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Gabaergic Systems and Age-Related Cognitive Decline

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Title:
Gabaergic Systems and Age-Related Cognitive Decline
Creator:
Banuelos, Cristina
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (164 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Medical Sciences
Neuroscience (IDP)
Committee Chair:
BIZON,JENNIFER L
Committee Co-Chair:
SETLOW,BARRY
Committee Members:
FOSTER,THOMAS C
FRAZIER,CHARLES JASON
SMITH,DAVID WILLIAM
Graduation Date:
5/3/2014

Subjects

Subjects / Keywords:
Cholinergics ( jstor )
Forebrain ( jstor )
Hippocampus ( jstor )
Interneurons ( jstor )
Memory ( jstor )
Neurons ( jstor )
Prefrontal cortex ( jstor )
Rats ( jstor )
Receptors ( jstor )
Working memory ( jstor )
Neuroscience (IDP) -- Dissertations, Academic -- UF
aging -- gaba -- hippocampus -- learning -- memory
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Medical Sciences thesis, Ph.D.

Notes

Abstract:
With advances in medical science contributing to increased longevity, it is becoming increasingly important to elucidate the neural factors that underlie age-related declines in cognitive functions and to develop strategies that can promote healthy cognitive outcomes across the full lifespan. The hippocampus and the prefrontal cortex are two brain regions implicated in cognitive function and both these regions are highly sensitive to changes in aging. The overarching goal of this dissertation was to investigate how aging alters inhibitory signaling in a rodent model of normal aging, and to determine how such changes impact both hippocampal- and prefrontal cortical- dependent cognition. In Chapter 2 western blotting was used to quantify GABAergic signaling protein expression in hippocampus and confocal stereology was used to quantify phenotypically-specific neuron populations in basal forebrain of young and aged rats that were first behaviorally characterized on a septohippocampal-dependent water maze task. While the expression of most GABAergic signaling proteins in hippocampus did not change with age, expression of glutamic decarboxylase (GAD67) was selectively reduced in aged rats with spatial learning impairment. Moreover, GABAergic projection neuron number in basal forebrain was increased in aged animals with spatial learning impairments. Together, these data support that altered GABAergic signaling in the septohippocampal system contributes to age-related memory decline. In Chapter 3, western blotting was used to assess the expression of GABAergic signaling proteins in the prefrontal cortex of young and aged rats that were first behaviorally characterized on an operant delayed-response test of working memory. Prefrontal cortical expression of the GABA synthesizing enzyme GAD67 was increased and the neuronal GABA transporter, GAT-1, was decreased with age. GABA(B) receptor (GABA(B)R) expression was also reduced in aged prefrontal cortex (PFC). GABA(B)R expression was significantly and inversely associated with working memory such that those aged rats with lower GABA(B)R expression exhibited better delayed response performance. These data suggest that aging is accompanied by increased GABA availability within PFC and that downregulation of GABA(B)R expression may preserve appropriate levels of tonic inhibition required for optimal working memory. Pharmacological studies were conducted which supported this hypothesis as administration of a GABA(B)R antagonist, both systemically and directly into the prefrontal cortex, significantly improved working memory performance in impaired aged rats, restoring cognitive function to a level on par with young. Together, the data presented in this dissertation demonstrate that GABAergic systems are significantly altered in aging and that these alterations play a causal role in age-related cognitive decline. In addition, pharmacologically targeting this system, specifically through GABA(B) receptors, improves working memory performance in aged rats indicating that this system may serve as a therapeutic target for treating cognitive decline in aging. ( en )
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.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: BIZON,JENNIFER L.
Local:
Co-adviser: SETLOW,BARRY.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-11-30
Statement of Responsibility:
by Cristina Banuelos.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
11/30/2014
Classification:
LD1780 2014 ( lcc )

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1 GABAERGIC SYSTEMS AND AGE RELATED COGNITIVE DECLINE By CRISTINA BAUELOS 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 2014

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2 2014 Cristina Bauelos

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3 To Dr. Luis Colom who started me on this journey

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4 ACKNOWLEDGMENTS I would like to thank all of the members of my dissertation committee for taki ng time out of their hectic schedules to help me get through this process. Specifically, I would like to thank Jennifer Bizon and Barry Setlow for providing outstanding mentorship over the last six years. I know I have driven you crazy more than a few tim es over the years. I really appreciate the time you took to teach me how to be a neuroscientist and for setting the example of what a successful mentor should be. I will always endeavor to achieve your level of success. I definitely would not have comple ted this project without the guidance and help from t he people working in the Bizon/Setlow lab. Candi and Karienn welcomed me into the lab and selflessly taught me the skills I needed to begin this journey. I will always remember late nights in lab with Karienn and how comforting it was to have company in trying times. Sofia was my roommate for half and lab mate for most of my graduate school experience and I will always appreciate her friendship, even the times that were not always friendly. Ryan Gilbe rt was a constant in lab thank you for teaching me most of what I know, always being up for anything and, of course, for keeping Doggy alive. I will always appreciate the calmness that Kristy brings to any situation. Caitlin personified the type of post d oc I aspire to be and it was just so much fun working with her. Also, I would like to thank Joe for always having the right words when I could not put a sentence together. Finally, I would like to thank my family and friends for all of their support. M om and Dad, thanks for making education such a top priority in our lives and making me feel like I could accomplish anything I put my mind on. My siblings Marko, Monica, Polo and Victor were always there to nudge me forward when I needed a good push.

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5 TA BLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 ABSTRACT ................................ ................................ ................................ ................... 13 C HAPTER 1 INTRODUCTION: SEPTOHIPPOCAMPAL AND PREFRONTAL CORTICAL GABAERGIC SYSTEMS IN COGNITION AND AGING ................................ .......... 16 Cognitive Decline in Aging ................................ ................................ ...................... 1 6 Hippocampus and Long Term Memory in Aging ................................ ..................... 17 Prefrontal Cortex and Working Memory in Aging ................................ .................... 20 Individual Variability in Long Term and Working Memory ................................ ....... 23 Animal Models of Cognitive Aging ................................ ................................ .......... 24 Assessing Spatial Memory: Morris Water Maze Task ................................ ...... 25 Individual Variability in Spatial Memory Ability ................................ .................. 26 Assessing Working Memory Delayed Response Task ................................ ..... 28 GABAergic Systems in Hippocampus and Prefrontal Cortex ................................ .. 29 GABAergic Systems in Aging ................................ ................................ ................. 34 Experimental Goals ................................ ................................ ................................ 37 2 AGE RELATED CHANGES IN SEPTOHIPPOCAMPAL GABAERGIC SYSTEMS: RELATIONSHIP WITH SPATIAL IMPAIRMENT ................................ 47 Introduction ................................ ................................ ................................ ............. 47 Methods ................................ ................................ ................................ .................. 50 Subjects ................................ ................................ ................................ ............ 50 Experiment 1: GABA sig naling Protein Expression and Spatial Memory Abilities ................................ ................................ ................................ .......... 51 Water Maze Task Procedures ................................ ................................ .... 51 Apparatus ................................ ................................ ................................ ... 51 Spatial reference memory (hidden platform) task ................................ ...... 51 Cued (visible platform) task ................................ ................................ ........ 52 Statistical a nalyses ................................ ................................ .................... 52 Western Blotting of GABAergic Signaling Proteins in the Hippocampus. ........ 53 Sample Preparation ................................ ................................ ................... 54 Immunoblotting ................................ ................................ .......................... 55 Statistical Analysis ................................ ................................ ..................... 55 Experiment 2: Stereological Quantification of Ros tral Basal Forebrain Cholinergic, GABAergic and Total Neurons ................................ .................. 56

