aged animals (97.9 7.98%, n = 5) (Fig. 45A). Furthermore, the specific peptide inhibitor of CaMKII, myr-AIP (5 M, 60 min), which was bat h applied prior to and during the application of DTT, effectivel y blocked (104.48 4.29%, n = 4) the DTT-mediated increase in NMDAR-fEPSP in aged animals (Fig. 4-5B). Finally, t -tests indicated no effect of 0.01% DMSO (96.38 7.64, n = 5) or CaM kinase inhibition per se (KN-62: 96.23 8.3%, n = 5; myr-AIP: 100.52 4. 42%, n = 4) on the baseline NMDAR-fEPSP slope in aged animals. An ANOVA comparison of the effect of D TT in the presence and absence of the all the pharmacological kinase inhibitors, indicated a significant effect of kinase inhibition on the DTT effect in aged animals [F (4, 33) = 5.85, p<0.01]. Post hoc comparisons (Fishers PLSD) indicated that the DTT-medi ated increase in the NMDAR response was blocked by H-7, myr-AIP, and KN -62 but not Bis-I (Fig. 4-5C). This conclusively proves that CaMKII underlies the DTT-mediated incr ease in the NMDAR-fEPSP slope in aged CA1 hippocampal neurons. 80

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DTT-Mediated Recovery of NMDAR Function in Aged Animals is Independent of Neuronal Protein Phosphatases It has been previously reported that neuronal kinases and phosphatases act in tandem to regulate the function of the NMDA Rs. While kinases are known to increase NMDAR function, phosphatases decrease NMDA R function. The activity of Ser/Thr phosphatases like calcineurin (CaN) and protein phosphatase 1 (PP1), is thought to contribute to altered synaptic plasticity during aging (Foster et al., 2001). Moreover, increase in phosphatase activity decreases NMDAR function through dephosphorylation of the cytosolic tails of the NMDARs (Lieberman and Mody, 1994; Wang et al., 1994). To test if DTT effects were mediated by Ca N, the CaN inhibitor FK-506 (10 M) (Norris et al., 2008) was bath applied for 45 minutes pr ior to and during the application of DTT on aged hippocampal slices, while simultane ously recording the NMDAR-fEPSP. Application of FK-506 per se did not affe ct the NMDAR-fEPSP slope (109.61 9.16%, n = 5). Importantly, FK-506 failed to block the DTT-mediated increase in the NMDAR response in aged animals such that the NMDAR-fEPSP slope increased to 148.61 16.42% (n = 5) (Fig. 4-6A). To examine the role of PP1, in mediating the DTT effect on aged NMDAR function, the PP1 inhibitor okadaic acid (OA) (1 M, 30 min) (Schnabe l et al., 2001) was (Schnabel et al., 2001) applied prior to and during application of DTT on aged hippocampal slices. Application of OA signi ficantly increased the NMDAR-fEPSP slope during the baseline recording period itself ( 121.46 9.19%, n = 5, p<0.05) (Fig. 4-6B). Therefore, a new stable baseli ne was recorded prior to the appl ication of DTT. Relative to the new baseline, DTT in creased the NMDAR-fEPSP slope (137.89 3.99%, n = 5) in the presence of OA (Fig. 4-6C). An AN OVA comparing the effect of DTT in the 81

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presence and absence of the phosphatase inhibi tors, indicated no significant effect of phosphatase inhibition on DTTs effect [F (2, 15) = 1.37, p>0.05]. A summary of the results obtained with phosphatase inhibitors is presented in Fig. 4-6D. Long-Term Potentiation is Enhanced in Aged Hippocampal Slices Exposed to DTT For studies on Long-Term Potentiation (LTP) and paired-pulse ratio, slices were bathed in normal ACSF, in order to record the AMPAR and NMDAR component of the synaptic response. LTP was induced by a single episode of high frequency stimulation (HFS) of 100Hz (1second). Hippocampal sl ices from aged animals were either incubated in normal ACSF (Aged Control) or incubated in ACSF containing 0.7 mM DTT (Aged DTT) for at least 45 min prior to t he delivery of HFS. In addition, a second pathway in the same slice that did not re ceive HFS (but received the baseline test pulses at 0.033 Hz, at baseline stimulation intensity) was used as the control pathway. The control pathway was used to monitor c hanges in slice health and to ensure stability of recording. In each case the fEPSP was re corded for 20 min prior to, and 60 min after, delivery of HFS. The magnit ude of LTP was greater in aged hippocampal slices preincubated with DTT for 45 min (136.53 2.77%; n = 10) (Fig. 4-7A), when compared to the aged controls not exposed to DTT (118.15 4.63%; n = 9) (Fig. 4-7B). Although the levels of LTP showed considerable variation (F ig. 4-7C), the LTP was significantly [F (1, 17) = 12.14, p<0.01] greater in AgedDTT group than the A ged-Control group. In order to evaluate the role of presynaptic transmitter release on the DTT mediated enhancement in aged LTP, the paired pulse ratio was computed. Examination of paired-pulses delivered at varying inter-pulse intervals ( t = 50 ms, 100 ms, 150 ms, 200 ms) (Fig. 4-7D), under control condition s and 45 min after the bath application of 82

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DTT, indicated no effect of tr eatment across the four interpulse intervals (Table. 4-1). This suggests that the increase in aged LTP under DTT recruits postsynaptic mechanisms, involving the NMDARs in the postsynaptic CA1 pyramidal neurons. Reducing Agent does not Alter Long-Te rm Potentiation in Young Hippocampal Slices DTT had selectively enhanced NMDAR func tion in aged, but not in young, hippocampal slices. In order to test w hether DTT had any age-dep endent effects in enhancing LTP, young hippocampal slices we re incubated with either normal ACSF (Young Control) or ACSF containing 0.7 mM DTT (Young DTT), bef ore delivering LTP inducing HFS. In contrast to the effect observed in the aged animals, there was no difference (p>0.05) in the levels of HFS-induced LTP between young controls (130.17 8.64%; n = 6) (Fig. 4-8A) and the young slices exposed to DTT (117.09 12.24%; n = 5) (Fig. 4-8B). Furthermore, the fEPSP obser ved in the HFS pathway was significantly higher than the fEPSP in the control pathway (Fig. 4-8C). CaMKII Activity is Enhanced in Aged Hippocampal CA1 Cytosolic Extracts Treated with DTT To determine whether DTT was directly influencing CaMKII activity, cytosolic extracts from CA1 region of the hippocam pus from aged animals were assayed for CaMKII activity by examining the phosphorylati on of the synthetic peptide, syntide-2, in the presence and absence of DTT. Relative to baseline control levels, cytosolic CaMKII activity was significantly enhanced (p<0.05) in the presence of 0.7 mM (113.11 3.47%, n = 3) and 1.4 mM DTT (120.46 3.14%, n=3) in the aged CA1 cytosolic extracts (Fig. 4-9A). Higher levels of DTT (2.8 mM) re sulted in a decrease in CaMKII activity, presumably due to denaturation of the enzyme. In addition, the CaMKII activity was 83

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significantly (p<0.05) blocked (Aged: 5.36 2.85%, n = 3) in the presence of Ca2+ chelator EGTA (2 mM) and CaMKII-specific peptide inhibitor myr-AIP (10 M). However DTTs effect on CaMK II activity from aged CA1 cytosolic extracts raised the possibility that DTT was acting on the CaMKII activity regulator calmodulin (CaM), and not exclusively on CaMKII. In this case the effect of DTT on CaMKII activity will be reduced by the addition of exogenous and un-oxidi zed CaM. To test this idea the assay was repeated in the absence of exogenously added CaM. The CaMKII activity from aged CA1 cytosolic extracts, in the presence of 0.7 mM DTT, an d in the absence of exogenous CaM, (112.11 3.91%, n = 3) was not enhanced beyond that observed following addition of exogenous CaM, suggesting that DTT effects were not mediated by reducing effect on CaM (Fig. 4-9B). DTT does not Alter CaMKII Activity in Young Hippocampal CA1 Cytosolic Extracts It was possible that DTTs effect were s pecific to the oxidiz ed CaMKII present in the aged CA1 cytosolic extracts, and not the re latively un-oxidized CaMKII present in the young CA1 cytosolic extracts. To test th is idea, the CaMKII activity was measured from the young CA1 cytosolic ex tracts, in the presence and abse nce of DTT. In contrast to the effect observed in aged animals, D TT had either no effect or decreased CaMKII activity in CA1 cytosolic extracts from young animals (Fig. 4-10A). However, as observed in aged animals, the CaMKII activity wa s inhibited (p<0.05) by the addition of Ca2+ chelator EGTA (2 mM) and CaMKII-spec ific peptide inhibitor myr-AIP (10 M) (Young: 19.99 9.01%, n = 3). Finally, addition of DTT to purified CaMKII (CycLex Co Ltd) decreased CaMKII activity (p<0.05) (0.7 mM DTT: 86.62 6.04%, n = 3; 1.4 mM DTT: 70.72 18.58%, n = 3) (Fig 4-10B), indicating that the DTT effects were specific for CaMKII present in the aged CA1 cytosolic extracts. 84