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6 Immunofluorescent Labeling of Cholinergic, GABAergic Projection and Total Rostral Basal Forebrain Neurons ................................ ......................... 56 Stereological Counts of ChAT, GAD67, and NeuN Immunopositive Neurons .. 57 Delineation of the rostral basal forebrain ................................ ................... 57 Estimation of neuron number using the optical fractionator ....................... 59 Statistical Analysis ................................ ................................ ..................... 61 Results ................................ ................................ ................................ .................... 62 Experiment 1: GABA signaling Protei n Expression and Spatial Memory Abilities ................................ ................................ ................................ .......... 62 Cognitive Performance in Young and Aged Rats ................................ ............. 62 Age and Cognitive Comparisons of GAD67, VGAT, GAT 1, GABA(B)R1a, GABA(B)R1b, and GABA(B)R2, expression in the hippocampus ................. 63 Experime nt 2: Stereological Quantification of Rostral Basal Forebrain Cholinergic, GABAergic and Total Neurons ................................ .................. 65 Cognitive Performance in Young and Aged Rats ................................ ............. 65 Distribution of ChAT GAD67 and NeuN Immunopositive Neurons ................. 67 Age and Cognitive Comparisons of ChAT, GAD67, and NeuN Immunopositive cell Numbers in the Rostral Basal F orebrain .......................... 68 ChAT immunopositive neurons ................................ ................................ .. 68 GAD67 immunpositive neurons ................................ ................................ 69 NeuN immunopositive neurons ................................ ................................ .. 70 Discussion ................................ ................................ ................................ .............. 70 Cholinergic (ChAT immunopositive) neurons ................................ ................... 71 GABAergic projection (GAD67 immunopositive) neurons ................................ 73 3 AGE RELATED CHANGES IN PREFRONTAL CORTICAL GABAERGIC SYSTEMS: RELATIONSHIP TO WORKING MEMORY IMPAIRMENT .................. 93 Introduction ................................ ................................ ................................ ............. 93 Methods ................................ ................................ ................................ .................. 95 Subjects ................................ ................................ ................................ ............ 95 Experiment 1: GABA signaling Protein Expression and Working Memory Abilities ................................ ................................ ................................ .......... 95 Delayed Response Task Procedures ................................ ......................... 95 Working Memory Assessment ................................ ................................ ... 97 Western Blotting Procedures ................................ ................................ ..... 99 Statistical analyses ................................ ................................ .................. 100 Experiment 2: Systemic Administration of the GABA(B) Receptor Antagonist CGP55845 ................................ ................................ ................ 102 Drug Administration Procedures ................................ .............................. 102 Data Analysis ................................ ................................ ........................... 103 Experiment 3: Intracerebral Microinjections of the GABA(B) Receptor Antagonist CGP55845 ................................ ................................ ................ 104 Cannulation Surgery ................................ ................................ ................ 104 Intracerebral Microinjections of the GABA(B) Receptor Antagonist CGP55845 ................................ ................................ ............................ 104 Histological Asse ssment of Cannulae Placement ................................ .... 106

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7 Data Analyses ................................ ................................ .......................... 106 Results ................................ ................................ ................................ .................. 107 Expe riment 1: GABA Signaling Protein Expression and Working Memory Abilities ................................ ................................ ................................ ........ 107 Working Memory Performance is Impaired in Aged F344 Rats ............... 107 Age Related Alterations in GABAergic Signaling Protein Expression in Aged mPFC ................................ ................................ .......................... 108 Experiment 2: Systemic Administration of the Selective GABA)B)R Antagonist CGP55845 Restores Working M emory Performance in Aged Rats ................................ ................................ ................................ ............. 110 Experiment 3: Intra mPFC Infusions of the Selective GABA(B) Receptor Antagonist CGP55845 Restores Working Memory Performance in Aged Rats ................................ ................................ ................................ ............. 112 Discussion ................................ ................................ ................................ ............ 114 Increased Inhibition in Aged Prefrontal Cortex ................................ ............... 114 GABA(B) Receptor Antagonist Administration Improves Working Memory in Aging ................................ ................................ ................................ ........... 117 4 SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS ............................... 125 APPENDIX A BASAL FOREBRAIN MAGNOCELLU LAR PREOPTIC AREA AN D HORIZONTAL DIAGONAL BAND OF BROCA GABAER GIC NEURONS INNERVATE INTERNEURO NS OF THE MEDIAL PRE FRONTAL CORTEX ...... 135 B DIRECT APPLICATION O F THE SELECTIVE M3 AGONIST CEVEMILINE O N BASAL FOREBRAIN IMPR OVES WORKING MEMORY PERFORMANCE IN YOUNG RATS. ................................ ................................ ................................ ..... 138 LIST OF REFERENCES ................................ ................................ ............................. 140 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 164

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8 LIST OF TABLES Table page 2 1 Sampling parameters used for estimat ing total number of rostral basal forebrain neurons. ................................ ................................ .............................. 91 2 2 Estimates of ChAT, GAD67 and NeuN immunopositive cells in rost ral basal forebrain of young and aged behaviorally characterized rats. ............................ 92

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9 LIST OF FIGURES Figure p age 1 1 Morris w ater m aze p erformance in y oung r ats a cross t raining and p robe t rials .. 42 1 2 Spatial memory performa nce of young and aged rats. ................................ ...... 43 1 3 Delayed response task perfo rmance in young and age d rats ............................. 44 1 4 Schematic of GABAergic synapse in the hippocampus and p re frontal cortex .... 45 1 5 Basal forebrai n projections to the hi ppocampus and prefrontal cortex ............... 46 2 1 Rostral basal forebrain boundaries used for cell count e s timatio ns .................... 79 2 2 Distribution of ChAT immunopositive c ells in rostral basal forebrain .................. 80 2 3 Distribution of GAD67 immunopositive c ells in rostral basal forebrai n ............... 81 2 4 Distribution of NeuN immunopositive cells in rostral basal forebrain. ................. 82 2 5 Antibody penetration throughout the tissue slice. ................................ ............... 83 2 6 Spatial learning in young and aged rats in Experiment 1. ................................ .. 84 2 7 Age related changes in GABAergic signaling protein expression and relationship to working memory ability. ................................ ............................... 85 2 8 Spatial learning in youn g and aged rats in Experiment 2 ................................ ... 86 2 9 High magnification immunofluorescent labeling of ChAT, GAD67 and NeuN immunopositive neurons ................................ ................................ ..................... 87 2 10 Cholinergic (ChAT immunopositive) cell number in the rostral basal f orebrain of young and aged rats ................................ ................................ ....................... 88 2 11 GABAergic (GAD67 immunopositive) cell number in the rostral basal forebrain of young and aged rats ................................ ................................ ........ 89 2 12 Total (NeuN immunopositive) cell number in the rostral basal forebrain of young and aged rats ................................ ................................ ........................... 90 3 1 Working m emor y is i mpaired in a ged F344 r ats ................................ .............. 121 3 2 Age related changes in GABAergic signaling protein ex pression and relatio nship to working memory ability ................................ ............................. 122

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10 3 3 Systemic GABA(B) receptor antagonist administration significantly improves working memory performance in aged rats ................................ ...................... 123 3 4 Intra mPFC GABA(B) receptor antagonist administration improves working memory performance in aged rats ................................ ................................ ... 124 4 1 Performance of s patially characterized r ats on d iscrimination l earning. ........... 132 4 2 GABA(B) a ntagonist R e ve rses a ge r elated o dor l earning deficits. .................. 133 4 3 Basal forebrain projections to the hippocampus and prefrontal cortex. ............ 134 A 1 Basal forebrain neurons project to the prefrontal cortex. ................................ .. 137 B 1 Systemic M3 muscarinic receptor agonist administration significantly improves working memory performance in young rats. ................................ .... 139

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11 LIST OF ABBREVIATIONS ACSF A rtificial cerebrospinal fluid AD BSA Bovine serum albumin CE Coefficient of error ChAT Choline acetyl transferase CV Coefficient of variance E East EDTA Ethylenediamine tetra acetic acid FA Formic acid GABA Gamma aminobutyric acid GABA(B)R GABA(B) receptors GAD Glutamic acid decarboxylase GAT GABA transporter H Hour hDB horizontal Diagonal Band LTP Long term potentiation NeuN Neuronal nucleic PBS Phosphate buffered saline PFA paraformaldehyde RIPA Radioimmunoprecipitation assay TBS Tris buffered saline TBST Tris buffered saline and Tween ACSF Artificial cerebral spinal f luid I.P. intraperitoneal

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12 ITI Intertrial i nterval IV Intravenous M PFC Medial prefrontal v ortex MS Medial NDS Normal donkey serum PFC Prefrontal cortex PB Phosphate b uffer S second SD Standard deviation SLI Spatial learning index vDB vertical Diagonal Band VGAT Vesicular GAB A transporter

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13 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 GABAERGEIC SYSTEMS AND AGE RELATED COGNITIVE DECLINE By Cristina Ba uelos May 2014 Chair: Jennifer Lynn Bizon Major: Medical Sciences Neuroscience With advances in medical science contributing to increased longevity, it is becoming increasingly important to elucidate the neural factors that underlie age r elated declines in cognitive function s and to develop strategies that can promote healthy cognitive outcomes across the full lifespan The hippocampus and the prefrontal cortex are two brain regions implicated in cognitive function and both these regions are highly sensitive to changes in aging. The overarching goal of this dissertation was to investigate how aging alters inhibitory signaling in a rodent model of normal aging and to determine how such changes impact both hippocampal and prefrontal corti cal dependent cognition In Chapter 2 western blotting was used to quantify GABAergic signaling protein expre ssion in hippocampus and confocal stereology was used to quantify phenotypically specific neuron populations in basal forebrain of young and aged rats that were first behaviorally characterized on a septohippocampal dependent water maze task While the expression of most GABAergic signaling proteins in hippocampus did not change with age, expression of glutamic decarboxylase (GAD67) was selectively reduced in aged rats with spatial learning impairment. Moreover, GABAergic projection

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14 neuron number in basal forebrain was increased in aged animals with spatial learning impairments. Together, these data support that altered GABAergic signaling in the se ptohippocampal system contributes to age related memory decline. In Chapter 3, western blotting was used to assess the expression of GABAergic signaling proteins in the prefrontal cortex of young and aged rats that were first behaviorally characterized on an operant delayed response test of working memory. Prefrontal cortical expression of the GABA synthesizing enzyme GAD67 was increased and the neuronal GABA transporter, GAT 1 was decreased with age GABA(B) receptor (GABA(B)R) expression was also reduce d in aged prefrontal cortex (PFC) GABA(B)R expression was significantly and inversely associated with working memory such that those aged rats with lower GABA(B)R expression exhibited better delayed response performance. These data suggest that aging is accompanied by increased GABA availability within PFC and that downregulation of GABA(B)R expression may preserve appropriate levels of tonic inhibition required for optimal working memory. Pharmacological studies were conducted which supported this hypoth esis as administration of a GABA(B)R antagonist both systemically and directly into the prefrontal cortex significantly improved working memory performance in i mpaired aged rat s, restoring cognitive function to a level on par with young Together, the d ata presented in this dissertation demonstrate that GABAergic systems are significantly altered in aging and that these alterations play a causal role in age related cognitive decline. In addition, pharmacologically targeting this system, specifically thr ough GABA(B) receptors, improves working memory performance in

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15 aged rats indicating that this system may serve as a therapeutic target for treating cognitive decline in aging

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16 CHAPTER 1 INTRODUCTION: SEPTOHIPPOCAMPAL AND PREFRONTAL CORTICAL GABAERGIC SYS TEMS IN COGNITION AND AGING Cognitive Decline in Aging The number of people in the U.S over the age of 65 is expected to double over the next 20 years. While medical advances and healthy lifestyle choices have significantly increased life expecta ncy, ther e is substantial risk for co gnitive decline at advanced ages Aging affects several aspects of cognition, however, two types of cognition that are particularly vulnerable to decline at a dvanced age are long term memory and working memory. A loss of these cognitive functions leads to decreased quality of life and loss of independence which in addition to causing personal distress is also associated w ith substantial financial costs (Freedman et al., 2002) As such, there is a tremendous need to understand t he neurobiological underpinnings of age relat ed learning and memory deficits in order to be able to detect early signs of failing cognitive ability and to develop effective treatments for age related cognitive decline. As humans age, there are two possib le cognitive trajectories, one leading t o pathological dementia due to disease s such as Alzh trajectory that leads to normal cognitive aging with relatively intact cognitive fu nction (Samson and Barnes, 2013) Approximatel y 14% of individuals over the age of 71 years follow the pa thological trajectory and are classified as demented while the other 86% of people over 71 follow t he normal cognitive aging path (Plassman et al., 2007) Notably, however, t he se normal cognitive agers do experience age related decline s in cognitive function and in many individuals, the degree of impairment can be sufficiently independence Both long term mem ory and working memory decline with advanced