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Discussion The DTT-mediated enhancement of NMDAR responses was specific to CaMKII activity because CaMKII inhi bitors, myr-AIP and KN-62, blocked the DTT-mediated increase in NMDAR-fEPSP in aged animals. The DTT effects were not blocked by inhibition of PKC, PP1, or CaN/PP2B. The re sults point to CaMKII as a critical link between the intracellular redox state and NMDAR hypofunction. The role of CaMKII was further confirmed by enzyme activity a ssays which established that DTT increased CaMKII activity only in CA1 cytosolic extrac ts from aged animals. In contrast, DTT did not increase CaMKII activity in CA1 cytosolic extracts from young animals. In fact, DTT decreased the CaMKII activity in a purifi ed sample of CaMKII, presumably due to enzyme denaturation by the r educing action of DTT. Upon comparative analysis of all results, an interesting observation arises based on one of our previous results in chapter 3. In Chapter 3, we demonstrated that the DTT-mediated increase in NMDAR-fEPSP persisted for more than 45 min after aborting DTT application (Fig. 3-4D). The results pr esented in this chapter complement the previous findings and suggest that the lasting increase in NMDAR function was sustained by DTTs effect on CaMKII. In fact DTTs effect on NMDARs expressed in heterologous and non-neuronal systems has a quick onset and immediate washout (Tang and Aizenman, 1993; Kohr et al., 1994; Choi et al., 2001). The persistent increase in NMDAR function of aged neur ons upon DTT application can be explained only when we invoke the neuronal CaMKII signaling mechanism, as proved by the results presented in here. Oxidation of methionine residues on CaM has been reported to decrease the ability of CaM to activate Ca MKII (Robison et al., 2007). Our results are not likely due to 85

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CaM methionine oxidation since DTT possesse s higher selectivity to reduce cysteine residues over methionine residues (Ciorba et al., 1997; Cai and Sesti, 2009; Long et al., 2009). In addition, DTT had equivalent effe cts in activating CaMKII, regardless of whether exogenous CaM was added to the CaMKII ac tivity assay. The results indicate that oxidation of CaMKII, rat her than CaM, underlies the redu ction in kinase activity and are consistent with a recent report demonstr ating that oxidative stress induced by ischemia results in disulfide linkages on the cysteine residues of CaMKII which decrease kinase activity (Shetty et al., 2008). While the data provide a link between age-related changes in intracellular redox st ate, CaMKII activity, and NMDAR function, the exact mechanism through which CaMKII r egulates the NMDAR response remains to be determined. In addition to regulating phos phorylation state of proteins, including AMPARs, synaptic CaMKII participates in pr otein-protein interactions with several proteins localized to the dendritic spine which could ultimately alter NMDAR location and function (Lisman et al., 2002; Robison et al., 2005). In this context an independent report suggests that reduced CaMKII activity is associated with a specific decrease in synaptic NMDARs and decreased LTP (Gardoni et al., 2009). In addition to a role for CaMKII, we obs erved that PP1 inhibition resulted in a modest increase in the NMDAR-fEPSP in aged hippocampal neurons. Age differences in the NMDAR response, which depend on kinas e/phosphatase activity, are reminiscent of the age-dependent effects of kinase and phosphatase inhibitors on the rapid component of synaptic transmission mediated by AMPARs (Norris et al., 1998b; Hsu et al., 2002; Foster, 2007) and suggest that PP1 activity contributes to a reduction in AMPAR and NMDAR components of synaptic transmission (Foster et al., 2001; 86

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Morishita et al., 2005), a characteristic specific to senescent CA1 synapses (Rosenzweig and Barnes, 2003). Our results indi cate that a shift in the intracellular redox state towards oxidizing conditions during aging may cause or magnify the imbalance in the kinase/phosphatase activity favoring phosphatases (Foster, 2007). Our results using the ROS-sensitive dy e provided us a direct and real-time indication of the intracellular redox state of the hippocampal neurons. Several points indicate that the readout of our ROS detection experiment was an accurate indication of the intracellular redox state. First the ROS sensitive dye c-H2DCFDA, is preferentially cleaved by intracellular esterases to yield a non-fluorescent product; a pre-requisite for subsequent oxidation by ROS. Second, upon oxidation by ROS, th e dye is converted into a fluorescent product which is memb rane impermeable; thus the fluorescent readout is primarily due to intr acellular signals. Finally, ex tracellular application of SOD and catalase did not affect c-H2DCFDA fluorescence, simply due to the fact that these proteins are relatively membrane imperm eable, and also because only intracellular events gave rise to c-H2DCFDA fluorescence. While quantifying ROS-derived fluorescence from aged neurons, a significantly large auto-fluorescent signal was detected in dye-unexposed slices, mainly fr om lipofuscin. Lipof uscin, a breakdown product of lipid oxidation, is reported to accumulate in the aged hippocampal neurons (Landfield et al., 1981; Oenzil et al., 1994) and is capable of emitting auto-fluorescent signals overlapping the upper end of green, and the lower end of the yellow emission spectra (Haralampus-Grynaviski et al., 2003). Hence the imaging for detection and quantification of c-H2DCFDA fluorescence was performed at 100 ms time-window in 87

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order to eliminate the auto-fl uorescent signals arising from lipofuscin accumulation in aged neurons. Finally, NMDAR function is critical to t he induction of LTP and we observed that DTT improved LTP in the CA3-CA1 synapses of aged animals. The interaction of NMDARs with CaMKII has been proposed as a m odel of memory (Lisman et al., 2002) and recent work indicates that disrupti on of the interaction between CaMKII and NMDAR impairs the induction of LTP and spat ial learning (Zhou et al., 2007). We have provided evidence to indicate that a more oxidized redox state is a biological mechanism that can progressively inhibit NMDAR function in the hippocampus during senescence. Together, the results suggest that age-related changes in the redox state contributes to a decline in CaMKII activity, wh ich ultimately leads to a decline in the NMDAR response. The outcome of such sene scent mechanisms is an alteration in the synaptic plasticity at the CA3-CA1 synapses which contributes to age-related cognitive decline. 88

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Figure 4-1. Detection of ROS in live hippocam pal slices. A) Bright field image of the CA1 region of the hippocampus from an aged F344 rat. Indicated are the various layers of hippocampal area CA1 stratum radiatum (s.rad), stratum pyramidale (s.pyr) and stratum oriens (s .or). B) The same image as in (A) was obtained with a green filt er. White arrow heads indicate few of the many CA1 pyramidal neurons that have oxidized the ROS detection dye into a green fluorescent product. C) Image obtai ned from one of the dye-unexposed slices from the same rat. Scale bars = 50 m. 89

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Figure 4-2. Detection of auto-fluorescenc e from dye-unexposed hippocampal slices. Shown are the images of the CA1 region of the hippocampus from aged ( top panel) and young ( bottom panel ) animals. Dye-unexposed slices were used to obtain the bright field image ( left), and the images with a filter designed to detect green fluorescence with exposure time set at 100 ms ( middle ) and 500 ms ( right ). Auto-fluorescent signals were detected, for a 500 ms exposure time, in the stratum radiatum of aged hippocampal slices ( white arrowheads ) but not in the young hippocampal slices. Scale Bar = 50 m. 90

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Figure 4-3. Enhanced ROS production is obser ved in hippocampal tissue from aged rats. A) Indicated above each image co lumn are the imaging conditions ( bright field or green emission) and the exposure time ( 100 ms ) for hippocampal slices that were either untreated or treated (Carboxy-H2DCFDA) with the dye. The rows are images of the CA1 region of young ( top row ) and aged ( second row) hippocampal slices. The lowermost image is a CarboxyH2DCFDA treated slice from an aged animal, which was incubated with SOD + catalase. The various layers of hi ppocampal area CA1stratum radiatum ( s.rad ), stratum pyramidale ( s.pyr ) and stratum oriens (s.or ) are indicated in the last set of images. Scale bars = 50 m. B) Quantific ation of the mean fluorescence intensity generated by the oxidation of c-H2DCFDA ( c-H2DCFDA Fluorescence) from young (n = 3) ( open bar ), aged (n = 3), and SOD + catalase exposed aged hippocampal slices (n = 3) ( gray bars ) expressed as percent of fluorescence in untreated (contro l) slices from the same animal. In this and subsequent figures error bars represent standard error of mean (S.E.M), asterisks indicate significant differenc e (p<0.05) between the groups indicated, and n.s indicates no significant difference. 91

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Figure 4-4. A Serine/Threonine (Ser/Thr) kinas e, but not protein kinase C, mediates DTT mediated increase in NMDAR func tion in aged hippocampus. A) Time course of the change in the normaliz ed NMDAR-fEPSP slope in the aged animals that were incubated with the broad spectrum Ser/Thr kinase inhibitor H-7 dihydrochloride (10 M n = 7) prior to and du ring DTT application. B) Time course of the change in the no rmalized NMDAR-fEPSP slope in the aged animals that were incubated with the PKC inhibitor Bis-I (500 nM, n = 6) prior to and during DTT application. 92

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Figure 4-5. CaMKII involvement in the D TT mediated enhancement of NMDAR synaptic responses in aged animals. A) Time c ourse of the change in the normalized NMDAR-fEPSP slope in the aged animals that were incubated with the CaMK inhibitor KN-62 (10 M, n = 5). B) Time course of the change in the normalized NMDAR-fEPSP slope in t he aged animals that were incubated with the specific CaMKII inhibitor myr-AI P (5 M, n = 4). C) Quantification of the mean percent change in the NMDAR-fEPSP slope for aged animals under DTT alone ( filled bar ), and DTT applied in the pr esence of H-7, KN-62, myrAIP and Bis-I ( gray bars ). Asterisk indicates a signi ficant difference (p<0.05) between the increases observed in presence of DTT alone relative to DTT applied in the presence of H-7, KN-62, and myr-AIP; n.s indicates no significant difference between the incr eases observed in presence of DTT alone and DTT applied in presen ce of Bis-I. Pound sign indicates a significant (p<0.05) increase in the response relative to baseline level of 100%, following DTT application. 93

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Figure 4-6. Calcineurin/ PP2B and PP1 are not involved in the DTT mediated enhancement of NMDAR synaptic responses in aged animals. A) Time course of the increase in the NM DAR-fEPSP slope in slices from aged animals that were incubated with FK506 (10 M), 45 minutes prior to and during the application of DTT (n = 5) B) The NMDAR-fEPSP slope exhibited a modest increase (121.46 9.19%) fo llowing a 30 minute incubation with OA (1 M) (n = 5). C) Following stabilizatio n of the response in OA, the baseline was recalculated. The figure illustrates t he time course for the increase in the re-normalized NMDAR-fEPSP slope following application of DTT (n = 5). D) Quantification of the mean percent change in the NMDAR-fEPSP slope for aged animals under DTT alone (filled bar ), and in the presence of FK-506 + DTT, OA + DTT and OA alone ( gray bars ). 94

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Figure 4-7. DTT enhances LTP in hippocam pal area CA1 of aged animals. A) Time course for the expression of LTP re corded in aged hippocampal slices bathed in control ACSF (n = 9) for at least 45 minutes prior to HFS. Baseline stimulation was applied to a control pathway ( open circles ) and to a second pathway that received HFS (100 Hz, 1s) ( filled circles ). B) Time course for the expression of LTP recorded in aged hippocampal slices bathed in ACSF containing DTT (n = 10) for at least 45 minutes prior to HFS. Baseline stimulation was applied to a control pathway ( open circles ) and to a second pathway that received HFS (100 Hz, 1s) ( filled circles ). Arrows in A and B denote HFS delivery. For purpose of cl arity, each point represents the mean of two consecutive responses. C) Distribution of the LTP magnitude for individual slices from aged animals bathed in control ACSF and ACSF+DTT. The rectangular boxes indicate the mean of each group. D) Quantification of the mean percent change in the fEPSP slope recorded from the control ( Cont ) and HFS ( HFS ) pathways from young slices bathed in ACSF or ACSF+DTT. E) Plot of the paired-pulse rati o obtained under control conditions ( black circles ) and after 45 minute bath application of DTT ( gray circles ) for four inter-pulse intervals (50, 100, 150, 200 ms). Inset : Responses obtained upon paired pulse stimulation (average of 5 consecutive traces; 50 ms inter-pulse interval) under control conditi ons and under DTT application. 95

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Figure 4-8. DTT does not alter the LTP in hippocampal area CA1 of young animals. A) Time course for the expression of LT P recorded in young hippocampal slices bathed in control ACSF (n = 6) for at least 45 min prior to HFS. Baseline stimulation was applied to a control pathway ( open circles ) and to a second pathway that received HFS (100Hz, 1s) ( filled circles ). B) Time course for the expression of LTP recorded in young hippocampal slices bathed in ACSF containing DTT (n = 5) for at least 45 min prior to HFS. Arrows in A and B denote HFS delivery. For purpose of cl arity, each point represents the mean of two consecutive responses. C) Quant ification of the mean percent change in the fEPSP slope recor ded from the control ( Cont ) and HFS ( HFS ) pathways of the young slices bathed in ACSF or ACSF+DTT. 96

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Figure 4-9. DTT enhances CaMKII activity in aged hippocampal CA1 cytosolic extracts. A) CaMKII activity measured from the hippocampal CA1 cytosolic extracts of aged F344 rats. CaMKII activity is repres ented as percent of control activity ( black bars ) in the presence of exogenous calmodulin. CaMKII activity was significantly enhanced in the presence of 0.7 mM and 1.4 mM DTT ( gray bars), and was blocked by the addition of EGTA (2 mM) + myr-AIP (10 M) ( white bars ). B) Removal of exogenous ca lmodulin did not further enhance the DTT (0.7 mM) effect on CaMKII acti vity suggesting that DTT is not acting through oxidized calmodulin in aged animals. Asterisk indicates a significant difference (p<0.05) from respective controls. Plus and minus represent the presence and absence (respectively) of the indicated component in the reaction mix. 97

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Figure 4-10. DTT does not enhance CaMKII activity in young hippocampal CA1 cytosolic extracts. A) CaMKII activi ty measured from the hippocampal CA1 cytosolic extracts of young F344 rats CaMKII activity is represented as percent of control activity ( black bars ) in the presence of exogenous calmodulin. Addition of 0.7 mM and 1.4 mM DTT did not increase CaMKII activity in hippocampal CA1 cytosolic extr acts of young F344 rats. B) Addition of 0.7 and 1.4 mM DTT decreased the activity of purified CaMKII. Asterisk indicates a significant difference (p<0.05) from respective controls. Plus and minus represent the presence and absence (respectively) of the indicated component in the reaction mix. 98

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99 Table 4-1. Paired-pulse ratios from aged animals Inter pulse Interval (ms) Paired pulse ratio Control (mean S.E.M) Paired pulse ratio DTT (mean S.E.M) 50 1.51 0.12 1.38 0.11 100 1.34 0.08 1.21 0.08 150 1.24 0.05 1.15 0.07 200 1.38 0.09 1.28 0.06 The table represents the paired pulse ratio obtained from control, aged hippocampal slices and aged hippocampal slices that were incubated with DTT for at least 45 minutes. The paired pulses ratio is the ratio of the slopes of two consecutive fEPSPs elicited apart by a time interval indicated by the inter pulse interval.

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CHAPTER 5 REDOX MODULATION MEDIATES REDUCTION IN NEURONAL AFTERHYPERPOLARIZATION OF AG ED HIPPOCAMPAL NEURONS Introduction An age-related decline in hippocampus-dep endent memory is thought to result from dysregul ation of Ca2+-dependent processes in CA1 pyramidal neurons including synaptic plasticity and neuronal excitability (Foster, 1999, 2007; Kumar et al., 2009; Burke and Barnes, 2010; Magnusson et al., 2010; Oh et al., 2010). The results presented in chapters 3 and 4 dealt with NMDAR hypofunction, a significant biomarker of aging in CA1 pyramidal neurons. One of the other well characterized markers of aging in CA1 pyramidal neurons is an increa se in the slow component of the Ca2+ activated, K+mediated afterhyperpolarization (sAHP) (Landfield and Pitler, 1984; Moyer et al., 1992; Kumar and Foster, 2004; Th ibault et al., 2007; Matthews et al., 2009) The exact mechanism that underlies the age-related increase in sAHP is unknown. The increase in the sAHP may be due to altered Ca2+ regulation, incl uding an increase in L-type voltage gated Ca2+ channels (L-type VGCC) (Thibault and Landfield, 1996; Veng and Browning, 2002) or increased release of Ca2+ from intracellular Ca2+ stores (ICS) (Kumar and Foster, 2004; Gant et al., 2006) or an increase in the function or density of K+ channels that mediate t he sAHP (Power et al., 2001; Power et al., 2002). Importantly, aging is associated with increased oxidative stre ss that could influence the highly redox sensitive RyRs, which mediate Ca2+ release from ICS (Eager and Dulhunty, 1998; Hidalgo et al., 2004; Bull et al., 2008; Huddleston et al., 2008). Moreover, aged neurons are characterized by a decrease in their redox buffering capacity (Parihar et al., 2008; Bodhinathan et al., 2010) and recent work from our lab 100