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17 age and there is tremendous individual variability in cognitiv agers. Understanding the neurobiological substrates that underlie cognitive individual variability among aged individuals is crucial to understanding age related cognitive decline and promoting healthy cognitive aging. Hippocampus and Long Term Memory in Aging Long ter m memory is the ability to store information for long periods of time ranging in duration from hours to years and, in some cases an entire lifetime. Long term memory includes declarative memory, the memory for specific personal events and their contents as well as general knowledge about the world. Spatial memory is also considered a su bset of declarative memory (Squire et al., 2004) Declarative memory is critically dependent on the hippocampus, a component of the med ial temporal lobe (Squire, 2004) The hippocampus is important for forming, retaining and recalli ng memories The first indications that the hippoc ampus played a crucial role in organizing memory emerged when Scovelle and Milner (1957) described memory deficits in H.M., an epileptic patient whose medial temporal lobe was bilaterally transected to treat severe recurring seizures. The surgery effectiv ely reduced the frequency and severity of the seizures, but H.M. experienced extensive post surgery memory impairments. H.M. was able to maintain and use well practiced information for a short time, however, he was not able to retain new information into long term memory. The study of H.M. demonstrated that short term and long term memory are distinct functions mediated by distinct regions of the brain (Squire, 2009; Clark and Squire, 2013) Other patients with damage more specific to the hippocampus als o displayed significant anterograde amnesia, lacking the ability to successfully form declarative memory after the time of brain damage further indicating the importance of the

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18 hippocampus in processing information into long term memory (Rempel Clower et a l., 1996) Bilateral hippocampal lesions also impair recognition memory, the ability to recognize, recently encountered material as familiar (Manns et al., 2003) Neuroimaging studies using positron emission tomography (PET) and functional magnetic reson ance imaging (fMRI) report that hippocampal activity is increased in the hippocampus, specifically in the right hemisphere, of humans when they navigate complex virtual environments (Smith and Milner, 1981; Maguire et al., 1996, 1997) Maguire et al. (200 0) also found that London taxi drivers, people who use extensive spatial navigation while performing their job, had significantly greater hippocampal volumes than control subjects. These data indicate that the hippocampus may be particularly important for and indeed, critically supports, spatial memory. In humans, long term memory is ass essed with a wide array of tests that require individuals to recall learned information after a period of time. One test that is used to assess long term memory is the ob ject recognition test. Test participants are presented with and asked to remember two different objects. After a time delay, they are then presented with two objects and asked to identify the novel object, testing their memory for the previous pair (Logo thetis and Sheinberg, 1996) Another assessment, the object location task, requires the subject to remember the physical location of an object on a two dimensional surface after a time delay. Spatial memory in humans is assessed by asking subjects to re member the location of objects in a virtual environment. With the advancement of technology, spatial memory in humans is increasingly being tested using virtual reality where subjects have the ability to move and navigate within

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19 computer generated environ ments and mazes (Burgess et al., 2002; Etchamendy et al., 2012) Aged humans consistently perform significantly worse than young counterparts on tests of long term memory, however, the exact pattern of decline varies depending on study parameters (Nilsso n, 2003; Hedden and Gabrieli, 2004; Samson and Barnes, 2013) In a cross sectional study of healthy adults ranging in age from 20 to 9 0 years old, Park et al. (2002) reported linear declines in several measures of long term memory across the lifespan. D ata from the Seattle Longitudinal Study, an ongoing study of cognition in healthy adults that begin in 1956, indicate that long term memory is relatively stable from the age of 20 until the age of 60, after which performance in verbal memory declines linea rly, at a rate similar to that seen in the cross sectional study (Schaie et al., 2004) In this study, verbal memory was assessed using tests of immediate and delayed recall as well as the Primary Mental Ability word fluency test which required subjects t o freely recall as many words as possible according to a lexical rule within a time period (Zelinski et al., 1993) Spatial orientation ability also declined with age in participants of the Seattle Longitudinal Study. Many studies using virtual environme nts have reported the decline in spatial memory in aged individuals (Uttl and Graf, 1993; Klencklen et al., 2012; Gyselinck et al., 2013) In a study of young (<45 years), middle aged (45 65 years) and aged (>65 years) individuals who were asked to search and reach a goal point in a virtual maze with unique wall textures and objects presented from a first person perspective on a computer screen, middle aged and aged subjects took significantly longer to reach the goal point and made more search errors than young participants (Moffat et al., 2001) In another study which investigated spatial

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20 knowledge acquisition in young (20 30 years), middle aged (40 50 years) and aged subjects (60 70 years) in a desktop virtual maze, aged subjects took significantly more trials to correctly navigate the maze than young subjects. Aged subjects also remembered significantly fewer landmarks than their young counterparts (Jansen et al., 2010) Prefrontal Cortex and Working Memory in Aging Executive functions, which includ e attention, working memory, and cognitive flexibility, represent critical control and planning mechanisms that mediate and guide goal directed behavior. Deficits in executive functions can profoundly disrupt a wide range of adaptive behaviors and the norm al activities of daily living (Robbins, 1996) One key aspect of P FC cognition is the support of working memory which may be defined as the ability to briefly store and act on a mental representation of information, even when that information is no longer associated with a persistent sensory input (Goldman Rakic, 1996; Bizon et al., 2012) Working memory is critically dependent upon the prefrontal cortex The critical role played by the prefrontal cortex in working memory was first described in neurophysi ological and lesion studies in monkeys. (Fuster, 1990; Goldman Rakic, 1990) In monkeys, working memory is typically studied using a delayed response or delayed match to sample task in which the subject is given a brief cue at the start of the trial and t hey have to keep that information in mind during a delay that can last for several seconds. At the end of the delay, the monkey must make a choice or differential response based on the cue previously given. Seventy eight years ago, C.F. Jacobson (1936) wa s the first to describe impairments in delayed response performance in monkeys after bilateral ablation of their dorsolateral prefrontal cortex. The monkeys in his study were able to successfully perform visual

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21 discriminations and manipulate problem boxe s, however, they were not able to remember the location of food (hidden under one of two identical cups) after a delay as short as a few seconds, a task they were able to perform with high accuracy before lesion surgery. Since then, many studies in monkeys found that the neurons in the prefrontal cortex are activated when a cue is presented and these neurons continue to fire during the delay phase (Fuster et al., 1985; Funahashi et al., 1989) Brain imaging studies using positron emission tomography (PET) and functional resonance imaging (fMRI) have described activation of the human prefrontal cortex in subjects performing tasks of working memory (Jonides et al., 1993; Petrides et al., 1993; McCarthy et al., 1996; Courtney et al., 1997) Many subclasses o f working memory have been described and distinct regions of the prefrontal cortex have been linked to these specific types of working memory in humans (Courtney et al., 1998) Visuospatial working memory is critically dependent on the dorsolateral prefro ntal cortex in humans and nonhuman primates (Wilson et al., 1993; Courtney et al., 1998) In humans, working memory is assessed with a number of tasks that require the subject to keep information in mind. Verbal working memory is assessed using the digit span test, where subjects are given a list of numbers then asked to recall them. The list is increased every time the subject correctly recalls the number series. Visuospatial working memory is typically assessed with the delayed match to sample task or the delayed response task. In the delayed response task, a piece of information such as spatial location or an object is presented to the subject. A short delay follows during which the piece of information is removed from the environment. After the del ay the information presented must be recalled or recognized. Neuroimaging studies of

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22 humans performing visuospatial delayed response or recognitions tasks report activation of the dorsolateral prefrontal cortex during the delay period of the tasks (McCar thy et al., 1996; Leung et al., 2002) Humans with bilateral lesions of the frontal lobe are impaired in spatial delayed response tasks (Freedman and Oscar Berman, 1986) Accumulating evidence indicates that during normal aging, executive functions suppor ted by the prefrontal cortex are among the earliest and most severely impaired cognitive abilities (Robbins et al., 1998; Salthouse et al., 2003; Glisky, 2007) Working memory in humans declines with age with aged individuals perform significantly worse t han young in tests of working memory (De Beni and Palladino, 2004; Holtzer et al., 2009; Nagel et al., 2009) Using a spatial delay response task, Lyons Warren and colleagues (2004) described a significant linear decline in working memory performance acro ss adulthood. In this study, participants sitting in front of a computer screen were presented with a cue in the form of a black dot flashing at a specific location on the screen for a short period of time. A 5 second delay followed the cue during which geometric shapes randomly flashed in the center of the screen. After the delay, subjects were asked to point to the location where the black dot had flashed during the cue presentation. The distance between the remembered location and the actual cue loc ation was calculated and used as the location error for that trial. Subjects completed 8 trials. In total, working memory data from 256 adult individuals ranging in age from 17 to 80 years old was analyzed. A significant positive relationship between ag e and location error was described such that older participants remembered the cue location with less accuracy after the 5 second delay than younger participants. Data from this

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23 study indicated that performance on this task becomes worse with age. Similar ly, Park et a l. (2002) reported linear declines in visuospatial and verbal working memory across the lifespan in a cross sectional study of healthy adults between the ages of 20 and 90 years. In this study, 345 participants were tested on a battery of me mory tests that included 4 tests of working memory (2 specifically testing visuospatial working memory and 2 testing verbal working memory). Performance on these tasks was transformed into a z score and plotted against age. Slopes were compared and analy zed. Performance on all working memory tasks declined similarly with age in this study. Individual Variability in Long Term and Working Memory A common finding in studies of human cognitive aging is that declines in memory are associated with increased va riability in performance among aged individuals (Ylikoski et al., 1999) Individual differences may be due to diverse life experiences, genetic differences and differences in performance strategies (Hedden and Gabrieli, 2004) In human studies of cogniti on in aging, individual differences among aged study participants can be very large with some aged individuals performing on par with young and others performing significantly worse (Rapp and Amaral, 1992; Hedden and Park, 2003) In the Seattle Longitudin al Study mentioned earlier, where long term memory was shown to decline with age, individual variability was measured by comparing how much the distribution of delay recall scores overlapped among age cohorts. Age groups with a mean age of 67 years or you nger showed a more than 90% overlap with the distribution of scores for cohorts with people in their 20s. The oldest group with a mean age of 88 years exhibited an almost 50% score distribution overlap with the scores of the youngest cohort (Schaie, 1988) In an assessment of delayed recall of a story from the Wechslor memory scale, a neuropsychological test designed to measure different

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24 memory functions in humans, a sample of healthy aged individuals displayed significant variability in performance (Alber t, 1993). Understanding the neurobiological substrates that underlie cognitive individual variability among aged individuals is crucial to understanding age related cognitive decline and promoting healthy cognitive aging. Animal Models of Cognitive Agin g E xploring neurobiological markers that correlate with age related cognitive decline in humans is often only possible post mortem. In order to explore the neurobiological underpinnings of individual differences in normal cognitive aging, animal models whi ch allow for the assessment of hippocampal and prefrontal cortical dependent learning and memory are essential. The anatomical organization of the hippocampus and its connectivity to associated cortices is highly conserved across humans, primates and rode nt s and hippocampal lesions across the three species result in impaired declarative memory (Clark and Squire, 2013) The prefrontal cortex of humans and non human primate is readily compared and focal lesions in the dorsolateral prefrontal cortex of huma n and nonhuman primates result in impaired working memory. However, there has been much debate regarding the existence of a rodent prefrontal cortex that can be compared to the primate dorsolateral prefrontal cortex (Uylings et al., 2003; Kesner and Churc hwell, 2011) This debate was largely fueled by the fact that the rodent cerebral cortex is significantly smaller and the rat displays less complex cognitive cerebral function (Uylings and van Eden, 1990) Based on similar connectivity patterns, function al properties and the distribution of neuronal populations, it is now accepted that the primate and rodent prefrontal cortex are comparable (Uylings et al., 2003; Kesner and Churchwell, 2011). Lesions to the rat medial prefrontal cortex impair performance on tests of working memory such as the

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25 delayed response task (Kolb et al., 1974). Primates exhibit similar working memory impairments following focal lesions to the dosrsolateral prefrontal cortex supporting that the medial prefrontal cortex is the roden t homolog of the primate dorsolateral prefrontal cortex (Uylings et al., 2003). Upon establishing that the hippocampus and prefrontal cortex of humans and rodents were functionally and anatomically comparable, rodent models have emerged that provide a rel iable, reproducible system in which to study brain ch anges in cognitive aging. A reliable rodent model of natural aging is the Fischer 344 (F344) rat. This inbred strain of rat is readily available from an aging colony maintained by the National Institut e of Aging. Inbred strains have increased reproducibility and behavioral predictability that may be a beneficial aspect of experimental design (van der Staay and Blokland, 1996) F344 rats hav e a mean life expectancy of 23 29 months in a laboratory (Hoff man, 1978) Rats in this strain do not develop senile neuritic plaques and neurofibrillary tangles in their brain as seen in the human neuropathologies of advanced a ge compared to young making this an ideal model of normal non pathological aging (van der Staay and Blokland, 1996). Previ ous work in our lab has demonstrated that these rats exhibit multiple aspects of cognitive decline at advanced age (LaSarge et al., 2 007; Bizon et al., 2009) Assessing Spatial Memory: Morris Water Maze Task Hippocampal dependent memory is often assessed in rodents with tests of spatial memory. Rats with hippocampal lesions show impairments in learning the t maze and radial arm maze, two tasks that assess spatial learning and memory (Olton et al., 1979; Becker et al., 1981; Rawlins and Olton, 1982; Jarrard, 1983; Murray et al.,

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26 1989; Bannerman et al., 2001) In the studies described in this dissertation the Morris water m aze was use d to assess spatial memory abilities in F344 rats. The water maze consists of a large circular tank filled with water in which a fixed platform is hidden beneath the waterline. The water maze is surrounded by black curtains affixed with geometric shapes that serve as extramaze spatial navigation cues. Over a period of 8 days, rats complete training and probe trials during which they are introduced to the pool from different start locations and they must navigate the pool for 90 seconds u sing the extramaz e cues to find the hidden platform and escape the water (Figure 1 1 A). Over the course of training, rats learn the location of the escape platform and the pathlength, the total distance traveled from the start position to the platform location decreases (Figure 1 1B). After 5 training trials, probe trials, during which the escape platform is not available to the rat for the first 30 seconds of the swim, are interpolated orm (Figure 1 1C). Performance on the Morris water maze is critically dependent on the hippocampus Young rats with hippocampal l esions took significantly longer to reac h the escape platform compared to rats with intact hippocampus and demonstrated severe, lasting impairments in place learning when the start locations vary (Morris et al., 1982; Morris et a l., 1986; Pearce et al., 1998). Interestingly, focal mPFC lesions do not impair performance in this task (Sloan et al., 1996). Individual Variability in Spatial Memory Ability The water maze is particularly sensitive to age related cognitive decline and aged rats perf orm similarly to hippocampal lesioned young rats. Age d rats demonstrate a more inaccurate search for the platform location compared to young rats, even after