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demonstrates that the shift in redox state contributes to altered Ca2+ regulation in CA1 neurons from aged animals (Bodhinathan et al ., 2010). Based on these observations we tested the hypothesis that the redox state of the aged neuron contributes to the increase in sAHP (Foster, 2007; Kumar et al., 2009). The results reveal that the sAHP is decr eased by the reducing agent dithiothreitol (DTT) in an age-dependent manner. Application of ryanodine, to block RyRs, prevented the DTT-mediated decrease of sAHP in t he aged neurons. Depletion of ICS by the application of thapsigargin also blocked the DTT effect on aged-sAHP. The DTTmediated decrease in aged-sAHP was independent of the activity of L-type VGCC or Ser/Thr kinase activity. Finally inhibition of the big conductance potassium (BK) channel activity did not influence DTT-mediated decreas e in aged-sAHP. The results point to an ICS-dependent and RyR-mediated mechanism t hat links oxidative redox state during aging and the enhanced sAHP in CA1 pyramidal neurons. Reversal of the redox state of aged hippocampal CA1 pyramidal neurons is a potential therapeut ic strategy to ameliorate Ca2+ dysregulation, decrease sAHP and re store normal functionality in aged neurons. Results Age Dependent Decrease in the s AHP Following DTT Application To study the effects of oxidative redo x state on the aged-sAHP the reducing agent DTT was applied to aged and young hippoc ampal CA1 pyramidal neurons while continuously recording the sAHP. In confirma tion of previous studies, the sAHP was significantly (p<0.05) increased in aged (6. 44 0.32 mV, n = 40) relative to young CA1 pyramidal neurons (4.23 0.17 mV, n = 12). The properties of the CA1 pyramidal neurons recorded from the young and aged animals are indicated in Table. 5-1. In a 101

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subset of these neurons, after a stable base line recording for 10 min, DTT was applied for 40 min. Application of DTT significantly (p<0.05) decreased the sAHP amplitude form the baseline levels in the aged (48 14% of baseline, n = 5), but not in the young animals (105 10%, n = 3) (Fig. 5-1A, 5-1B ). The DTT-mediated reduction in sAHP of aged neurons does not appear to be due to al tered membrane properties, since the holding current required to maintain the me mbrane potential at -63 mV did not differ (p>0.05) between baseline and 45 minutes a fter DTT application. The DTT-mediated decrease, specific to the aged-sAHP, suggest s a link between oxidative redox state and the increased sAHP am plitude in aged neurons. DTT Mediated Decrease in Aged-sAHP Invo lves Intracellular Calcium Stores and Ryanodine Receptors To test the hypothesis that the DTT-mediated decrease in the aged-sAHP was due to decrease Ca2+ mobilization from ICS, ICS we re depleted by the application of thapsigargin prior to and during the applic ation of DTT to aged hippocampal slices. Application of thapsigargin fo r 30 min significantly decreas ed (p<0.05) the amplitude of aged-sAHP to 58 8% (n = 7) of the baseline levels (Fig. 5-2A). In a subset of these cells (n = 4), a new baseline was establis hed and DTT was applied. Application of DTT for 50 min failed to decrease the aged-sAHP amplitude (104 23%) (Fig. 5-2B, 5-2C). The results suggest that ICS provide a redox sensitive Ca2+ source that contributes to the age-related increase in the sAHP. RyRs mobilize Ca2+ from the ICS and are highly redox sensitive. To test whether RyRs were involved in the DTT-mediated de crease in aged-sAHP, RyRs were block by ryanodine prior to and during the application of DTT. Applic ation of ryanodine for 40 min, significantly (p<0.05) decreased t he aged-sAHP amplitude to 47 10% (n = 4). 102

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Application of DTT for 50 min failed to fu rther decrease the aged-sA HP amplitude such that it was 54 7% (n = 4) of the original baseline (Fig. 5-3A, 5-3B). Both DTT (Fig. 5-1) and ryanodine (Fig. 5-3) decreased the aged-sAHP to ~50% of baseline. The similar magnitude effect rais es the possibility of a floor effect of ryanodine, which may have masked DTT influences on the sAHP. To address this issue, the sAHP of aged neurons was enhanced by increasing the extracellular Ca2+ concentration from 2 mM to 4 mM. Increasing the extracellular Ca2+ to 4 mM increased the sAHP almost two fold, from 6.71 0.79 mV (n=11) to 11.06 1. 09 mV (n = 6) (Fig. 5-4A, 5-4B). In five cells a baseline was recorded in 4 mM Ca2+, followed by application of ryanodine for 40 min, which was then follo wed by the application of DTT for 50 min (Fig. 5-4C). Application of ryanodine decre ased (p<0.05) the aged-sAHP amplitude to 53 6% (5.31 0.86 mV) and application of DTT for 50 min failed to further decrease the aged-sAHP amplitude (51 7% 5.06 1.05 mV) of the orig inal baseline (Fig. 5-4B, 5-4C, 5-4D). Thus, DTT failed to r educe the sAHP amplitude under high Ca2+ and ryanodine application, despite the fact that sAHP am plitude was similar to the baseline under normal 2 mM Ca2+ conditions (Fig. 5-4B). The resu lts indicate that the ryanodine blockade of the DTT-mediated decrease in the sAHP was not due to a floor effect of the sAHP during ryanodine application. Rather, these data suggest that the DTT effect on sAHP in aged animals is mediated by RyRs. DTT Mediated Reduction in the Aged -sAHP is Independent of L-VGCC L-type VGCCs are another major source of Ca2+ for the sAHP. To test the hypothesis the DTT-mediated decrease in the aged-sAHP in volves the L-type VGCC, nifedipine was applied prior to and during the application of DTT to aged hippocampal slices (Fig. 5-5A). Application of nifedipi ne for 20 min decreased the sAHP to 68 4% 103

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(n = 5) of the baseline. Subsequent applicat ion of DTT for 30 min further decreased the amplitude of aged-sAHP to 34 4% (n = 5) of the original baseline. The results suggest that the effects of nifedi pine and DTT may be independent. In fact, using the sAHP responses recorded in nifedipine (bath applicat ion for at least 20 min) as the baseline, application of DTT decreased t he amplitude of the aged-sAHP to 48 6 % (p<0.05; n = 6) (Fig. 5-5B), a decrease comparable to t hat observed following D TT application in the absence of nifedipine (Fig. 5-7B). The activity of BK channels is sensitive to oxidation (DiChiara and Reinhart, 1997). Moreover, an increase in BK channel activi ty can reduce the sAHP amplitude by decreasing the action potential spike width (Giese et al., 1998; Shao et al., 1999; Murphy et al., 2004). To test the hypothesis that the DTT-mediated decrease in agedsAHP involves the BK channels, paxilline firs t applied to inhibit BK channel activity (Sanchez and McManus, 1996). Aged hippocampal slices were incubated in paxilline for at least 60 min prior to record ing the sAHP and applying DTT. Paxilline failed to block the DTT-mediated decrease in aged-sAHP, such that DTT application was still able to decrease the amplitude of the aged-sAHP to 28 11 % (n = 3) of the baseline (Fig. 56A, 5-6B). Furthermore, the DTT-mediated decrease in the presence of paxilline was not significantly (p>0.05) different from the decrease observed in the presence of DTT alone. Serine/threonine kinases provide another pot ential mechanism for regulating RyRs and the K+ channels that mediate the sAHP. Protein kinase A increases the activity of cardiac RyRs (RyR subtype 2) (Yoshida et al., 1992; Danila and Ha milton, 2004; Xiao et al., 2007; Morimoto et al., 2009), and kinase ac tivity inhibits the sAHP (Madison and 104

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Nicoll, 1986; Malenka et al., 1986; Mulle r et al., 1992; Pedarzani and Storm, 1993; Melyan et al., 2002). In order to test whether the changes in kinase activity underlies the decrease in sAHP of aged neurons upon app lication of DTT, the broad spectrum serine/threonine kinases inhibitor H-7 was app lied prior to and during the application of DTT. Aged hippocampal slices were incubated with H-7 for at least 60 minutes before recording the sAHP. In the presence of H-7, application of DTT significantly (p<0.05) decreased the aged-sAHP to 53 14 % (n = 3) of the baseline (Fig. 5-7A). The results suggest that DTT is not altering the sAHP through modulation of kinase activity. Fig. 5-7B summarizes the change in the sA HP amplitude following DTT application under various conditions. In each case, t he response was normalized to the pre-DTT application baseline. In additi on, the percent change for applic ation of nifedipine-alone or ryanodine-alone relative to the pre-drug baseline is illustrated for comparison. Treatments that blocked Ca2+ release from ICS (thapsigar gin, ryanodine) blocked the DTT-mediated reduction in the sAHP. In a ll cases in which DTT reduced the sAHP; including in the presence of nifedipine, t he reduction was ~50%. A similar reduction was observed following treatment wit h ryanodine-alone, consistent with previous reports in aged animals (Kumar and Foster 2004; Gant et al., 2006). Application of nifedipinealone decreased the sAHP by ~30%, consist ent with previous reports in young and aged animals (Power et al., 2002; Disterhoft et al., 2004). Discussion The results demonstrate a link between the age-related increase in the sAHP and redox state, through the release of Ca2+ from ICS. A shift in Ca2+ regulation and altered Ca2+ channel function is a characteristic of certain aging neurons (Foster, 1999, 2007; Kumar et al., 2009; Burke and Barnes, 2010; Magnusson et al., 2010; Oh et al., 2010). 105

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Recently we demonstrated that DTT could reverse an age-related decrease in NMDA receptor function in region CA 1 (Bodhinathan et al., 2010). In the current study, the reducing agent, DTT, decreased the sAHP in aged, but not in young CA1 neurons. The data are consistent with t he weakened redox buffering in aged animals as a mechanism contributing to Ca2+ dysregulation and electrophysiological changes observed in aged neurons. Redox modulation has been observed for several ion channels including K+ and Ca2+ channels (Ruppersberg et al., 1991; Ch iamvimonvat et al., 1995; Stephens et al., 1996; DiChiara and Reinhart, 1997; Hidalgo et al., 2004), which could contribute to the sAHP. The identity of the K+ channel that underlies the sA HP is unknown (Furuichi et al., 1994; Sah and Faber, 2002); however, the amplitude to the sAHP is reduced by activation of Ser/Thr kinases, including PKA (Madison and Nicoll, 1986; Pedarzani and Storm, 1993), CaMKII (Mulle r et al., 1992), and PKC (Malenka et al., 1986). In the current study, the broad s pectrum Ser/Thr kinase inhibi tor H-7 had no influence on the DTT-mediated decrease in aged-sAHP indicati ng that the reduction was not mediated through kinase activity. In the case of K+ channels, previous reports indi cate cysteine specific oxidation decreases BK channel activity (Tang et al., 2001; Tang et al., 2004), and that the reducing agent DTT increases BK channel activity (DiChiara and Reinhart, 1997). The BK channel is involved in repolarization of action potential, and an increase in BK channel activity will reduce the width of the action potential (Shao et al., 1999). Moreover, a decrease in the spike width can decrease the sAHP, by limiting the duration of depolarization-induced Ca2+ entry through L-type VG CCs (Giese et al., 1998; Murphy et al., 2004). Thus, DTT could be acting on the BK channels to decrease 106

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L-type VGCC activity and the sAHP amplitude. However several pieces of evidence suggest that this might not be the case. Firs t, blockade of BK channels with paxilline did not block the DTT-mediated decrease in agedsAHP. Second, blockade of VGCCs with nifedipine did not influence the DTT-mediat ed decrease in aged-sAHP. Finally, the fast AHP, which is mediated by the BK channel, is not altered with age (Matthews et al., 2009) The amplitude of the sAHP is dependent on the level of cytosolic Ca2+. L-type VGCCs play a role in determining the amplit ude of the sAHP (Landf ield and Pitler, 1984; Moyer et al., 1992; Norris et al., 1998a) and contribute to the increase in the sAHP during aging (Thibault and Landfield, 1996; Veng and Browning, 2002). However, it does not appear that the DTT-m ediated reduction in the agedsAHP is acting through Lchannels. The DTT-mediated reduction in the sAHP was larger than that observed for Lchannel blockade, and was specific to aged anima ls. Previous research indicates that the decrease in the AHP following blockade of L-channels is quantitatively larger in aged animals; however, the percent decrease (~ 30%) is similar across ages, suggesting other mechanisms contribute to the age-relat ed increase in the AHP animals (Power et al., 2002; Disterhoft et al., 2004). In the cu rrent study, blockade of the L-channel reduced the aged-sAHP ~30%, cons istent with previous repo rts (Power et al., 2002). Regardless of L-channel function, DTT reduc ed the aged-sAHP by ~50% and the effect of DTT was specific to aged animals. The results suggest that DTT is acting on mechanisms other than the L-channel, which may mediate the age-related increase in the sAHP. 107

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Release of Ca2+ from ICS, through RyR activation, plays a role in determining the sAHP amplitude (Sah and McLachlan, 1991; Usachev et al., 1993; Davies et al., 1996; Borde et al., 2000; van de Vrede et al., 2007). Ca2+ from ICS contributes to altered physiology during aging (Kumar and Foster 2004, 2005; Gant et al., 2006). In addition, the RyRs are highly redox sensitive (Bull et al., 2008), such that oxidation of the cysteine residues increases the Ca2+ sensitivity and activity of RyR (Eager and Dulhunty, 1998; Hidalgo et al., 2004; Huddlest on et al., 2008). In th e current study, the DTT-mediated decrease in the sAHP was blocked upon depletion of ICS by thapsigargin or blockade of RyRs by ryanodi ne indicating the involvement of ICS and RyRs in the decreased Ca2+ mobilization by DTT applicat ion. Together the results suggest that the increase in the sAHP in a ged neurons is related to redox sensitive Ca2+ mobilization from the ICS thr ough the RyRs. Interestingly, decreased sAHP is observed in aged memory-unimpaired and young rats but not in aged memory-impaired rats (Moyer et al., 2000; Tombaugh et al., 2005; Mur phy et al., 2006; Matthews et al., 2009). It is interesting to speculate that the dec rease in the sAHP of memory unimpaired animals may result from a shift in redox st ate associated with learning (Shvets-TenetaGurii et al., 2007). Alternativel y, treatments that modify in tracellular redox state may provide a novel therapeutic strategy to restore Ca2+ homeostasis in the aged neurons. 108

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Figure 5-1. Age-dependent reduc tion in the sAHP by DTT. (A) Time course of the change in the normalized sAHP amplitude in the aged (filled circles) (n = 5) and young (open triangles) animals (n=3), following application of DTT for 40 minutes. (B) Representative traces illust rating the change in the AHP of aged (left) and young (right) animals under c ontrol conditions and at the end of a 40 minute application of DTT. The line beneath the tr aces indicates the onset and offset of the step current used to elicit a train of 5 action potentials. Calibration bars: 200 ms, 10 mV. 109

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Figure 5-2. Intracellular calcium stores underlie DTT-mediated decrease in aged-sAHP. (A) Time course of the change in the normalized sAHP amplitude in the aged animals that were incubated with thapsig argin (n = 7). (B) Time course of change in the normalized sAHP amplitude in cells (n = 4) incubated with thapsigargin prior to and during DTT appl ication. (C) Representative traces illustrating the AHP of aged animals under control condition (black trace), and at the end of a 40 minute application of thapsigargin (gray trace) and at the end of 50 min application of thapsigargin+DTT (gray trace). Calibration bars: 200 ms, 10 mV. 110

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Figure 5-3. RyR blockade inhibits DTT mediated decrease in aged-sAHP. (A) Time course of the change in the normalized sAHP amplitude recorded from aged animals that were incubated with the ryanodine receptor antagonist ryanodine (n = 4) prior to and during application of DTT application. (B) Representative traces illustrating the change in the AHP of aged animals under control condition (black trace), at the end of a 40 minute application of ryanodine (black trace), and at the end of 50 mi n application of ryanodine+DTT (gray trace). Calibration bars: 200 ms, 20 mV. Inset: Magnified representation of change in the aged AHP under control c ondition, ryanodine, and ryanodine + DTT. 111

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Figure 5-4. RyR blockade inhibits the DTT -mediated decrease in aged-sAHP when the AHP is increased by increasing calcium in the recording medium. (A) Representative traces illustrating the AHP from aged neurons recorded under conditions of 2 mM or 4 mM Ca2+in the ACSF. Calibration bars: 200 ms, 10 mV. (B) Quantification of the mean sA HP amplitude in aged neurons recorded under 2 mM Ca2+ (n = 11, open bar), under 4 mM Ca2+ (n = 6), under 4 mM Ca2+ with ryanodine (n = 5), and under 4 mM Ca2+ with ryanodine + DTT (n = 5); all values under 4 mM Ca2+ are represented as filled bars. (C) Time course of the change in the normalized sAHP amplitude recorded in 4 mM ACSF from aged animals and incubated with ryanodine (n = 5) prior to and during DTT application. (D) Representative traces illustrating the AHP of aged animals under control condition, and at the end of a 40 mi nute application of ryanodine and at the end of 50 min application of ryanodine + DTT. Calibration bars: 200 ms, 20 mV. 112

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Figure 5-5. DTT mediated decrease is indep endent of L-type calcium channel function. A) Time course of the change in the normalized sAHP amplitude in the aged animals (filled circles) that were inc ubated with nifedipine (n = 4). B) Time course of the change in the normalized sAHP amplitude in the aged animals (filled circles) that were incubated with nifedipine (n = 6) for at least 45 min prior to the application of DTT for 30 min. Calibration bars: 200 ms, 10 mV. C) Representative traces illustrating the change in the AHP of aged animals under control condition (black trace), and at the end of a 20 min application of Nifedipine (gray trace) and at the end of 30 min app lication of Nifedipine + DTT (black trace). 113

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Figure 5-6. DTT effects on aged-sAHP are i ndependent of BK channel function (A) Time course of the change in the normalized sAHP amplitude in the aged animals (filled triangles) that were incubated with paxilline (n = 3) for at least 60 min prior to DTT application. (B) Represent ative traces illustrating the change in the AHP of aged animals under paxilline (black trace), and at the end of 45 min under paxilline+DTT (gray trace) Calibration bars: 200 ms, 10 mV. 114

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Figure 5-7. Ser/Thr kinase activity does not mediate DTT effe cts on aged-sAHP. (A) Time course of the change in the normalized sAHP amplitude in the aged animals that were incubated with the broad spectrum Ser/Thr kinase inhibitor H-7 (n = 3) for at least 60 min prior to DTT application. (B) Summary diagram representing the mean percent ch ange in the sAHP amplitude of aged neurons following DTT application under various conditions. In each case, the response represents the percent change re lative to the pre-DTT application baseline (dashed line). The effect of nifedipine or ryanodine (open bars) on the pre-drug baseline is presented fo r comparison. The numbers above each bar represents the number of ne urons recorded in each condition 115

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116 Table 5-1. Physiological properties of CA1 neurons from young and aged animals IR (M ) RMP (mV) Sp Amp (mV) Young (n = 12) 37.0 2.5 -62.8 1.5 80.6 1.9 Aged (n= 40) 37.8 1.1 -61.9 0.6 82.8 0.7 The values for input resistance (IR), resting membrane potential (RMP), and spike amplitude (Sp Amp) are indicated as mean S.E.M. The values of holding current (HC) is presented as a range. The number in parentheses indicates the number of cells from the young and aged animals.