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27 several days of training (Gallagher and Burwell, 1989; Lindner, 1997) Numerous studies have described significant individual variability in water maz e performance among aged rats with some aged rats performing on par with young and other aged rats displaying impairment (Gage et al., 1984; Gallagher and Burwell, 1989, Rapp et al., 1987; Gallagher et al., 1993). A spatial learning index (SLI) score can be calculated from interpolated probe trial data in order to provide a graded measure of spatial learning ability for individual aged rats. The SLI, first described by Gallagher et al. (1993) and modified for F344 rats by Bizon et al., (2009) is calculate d by summing weighted mean search error measures from each interpolated probe trial. The weight assigned to each probe trial was determined by data from a large cohort of F344 rats behaviorally characterized on the spatial reference memory task in the Mor ris water maze (Bizon et al., 2009). The weights are derived from probe trial performance of young rats and are meant to favor rapid acquisition of platform location since age related differences in water maze performance are most pronounced at the early phases of training when young rats develop an accurate search strategy quicker than aged rats. Higher SLI scores represent worse performance on the water maze during probe trials. Previous work in our lab has reliably shown a significant difference in spa tial learning performance between young and aged male F344 rats as assessed by the Morris water maze (LaSarge et al., 2007; Bizon et al., 2009). Figu re 1 2A shows water maze performance as measured by pathlength (total distance traveled from the start po sition to the platform). As a group, aged rats performed significantly worse than young rats. Figure 1 2B shows the SLI distribution of rats characterized in the water maze

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28 task in our lab and illustrates how half of the aged rats performed on par with t h e young cohort (aged spatially unimpaired); whereas the other half of aged rats performed outside the range of young (ag ed spatially impaired) To relate neurobiological features to cognitive outcomes, those animals that perform on par with young are class ified as aged spatially unimpaired and those performing significantly worse than young animals are classified as aged spatially impaired. The ability to detect individual variability among aged rats in this model of natural aging lends validity to human a ge related cognitive decline and allows for the use of these individual differences to understand and uncover the neurobiological factors driving differences in cognition. Assessing Working Memory Delayed Response Task In healthy adult humans and non huma n primates, the dorsolateral prefrontal cortex is crucial to working memory function (Mishkin, 1957; Butters and Pandya, 1969; Goldman and Rosvold, 1970; Passingham, 1985; Freedman and Oscar Berman, 1986; Funahashi et al., 1993; Goldman Rakic, 1996; D'Espo sito et al., 2000) To date, most rodent studies assessing working memory utilize behavioral tasks that heavily involve spatial information (i.e. t maze and radial arm maze) and are, therefore, mediated by both the prefrontal cortex and hippocampus (Bizon et al., 2012) In the studies described in this dissertation, working memory is assessed in F344 rats using a Dela yed Response Task (Figure 1 3 A) based on Sloan et al. (2006) Rat performance on this task is specifically mediated by the medial prefront al cortex the rodent homolog of the dorsolateral prefrontal cortex. Sloan et al. demonstrated that focal ibotenic acid induced excitotoxic lesions of the mPFC result in impaired performance on this task compared to unlesioned control rats. Focal lesions to the hippocampus did not affect performance in this task (Sloan et al., 2006)

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29 In this task performed in an operant chamber, rats are prese nted with and trained to press lever s to obtain a food reward. Rats perform trials with 3 phases. During the fi rst phase, the sample phase, rats are presented with either a left or right lever which they are trained to press (rats press whichever lever is extended). The sample phase is followed by a delay phase which lasts from 0 to 24s and during which both lever s are retracted. The choice phase follows the delay phase and both levers are extended. Rats must press the same lever as in the sample phase to earn a food reward. Performance on this task is measured as the percentage correct choices averaged across 5 daily sessions at each delay. Young and aged rats perform similarly at no delay, exhibiting a high degree of choice accuracy, however, as the delays young and age rats p erform with less accuracy. Reliably in our lab aged rats are significantly impaired in this task compared to young, particularly at long delays (Beas et al., 2013) (Figure1 3 B). Human and non human primate working memory ability is assessed similarl y suggesting that t he delay response task used in this study may allow for a more p recise cross species comparison of working memory. This task may be an effective tool for iso lating the effects of age on medial prefrontal cortical mediated cognition. GA BAergic Systems in Hippocampus and Prefrontal Cortex The balance between excitation and inhibition is crucial for optimal cognitive function and maintaining network oscillations that support cognitive function (Turrigiano and Nelson, 2000) Impaired inhibi tory signaling has been implicated in neuropsychiatric disorders such as schizophrenia (Di Cristo, 2007; Gonzalez Burgos and Lewis, 2008; Lewis and Gonzalez Burgos, 2008; Goto et al., 2009; Yoon et al., 2010; Braakman et

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30 al., 2011; Gonzalez Burgos et al., 2011; Yizhar et al., 2011; Rowland et al., 2013) autism (Kubas et al., 2012) insomnia (Morgan et al., 2012) and panic disorder (Long et al., 2013) Experimental disruption of GABAergic signaling can reproduce some forms of executive function impairment t hat occur in normal aging (Stefani et al., 2003; Korotkova et al., 2010; Enomoto et al., 2011; Murray et al., 2011) D espite these findings, surprisingly little is known about GABAergic alterations in the hippocampus and prefrontal cortex that accompany no rmal aging and whether such changes contribute to age associated impairments in spatial memory and working memory. In hippocampal and prefrontal cortical interneurons, GABA is synthesized from glutamate with the GABA synthesizing enzyme glutamic acid deca rboxylase (GAD). GABA is transported to the membrane by the vesicular GABA transporter (VGAT). At the synapse, GABA is released and binds to ionotropic GABA(A) receptors which mediate fast inhibitory synaptic neurotransmission and to metabotropic GABA(B) receptors which are localized primarily extrasynaptically and mediate tonic inhibition (Pinard et al., 2010) Notably, an important regulator of GABA(B) receptor occupancy is the neuronal GABA transporter, GAT 1. This transporter is localized near GABA( B)Rs and its exp ression and activity contribute to how much GABA is available extrasynaptically to act upon GABA(B) receptors (Vitellaro Zuccarello et al., 2003; Conti et al., 2004; Gonzalez Burgos et al., 2009) (Figure 1 4 ). Activation of GABA(A) and GAB A(B) receptors by GABA results in hyperpolarization of the postsynaptic membrane and has an inhibitory effect on mature postsynaptic neurons (Druga, 2009). The net effect of this process is suppression and modulation of principal neuronal activity (Cherub ini and Conti, 2001)