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CHAPTER 6 CONCLUSION AND FUTURE DIRECTIONS Conclusion While numerous mechanisms could contri bute to dysfunctional hippocampal synaptic transmission during aging, we were primarily interested in the role of increased oxidative stress and oxidative redox state in mediating NMDAR hypofunction, altered synaptic plasticity, increased sAHP, and altered Ca2+ homeostasis. The results presented in Chapter 3 describe the age-re lated changes in the baseline NMDAR mediated synaptic transmission, and indicate how these changes are linked to the redox state of the aged neurons. Briefl y, measurement of the NMDAR-fEPSP amplitude in the young and aged neurons, and comparison across 0.4 mV bins of PFV amplitude, indicated a significant age-related decrease in the NMDAR function. The use of oxidizing agent X/XO decr eased NMDAR function in t he young but not in the aged neurons; and the use of reducing agent DTT, increased the NMDAR function in aged but not in the young animals. The effect of X/XO was washed out in young animals, even under higher concentrations of X/XO, indicating a transient effect on young NMDAR function. This effect could possibl y be due to a robust antioxidant capacity or better redox buffering capacity in the young neurons In contrast, the DTT effect in aged animals could not be washed out even 45 minut es after switching-off DTT application. This observation predicted secondary signaling mechanisms underlying the DTTmediated increase in NMDAR function. Quite remarkably, this predi ction was accurate and the data presented in Chapter 4 delineat es the signaling mechanism underlying DTT-mediated increase in NM DAR function of aged animals. 117

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Results presented in Chapter 4 describe the age-related increase in the rate of ROS production in the hippocampal slices. The use of dye-based ROS detection technique in live hippocampal neurons suggested that the rate of oxyradical production is higher in the aged neurons, when compared to young neurons. Moreover the use of membrane impermeable antioxida nt enzymes (SOD and catala se) in the extracellular solution did not affect the dye-based fluor escence, suggesting t hat the source of oxyradical production is intrac ellular. Furthermore, in Chapter 4, the use of kinase and phosphatase inhibitors localized the specif ic effects of DTT to CaMKII, a Ca2+ sensitive Ser/Thr kinase that participates in regulat ing the NMDAR function and is also redox sensitive. First, physiological studies i ndicated that the D TT effect on aged NMDAR function was blocked by CaMKII inhibitors KN-62 and myr-AIP. Second, biochemical assays suggested that DTT increases CaMKII activity in aged but not in young CA1 cytosolic extracts of hippocampal neurons. In contrast no effect of phosphatase inhibition was observed on the DTT-mediated increase in NMDAR function. Neither the inhibition of CaN with FK-506 nor the inhibi tion of PP1 with OA had any effect on the DTT-mediated increase in aged NMDAR functi on. Finally, DTT treatment increased LTP in aged neurons following a single episode of 100 Hz stimulation. Taken together the data presented in Chapter 4 is indicative of the link between oxidative redox state and oxidized CaMKII signaling pathways in the aged neurons, which ultimately contributes to the decreased NMDAR function observed in Chapter 3, and weakened hippocampal function during aging. This complex relationship is summarized by the schematic in Fig. 6-1. 118

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NMDARs in aged neurons can be subject to numerous post-translational changes that could mediate the observed deficit in ac tivation. Results provided in Chapters 3 and 4 suggest a role for oxidative redox state. However numerous alternative mechanisms could mediate deficit in NM DAR function. Altered mRNA and protein expression of specific NMDAR subunits is proposed as a potential mechanism for the observed decrease in NMDAR function (Magnusson, 2000). Significant decreases have been observed in the expression of NR1 protein (E ckles-Smith et al., 2000; Mesches et al., 2004; Liu et al., 2008) and NR1 mRNA (Adams et al., 2001) in aged hippocampus. In contrast, other studies report no age-related decrease in NR1 protein expression in the whole hippocampus (Sonntag et al., 2000; Zhao et al., 2009). These studies point to a lack of congruent changes in hippocampal NR1 subunit expression. Some studies indicate age-related changes in the modulatory NR2 subunits. A decrease in the NR2A protein expression has been observed in the hippocampus (Sonntag et al., 2000; Liu et al., 2008), which is not observed in the frontal cortex (Sonntag et al., 2000). Furthermore, NR2A mRNA expression was reported to decline in the ventral hippocampus (Adams et al., 2001) In contrast, other studi es report no significant change in the NR2A protein expression levels in the hippocampus and cortex (Sonntag et al., 2000; Martinez Villayandre et al ., 2004). Age-related changes have also been reported for NR2B subunit of the NMDAR; in particular the expression of NR2B protein (Mesches et al., 2004; Zhao et al., 2009) an d NR2B mRNA (Adams et al., 2001; Magnusson, 2001) declines in the aged hip pocampus. This effect may be region specific since a decline in NR2 B protein is not observed in the frontal cortex (Sonntag et 119

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al., 2000). In contrast, NR2B mRNA decreases in the frontal cortices of aging macaque monkeys, but not in the hi ppocampus (Bai et al., 2004). In conclusion, a lack of a clear model describing mRNA and protein changes of various NMDAR subunits in aged hippocampus gives rise to another alternative mechanism for reduced NMDAR activation in these neurons. The other possible mechanism is that alterations in the NMDA R localization, through the insertion of receptors into the membrane or recruitment of extra-synaptic receptors into the synapse, may have important effects on NM DAR function during aging. It has been suggested that NR2B containi ng receptors may be more pr evalent at extra-synaptic sites (Massey et al., 2004), before being interna lized into the cytoplasm (Blanpied et al., 2002; Lau and Zukin, 2007). A decrease in the NR2B protein expression in the synaptic membrane fraction, but not in whole homoge nates (Zhao et al., 2009) suggests an agerelated sequestration of NR2B in the extra-synaptic sites. Recent work indicates that extra-synaptic NMDARs couple to different signaling cascades, and initiate mechanisms that oppose synaptic potentiation, by shutting o ff the activity of cAMP response element binding protein and decreasing expressi on of brain-derived neurotropic factor (Hardingham et al., 2002; Vanhoutte and B ading, 2003). However, it remains to be determined whether altered localization of the NMDARs (specifically extra-synaptic localization) is the mechanism by wh ich the NMDAR function declines during senescence. The other likely candidate mechanism fo r regulating NMDAR function during aging is posttranslational modification of the rec eptor and/or its associated signaling cascades (investigated in Chapters 3 and 4). NM DAR function can be altered by the 120

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oxidation/reduction of sulfhydryl moieties on their structure. Previous research demonstrates that oxid izing agents like 5,5 -dithiobis(2-nitrobenzoic acid) (Aizenman et al., 1989), hydroxyl radicals ge nerated by xanthine / xanthi ne oxidase (Aizenman, 1995) and oxidized glutathione (Sucher and Lipton 1991) decrease NMDAR function in the neuronal cell cultures. The decrease in NMDA R function under oxidizing conditions is thought to result from the formation of disu lfide bonds on the sulfhy dryl group containing amino acid residues in NMDARs (Aizenman et al., 1990; Sullivan et al., 1994; Choi et al., 2001); specifically the cystei ne residues are more susceptible to oxidation over the methionine residues (Shacter, 2000). The aging brain is associated with an increase in the levels of oxidative stress and/or a dec rease in redox buffering capacity, which contributes to a shift in the redox state fa voring an oxidative stat e (Foster, 2006; Poon et al., 2006; Parihar et al., 2008). Thus conditions during aging should promote a decrease in NMDAR function. Another candidate mechanism associated with posttranslational modification is altered phos phorylation state of the receptor. In particular, the function of the NMDAR is influenced by its phosphorylation state. Activation of the tyrosine kinase (Wang et al., 1994; Heidinger et al., 2002), protein kinase C (Ben-Ari et al., 1992; Chen and Huang, 1992) and protein kinase A (Raman et al., 1996) increases NMDAR mediated currents. In contrast, protein phosphatases, including calcineurin and protein phosphatas e 1, decrease NMDAR currents (Lieberman and Mody, 1994; Wang et al., 1994; Raman et al., 1996). Interestingly, Ser/Thr kinases promote NMDAR trafficking from endoplasmic reticulu m and insertion into the postsynaptic membrane (Scott et al., 2001; Carroll and Zukin, 2002), while phosphatases promote internalization of NM DARs into the cytoplasm (Snyder et al., 121