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31 Throughout the brain, a common feature is the integration of excitatory and inhibitory circuitry to facilitate information processing. How these excitatory and inhibitory neuronal populations are organized is unique to specific regio ns of the brain. The hippocampus and the prefrontal cortex are comprised primarily of glutamatergic principal neurons. In addition to these excitatory cells, 20 30% of the cells in the hippocampus and prefrontal cortex are inhibitory interneur ons that sy nthesize GABA (Druga, 2009). The hippocampal formation consists of the subiculum, the dentate gyrus and the hippocampus proper which can be further divided into the three regions, the CA1, CA2 and CA3. Connectivity between these regions is largely unidir ectional (Witter and Amaral, 2004). In CA1, CA2, and CA3 areas of the hippocampus, the principal cellular layer consists primarily of excitatory glutamatergic pyramidal cells with extensive dendritic and axonal arborizations. Within the pyramidal cell la yer, there are also inhibitory GABAergic basket cells whose axons extend transversely innervating the cell bodies of the hippocampal pyramidal cells by forming a basket like plexus (Freund and Buzsaki, 1996). These GABAergic basket cells are heterogeneous in nature with varying cell shapes and sizes. In addition to the basket cells in the pyramidal cell layer, there is a diverse population of GABAergic interneurons in the cell strata above and below the pyramidal cell layer that make up the local interneu ron circuits of the hippocampus (Witter and Amaral, 2004). Some of these cells predominantly or exclusively innervate the dendritic tree of pyramidal cells as well as other interneurons (Freund and Buzsaki, 1996). While hippocampal interneurons are GABA ergic and contain the GABA synthesizing enzyme glutamic acid decarboxylase (GAD), interneurons can be subclassified by the calcium binding proteins and neuropeptides

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32 they produce. Hippocampal interneurons represent a diverse group of cells that vary in sh ape, morphology, connectivity and functional properties (Kullmann, 2011 ; Maccaferri and Lacaille, 2003 ). GABAergic interneurons in the hippocampus project locally onto principal cells as well as onto other interneurons forming multiple contacts with their postsynaptic targets (Fruend amd Gulyas, 1997) These interneurons provide intrinsic inhibitory signaling in the h ippocampus that is crucial for regulating firing activity of principal cells (Freund and Buzsaki, 1996; Freund and Gulyas, 1997; Wang et al. 2004) The principal cells of the hippocampus also receive inhibitory input from extrinsic sources, regions of the brain whose GABAergic neurons project to the h ippocampus The basal forebrain provides GABAergic inner vation to the hippocampus via the s eptohippocampal pathway. This projection system has been heavily implicated in hippocampal dependent cognition, particularly spatial memory (Albert, 1993; Sarter and Bruno, 2002; Fisk and Sharp, 2004; Colom et al., 2006) The basal forebrain is made up of heterogeneous structures of the telencephalon on the medial and ventral aspects of the cerebral hemisphere that provide critical projections to the cortex and medial temporal lobe (Risold, 2004; Colom et al., 2006) The septohippocampal pathway originate s in the medial septum (MS) and vertical (vDB) and horizontal (hDB) limbs of the diagonal band of Broca r egions of the rostral basal for ebrain (BF) providing subcortical cholinergic, GABAergic and glutamatergic innervation of the hippocampus v ia the fimbri a fornix (Figure 1 5 ). Estimates indicate 30 50% of neurons from MS nuclei and 50 75% neurons from DB nuclei that innervate the hippocampus are cholinergic, with GABAergic neurons comprising a large portion of the remaining

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33 projection (Witter and Amaral, 2004) GABAergic basal forebrain projection neurons primarily innervate interneurons of the hippocampus potentially disinhibiting hippocampal pyramidal cells (Freund and Antal, 1988; Toth et al., 1997) The septohippocampal pathway is crucial for normal s patial learning and fimbria fornix lesions that disrupt this pathway result in impaired spatial learning performance in young rats (Becker et al., 1980; McDonald and White, 1994; Everitt and Robbins, 1997; Parent and Baxter, 2004) In fact, many of the e arly lesion studies conducted to investigate the role hippocampal systems in water maze performance included rats with fimbria fornix lesions (Olton et al., 1979) While fimbria fornix lesions impair performance on the water maze, s elective immunotoxic le sions of basal forebrain cholinergic and GABAergic neurons in young animals did not result in spatial memory deficits (Baxter et al., 1995; Pang et al., 2001) These GABAergic cells, however, are not well characterized in relation to aging nor the cognitive deficits ass ociated with the aging process. In the prefrontal cortex, a distinction can be made between the medial prefrontal cortex (m PFC) and orbitofrontal cortex (O FC) based on connectivity (Kolb and Cioe, 2004). As mentioned earlier, the mPFC is the rodent homolog of the primate dorsolateral prefrontal cortex. It consists of the anterior cingulate cortex, the prelimbic cort ex and the infralimic cortex. The medial prefrontal cortex consists of 6 cell layers although they are not as defined in the ra t mPFC as in primate cortex. The principal cells in the prefrontal cortex are excitatory glutamatergic pyramidal cells. As in the hippocampus, in addition to these cells, 20 percent of cells in the medial prefrontal cortex are inhibitory interneurons (M einecke and Peters, 1987) Prefrontal cortical

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34 interneurons make inhibitory contacts with principal cells and other interneurons. These neurons are incredibly diverse displaying varying morphologies, intrinsic electophysiological properties, synaptic cha racteristics and protein expression patterns (Kawaguchi and Kubota, 1998; Kawaguchi and Kondo, 2002; Isaacson and Scanziani, 2011) Electrophysiological studies in awake primates first provided evidence for the crucial role played by cells in the prefronta l cortex in mediating working memory during performance on a delayed response task. In these studies, cells began firing when a cue was presented and some of those cells persisted during the delay phase, when the cue was no longer in the environment. Cell firing during the delay phase was directly related to performance on the task. GABAergic interneurons provide lateral inhibition needed for the spatial tuning of pyramidal cells in the prefrontal cortex (Rao et al., 1999; Constantinidis and Goldman Rakic, 2002) GABAergic Systems in Aging T o date, cognitive aging research has focused primarily on excitatory neurons that synthesize acetylcholine (ACh) This is largely due to the fact that cholinergic neurons are significantly reduced in the age related di a disease whose hallmark symptom is cognitive decline. Stereological studies have shown that there is no gross neuronal loss associated with normal non pathological aging and that cholinergic and principal cell populations are preserved in aging across species (Calhoun et al., 1998; Rapp and Gallagher, 1996; Rasmussen et al., 1996; Rapp, 1995; Peters et al., 1996). To date, m ost currently available treatments for age related cognitive decli ne target excitatory circuitry a nd while p atients with mild cognitive impa irment show improved performance on explicit memory tasks, this therapeutic approach appears to be effective for a relative short time after initiation of treatment