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2005). Thus, the kinases and phosphatases act like molecular switches which increase or decrease NMDAR function, respectively. Du ring aging there is a shift in the balance of kinase/phosphatase activity, favoring an incr ease in the phosphatase activity (Norris et al., 1998b; Foster et al., 2001; Foster, 2007) Thus alterations in the phosphorylation state of the NMDARs could mediate the decreased NMDAR activation in aged neurons (Coultrap et al., 2008). In this study we have presented evidence that suggests that age-related increase in oxidative stress or oxidative redox state contributes to the decrease in NMDAR function. Alternatively it is also possible that increased nitrosative stress affects NMDAR function. In the hippocampal neurons, nitric oxide is produced by neuronal nitric oxide synthase (nNOS), which is activated upon stim ulation of NMDARs. In memory impaired, aged F344 rats there is no loss of NOS containing neurons, rather a decreased production of NO (Meyer et al., 1998), pr obably due to decreased activation of NMDARs themselves (Barnes et al., 1997; Billar d and Rouaud, 2007; Bodhi nathan et al., 2010). Subsequently, nitric oxide reacts with super oxide to produce peroxynitrite, a potent oxidant and nitrating agent (Squadrito and Pryor, 1998), which has been reported to decrease NMDAR function (Lipton and St amler, 1994; Lipton et al., 1998); and proposed to inhibit NMDAR-dependent LTP (Wang et al., 2004). The nitrosative stress mediated decrease in NMDAR function is thought to occur by S-nitrosylation of the receptor (Lipton and Stamler, 1994; Choi et al., 2000; Takahashi et al., 2007). Thus, excess amounts of superoxide plays an import ant role in mediating the effects of nitrosative stress, by contributing to the production of the per oxynitrite, which leads to Snitrosylation of the NMDARs. Although we c annot completely discount the effect of 122

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nitrosative stress on age-related decrease in NMDAR function, it is likely that the DTT mediated increase in NMDAR function does not involve the removal of S-nitrosyl groups on NMDARs. The results presented in Chapter 5 descr ibe the link between a more oxidized redox state and increased sAHP during aging. Increased sAHP is a physiological marker of aging, which makes it harder for the CA1 pyramidal neurons to reach the threshold for action potential firing. Our result s indicate that application of the reducing agent DTT significantly decreases the sAHP in aged but not in young CA1 pyramidal neurons. Moreover, the DTT-meditated dec rease in sAHP amplitude involves decreased Ca2+ mobilization from the ICS through a decrease in RyR function. Although the L-type VGCCs and the BK channels contain redox sensitive cysteine residues, they do not contribute to the DTT-mediated dec rease in aged-sAHP. Furthermore, DTTs effects are mediated by a direct reducing action on the cysteine residues of RyR, and not due to redox modulation of Ser/Thr kinases which are k nown to regulate the function of RyRs. In summary, our results suggest that during aging there is enhanced Ca2+ mobilization from the ICS thr ough the RyRs, which leads to increased activation of the Ca2+ dependent K+ current that underlies sAHP. The age-related increase in RyR function does not appear to be due to increas ed RyR expression (Martini et al., 1994), rather an oxidative stress induced shift in th e intracellular redox state may enhance the responsiveness of RyRs during aging (Hidalgo et al., 2004; Bull et al., 2007; Gokulrangan et al., 2007). Hence posttrans lational changes (primarily redox modification) of the RyRs are thought to underlie age-related increase in sAHP. Previous reports indicate that RyR cont ributes negatively to t he induction of LTP and 123

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interferes with normal spatial l earning (Futatsugi et al., 1999). In fact blockade of RyRs using ryanodine ameliorates hippocampal mark ers of aging (Gant et al., 2006). Thus our model indicates that decreasing RyR function in aged hippocampal neurons, by shifting the redox environment to a more reductive state would enhance LTP and memory (as indicated in chapter 3 and 4), and decrease the sAHP amplitude (as demonstrated in Chapter 5). Therapeutic Potential of the Current Study The fact that functional lesion of the hippocampus during normal aging can be reversed to a certain extent, is an exci ting starting point for the development of therapeutic strategies aimed to treat me mory loss and cognitive dysfunction. The therapeutic potential of the current study is highlighted in the following points1). normal memory function could be restored in aging brains by reversing the subtle physiological, biochemical and posttr anslational changes to the neurons. 2). the ease of using antioxidant or pill-bas ed therapeutics far outweighs the complexity of the therapeutics based on ce ll replacement strategies. Therapeutics based on stem cell would be more suited to treat neurodegenerative disorders that are characterized by neuron loss; as opposed to treating memory dysfunction during normal aging that is characterized by posttranslational changes like oxidation/reduction. In fact, antioxidants (Socci et al., 1995; Cotman et al., 2002; Zhang et al., 2007; Li et al., 2009) and antioxidant mimetics (Stoll et al., 1993; Liu et al., 2003) have been reported to ameliorate age-related learning and memory deficits by reducing oxidative damage. Antioxidants are also effective in improving s patial memory in rats following brain injury (Long et al., 1996; Koda et al., 2008) point ing at a general protective role for antioxidants in learning and memory. 124

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3). the possibility of a nongenetic approach for the treatm ent of memory loss during aging eschews the complexiti es of gene based therapeutics. The results presented here indicate that a majority of neurons lose their function during aging; however it is still possible that some neurons retain normal functionality in the aged brain. If we assume that the normal functionality of these aged neurons derives from proper functioning of certain mechanisms, then the id entification of such mechanisms is critical to treating cogniti ve impairment and memory dysfunction. The strategy to prevent a normal neuron from becoming dysfunctional can be achieved by creating suitable therapeutic barriers (Fig. 6-2). In this context, therapeutic barriers could be strategies that keep intracellular Ca2+ concentrations within tolerable levels or strategies that decrease oxidative damage to neurons li ke spin trap agents that scavenge ROS, nutritional supplements, regular exercise, or caloric restriction. In the provided conceptual framework (Fig. 6-2) integrated Ca2+ levels is considered as the biomarker upon which a hypothetical therap eutic barrier could be applied. As an extension of this idea, potentially any age-rela ted biomarker, that ma rks the transition of the neurons from a functional to a dysfuncti onal stage, could be subjected to a tailor made therapeutic barrier. Based on the findi ngs of the studies presented here, the function of the NMDARs and/or the amplitude of sAHP of the CA1 pyramidal neurons could be a biomarker for cognitive impairm ent and memory decline; and the therapeutic barrier could be strategies that prevent an oxidative redox state or increased oxidative stress. Future Directions Understanding the age-re lated changes in the Ca2+ handling mechanisms of the neurons is critical to the development of therapeutics aimed to ameliorate cognitive 125

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dysfunction and memory loss. Amongst the key players that maintain Ca2+ homeostasis in the neurons (Fig. 6-3), NMDARs stand out for their significant role in synaptic plasticity, learning and memory The findings presented in th is dissertation delineate the biochemical and physiological changes to the NMDARs during aging. Debate surrounding NMDAR function during aging a nd neurodegeneration are at the heart of developing suitable therapeutics that reverse the biochemical and physiological changes and reinstate normal function to dysfunctional aged neurons. One of the important goals for future research is to distinguish between situations that demand an increase in NMDAR function ( like improving neuronal function) versus situations that demand a decrease in NMDAR function (like preventing neuron death) The decrease in the NMDAR function of aged neurons might represent a compensat ory neuroprotective mechanism associated with inappropriate recept or activity. It is well documented that NMDAR associated Ca2+ influx triggers neurotoxicity and activates cell death programs in neurons (Chen et al., 1992; Lei et al., 1992; Pivovarova et al., 2004). Thus aged neurons could progressively down regulate neurotoxicity-associated NMDAR activation for purposes of cell preservation (Foster, 1999). However one of the consequences of down regulating NMDAR function is impaired NMDAR-dependent synaptic plasticity and memory. Thus treatment strategies that deal with NMDARs in aged neurons have to reconcile the opposing features of functional rescue by NMDAR activation and over expression, with neuroprotection by NMDAR blockade and down regulation. Interestingly, over expression of NR2B su bunit improves synaptic plasticity and memory in aged mice (Cao et al., 2007). However, NMDAR blockade by memantine improves cognition and synaptic plasticity (B arnes et al., 1996; Norris and Foster, 1999; 126

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Pieta Dias et al., 2007), possibl y by blocking inappropriate NMDAR activation (Rosi et al., 2006; Matute, 2007; Chang and Gold, 2008); thus indicating a func tional rescue. In the case of neurodegenerative disease, dec reased expression of NR1 mRNA has been observed in brain regions that are most at risk for cell death, including Huntington's disease, wherein a decrease in NR1 mRNA expression is observed in the neostriatum (Arzberger et al., 1997). Furthermore, t here is evidence for decreased NMDAR expression in the hippocampus during the early stages of Alzheimers disease (Mishizen-Eberz et al., 2004; Jacob et al ., 2007), hinting at neuroprotection mechanisms employed by those neurons. Thus it will be important for future research to determine whether enhancing or inhibi ting NMDAR function will be beneficial in preserving hippocampus dependent learning and memory function during normal aging and in the face of neurodegenerative diseas e. A sound strategy would be to find a balance between the degree of functional re scue and the extent of neuroprotection needed for successful aging and pr eserved neuronal function. 127

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Figure 6-1. The biochemical model of brain aging and hippocampal dysfunction. The proposed model linking increased oxidat ive stress and decreased NMDA receptor activity during normal aging [e ither directly or indirectly through kinases and phosphatases]. The outcome would be enhanced LTD and impaired LTP for neural activity occurring at the modification threshold. If the proposed model turns out to be true, it could explain the age related weakening of synaptic connec tions in the hippocampus 128

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Figure 6-2. Conceptual framework fo r age-related neuronal dysfunction based on intracellular calcium levels. Integrated Ca2+ levels within the neurons can be used as an effective marker for differ entiating the event s associated with normal aging and neurodegenerati on. The integrated Ca2+ levels follow a sigmoid pattern of increase as neurons tr ansition from normal (light blue) to aged category (dark blue). Apoptosis can be observed at these stages. Sustained increase in integrated Ca2+ levels activates neurotoxic pathways and leads to necrosis or neuron death observed in neurodegeneration (darker shades of red). From a t herapeutic standpoint, a thera peutic barrier could be erected, in the form of nutritional or therapeutic interv ention, which will potentially prevent the normal neurons fr om exhibiting the intracellular Ca2+ profile of aged neurons. This crosso ver is proposed to precede neuronal dysfunction and ultimately memory dysfunction. 129

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130 Figure 6-3. Integrative model of th e impact of aging on the calcium handling mechanisms and physiological processes. During aging there is an interaction between increased oxidative stre ss and decreased neuron health with mechanisms for Ca2+ regulation that includes t he NMDA receptors (NMDAR), voltage-dependent Ca2+ channels (VDCC), intracellular Ca2+ stores (ICS), and Ca2+ buffering and extrusion mechanisms. An indication of regional specificity (hippocampus, frontal cortex, cortex, bas al forebrain) and the direction of change (increase red arrow and decrease green arrow) for each mechanism are also provided. The sh ift in Ca2+ homeostatic mechanisms may represent neuroprotective mechani sms to decrease further rise in intracellular Ca2+ by decreasing neuron activity. These changes impair the function of the neuron (Adapt ed from Kumar A, Bodhi nathan K, and Foster T C, Front Ag Neurosci 2009)

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APPENDIX A DRUGS, SOLUTUIONS, AND SUPPLIERS AP-5 (DL-2-Amino-5-phosphonoval eric acid); NMDAR antagonist; Sigma, St. Louis, MO ATP-Na+ lyophilized salt (Adenosine-5'-triph osphate); phosphate donor in kinase reactions; CycLex Co Ltd, Nagano, Japan Bis-I (bisindolylmaleimide-I); specific inhibitor of protein kinase C; Calbiochem, San Diego, CA c-H2DCFDA (5-(and-6)-carboxy-2 7 -dichlorodihydrofluorescein diacetate); fluorescent dye used to detect reactive oxygen spec ies; Molecular Probes Inc, Eugene, OR CaM (Calmodulin, purified from bovine brain); Ca2+ binding protein and co activator of CaM-Kinase II; CycLex Co Ltd, Nagano, Japan CaMKII (Ca2+/calmodulin-dependent protein kinase II) ; Ser/Thr kinase; CycLex Co Ltd, Nagano, Japan Catalase (purified from hum an erythrocytes); ant ioxidant enzyme converts hydrogen peroxide to water; Sigma, St. Louis, MO DMSO (dimethyl sulfoxide); non-aqueous solvent for various drugs; Sigma, St. Louis, MO DNQX (6, 7-Dinitroqui noxaline-2, 3(1H, 4H)-dione); AMPAR antagonist; Sigma, St. Louis, MO DTNB (5, 5 -Dithiobis (2-nitrobenzoic acid); oxid izing agent; Sigma, St. Louis, MO DTT (Dithiothreitol); reducing agent; Sigma, St. Louis, MO EGTA (Ethylene glycol-bis-(2-aminoethyl)-N, N, N, N-tetraacetic acid); chelating agent; CycLex Co Ltd, Nagano, Japan Ethanol; non-aqueous solvent for various drugs ; Fisher Scientific, Pittsburgh, PA FK-506; Calcineurin/Protein Phosphates 2B inhibitor; LC Laboratories, Woburn, MA H-7 (()-1-(5-Isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride); broad spectrum Serine/Threonine kinase inhibito r; Tocris Bioscience, Ellisville, MO HCl (Hydrochloric acid); general acid; Fisher Scientific, Pittsburgh, PA HRP conjugated anti-phospho-Syntide-2 antibody; CycLex Co Ltd, Nagano, Japan 131

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KN-62 (4-[(2S)-2-[(5-isoquinolinylsulfony l) methylamino]-3-oxo3-(4-phenyl-1piperazinyl) propyl] phenyl Isoquinolinesul fonic acid ester); specific CaMKII inhibitor; Tocris Bioscience, Ellisville, MO L-Glutathione reduced form (L-GSH); biologically available reduc ing agent; Sigma, St. Louis, MO Myr-AIP (myristoylated autocamtide-2 relat ed inhibitory peptide); specific peptide inhibitor of CaMKII with the following sequence [Myr-N-Lys-Lys-Ala-Leu-Arg-ArgGln-Glu-Ala-Val-Asp-Ala-Leu-OH]; Calbiochem, San Diego, CA Nifedipine; L-ty pe Voltage-gated Ca2+ Channel Antagonist; Tocris Bioscience, Ellisville, MO NaOH (Sodium Hydroxide); used to adjust the pH of solutions; Sigm a, St. Louis, MO OA (Okadaic acid); Protein Phosphatase 1 inhi bitor; Tocris Bioscience, Ellisville, MO Picrotoxin (PTX); GABAA receptor antagonist; Tocris Bi oscience, Ellisville, MO Ryanodine (RyR); Ryanodine receptor antagonist; Calbiochem, San Diego, CA Superoxide Dismutase (SOD, from human erythr ocytes); antioxidant enzyme converts superoxide anion to hydrogen peroxide or oxygen; Sigma, St. Louis, MO TMB (Tetra methyl-benzidine); chromogenic substrate for horseradish peroxidase; CycLex Co Ltd, Nagano, Japan Xanthine (X); substrate for Xanthine Ox idase which react together to produce superoxide radicals; Calbiochem, San Diego, CA Xanthine Oxidase (XO); an enzyme which reac ts with Xanthine to produce superoxide radicals; Roche Diagnostics, Indianapolis, IN 132

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APPENDIX B DRUG CONCENTRATIONS USED IN THE EXPERIMENTS AP-5 (100 M) ATP-Na+ lyophilized salt (62.5 M) Bis-I (500 nM) c-H2DCFDA (10 M) Ca2+/Calmodulin-dependent protei n kinase II (15 to 30 mU) Calmodulin (200 ng/mL) Catalase (260 units/mL) DMSO (final solvent concentration of < 0.01%) DNQX (30 M) DTNB (500 M) DTT (700 M) EGTA (2 mM) Ethanol (final solvent concentration of < 0.0001%) FK-506 (10 M) H-7 (10 M) KN-62 (10 M) L-Glutathione reduced form (700 M) Myr-AIP (5 M) Nifedipine (10 M) Okadaic acid (1 M) Paxilline (10 M) Picrotoxin (10 M) Ryanodine (20 M) Superoxide Dismutase (121 units/mL) 133

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134 Thapsigargin (1 M) Xanthine (20 g/mL) Xanthine Oxidase (0.25 to 1.00 units/mg of Xanthine)

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BIOGRAPHICAL SKETCH Karthik was born in Madurai, India, to Rajeswari Bodhinathan and Bodhinathan Sundarapandian. Karthiks primary schooling was at the Air Force School, Bangalore, situated in the midst of Indias scientific co mplex on Sir C. V. Raman road (named after the late Nobel Laureate Sir C. V. Raman) Karthik graduated from T.V.S. Lakshmi Matriculation Higher Secondary School in 2001. He then graduated first-class honors in 2005 with a Bachelor of Technology (Major: Biotechnology) from P.S.G. College of Technology in Coimbatore, affiliated with Anna University, one of Indias eminent engineering universities. His initial scient ific pursuits were shaped by a Summer Research Fellowship (2003 and 2004) awarded by The Jawaharlal Nehru Center for Advanced Scientific Research in Bangalo re, India. Consequently, the fellowship helped him pursue his undergraduate research work in the lab of Dr. Saumitra Das at the Indian Institute of Science in the spri ng of 2005. Karthik was recruited by the Interdisciplinary Program for Bi omedical Research at the Univer sity of Florida, in the fall of 2005, with the Alumni Fellowship. The exciting and unanswered questions of brain function and dysfunction led him, in the summer of 2006, to join the lab of Dr. Thomas C. Foster, the McKnight Chair for Research on Aging and Memory in the Department of Neuroscience at the University of Florida. 162