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35 (Jacqueline and Leon, 2006; Raschetti et al., 20 07) As such there is a great need to develop novel therapeutic approaches to treating age related cognitive decline. Targeting GABAergic systems may be such an approach. GABAergic interneurons are well positioned to critically mediate and support hi ppocampal and prefrontal cortical dependent cognition, the same cognitive functions that are known to decline at advance age. Very little is known about how GABAergic systems in the hippocampus and the prefrontal cortex change with age or whether targetin g these systems can improve long term memory or working memory. Hippocampal systems an d the cerebral cortex undergo extensive transformation in aging (Risold, 2004; Allard et al., 2012) In the hippocampus, n ormal aging is not associated with widespread cell loss in the rat (Pugnaloni et al., 1998) or human hippocampus and cortex (Terry et al., 1987; Haug and Eggers, 1991) however, there is abundant evidence that s ignificant changes occur in aging in these regions including neuronal atrophy (Allard et al ., 2012) dendritic regression (de Brabander et al., 1998) and synaptic loss (Wong et al., 2000; Jacobs et al., 2001; Uylings and de Brabander, 2002; Duan et al., 2003) Ther e is clear evidence that aging a ffects how information is processed by the hippo campus Studies in aged F344 rats have demonstrated that GABAergic interneurons in the hippocampus are susceptible to age related alterations. Glutamic acid decarbozylse (GAD), the rate limiting enzyme for GABA synthesis, has been used as a reliable mark er for inhibitory interneurons (Esclapez, 1994; Dupuy and Houser, 1996; Fukada et al ., 1998; Shetty and Turner 1998), and GAD immunoreactive cells are distributed throughout the rodent hippocampus (Babb et al ., 1988; Woodson et al., 1989). Decreases in th e number of interneurons expressing GAD67 and certain

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36 calcium binding proteins in aged rats have been reported (Shetty and Turner, 1998). This reduction of neurons expressing GABAergic markers is not associated with the degeneration or loss of interneuron s (Stanley and Shetty, 2004). Hippocampal synaptic marker intensity has been correlated to spatial learning performance in aged animals such that worse performance is associated with regionally specific synaptic marker intensity reduction (Smith et al., 2 000) There are a number of studies that demonstrate an inc reased excitability of principal cells in the hippocampus and loss of functional inhibition is associated with learning and memory dysfunction reported in aging rodent models (Potier et al ., 1992 ; F rick et al ., 2002; Vela et al., 2003) Age related alterations of GABAergic septohippcampal projections could significantly impact hippocampal dependent spatial memory at advanced age, yet, this component of the projection system has not been characterize d in the context of cognitive aging. Numerous studies have reported a decrease in prefrontal cortical volume in aged humans, non human primates and rodents (Peters et al., 1998) Cell counting studies in humans and primates have shown that, as seen in the hippocampus, total cell numbers are preserved in aging (Huag et al., 1981; Terry et al., 1987, Huag and Eggers, 1991; Peters et al., 1996). There is, however, evidence of significant age related changes in neuronal morphology including apical dendritic r egression, loss of synapses and a decrease in spine number across species (Peters et al., 1996; Jacobs et al., 1997; Markham and Juraska, 2002; Dickstein et al., 2007; Peters et al., 2008; Dumitriu et al., 2010; Bloss et al., 2013) Previously, our labora tory repor ted that expression of GABA(B) receptors is preserved in hippocampus and reduced in aged prefrontal cortex (McQuail et al., 2012) of rats characterized in the hippocampal

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37 dependent Morris water maze. The relationship between the reduction in pre frontal cortical receptor expression and working memory was not evaluated. V ery little is known about age re lated changes to the GABAergic s ystem in the prefrontal cortex and whether targeting this system could improve working memory in aging. T he balance between excitation and inhibition is critical for optimal cognitive performance and the inhibition generated in cortical networks is proportional to local and incoming excitation. During spontaneous cortical activity, increases in excitation results in i ncreases in inhibition (Haider et al., 2006; Okun and Lampl, 2008; Atallah and Scanziani, 2009) Changes in the magnitude of excitation or inhibition elicit compensatory effects that preserve the excitability of cortical networks emphasizing the importanc e of homeostasis (Turrigiano and Nelson, 2000) Emerging literature has demonstrated that, in aging, significant changes in both excitatory and inhibitory synaptic substrates may be relat ed to cognitive impairments (Luebke et al., 2004; Bories et al., 201 3) Experimental Goals The overarching goal of this dissertation is to begin to characterize age related changes to GABAergic systems in the basal forebrain, hippocampus and prefrontal cortex and to assess the how these changes relate to cognitive decli ne in a rodent model of aging. Specifically experiments in this dissertation will explore whether targeting GABAergic systems will improve cognition in aged rats. The main objectives of this r esearch project are as follows: Objective 1: To characterize changes in GABAergic signaling p roteins in the hippocampus of young and aged F344 rats and to determine if the number of basal forebrain projecting cholinergic and GABAergic neurons change as a function of age and/or cognitive impairment in spatially char acterized young and aged Fischer 344 rats.

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38 GABAergic interneurons in the hippocampus critically regulate firing activity of pyramidal cells. A vast literature describes age related alterations in GABAergic systems in the hippocampus. The basal forebrai n is an integral part of a complex circuitry that controls cognitive functions such as emotions, learning, and memory in the mammalian brain. Both cholinergic and GABAergic projections from the rostral basal forebrain have been implicated in hippocampal function and mnemonic abilities. While dysfunction of cholinergic neurons has been heavily implicated in age related memory decline, significantly less is known regarding how age related changes in co distributed GABAergic projection neurons contribute to a decline in hippocampal dependent spatial learning. In the current study, GABAergic signaling protein (GABA(B)Rs, GAT 1, and VGAT) expression in the hippocampus of young and aged rats that were first characteri zed on a spatial learning task was assessed using western blot. confocal stereology was used to quantify cholinergic (choline acetyltransferase (ChAT) immunopositive) neurons, GABAergic projection (glutamic decarboxylase 67 (GAD67) immunopositive) neurons, and total (NeuN immunopositive) neurons in the rostral basal forebrain ChAT immunopositive neurons were significantly but modestly reduced in aged rats. Although ChAT immunopositive neuron number was strongly correlated with spatial learning abilities among young rats, the reduction of ChAT immu nopositive neurons was not associated with impaired spatial learning in aged rats. In contrast, the number of GAD67 immunopositive neurons was robustly and selectively elevated in aged rats that exhibited impaired spatial learning. Interestingly, the total number of rostral basal forebrain neurons was comparable in young and aged rats, regardless of their cognitive status. In the hippocampus expression of GABA(B) receptors, the GABA

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39 tranporter GAT 1 and the vesicular GABA transporter, VGAT, did not change w ith age or cognitive status. Interestingly, GAD67 was significantly reduced in the hippocampus of aged spatially impaired rats compared to young and aged spatially unimpaired counterparts. These data demonstrate differential effects of age on phenotypical ly distinct rostral basal forebrain projection neurons, and implicate dysregulated cholinergic and GABAergic septohippocampal circuitry in age related mnemonic decline. Objective 2: To characterize changes in GABAergic signaling proteins in the prefrontal cortex of young and aged F344 rats and how these changes relate to performance on a test of PFC mediated working memory. Impairments in working memory functions supported by the prefrontal cortex (PFC) are a common feature of normal aging. Working memo ry critically involves GABAergic signaling in PFC; yet, surprisingly little is known about GABAergic alterations in PFC normal aging or whether such changes contribute to age associated impairments in working memory. To investigate this, young adult and ag ed male F344 rats were characterized on an operant based delayed response test of working memory. Aged rats performed comparably to young at no delay but exhibited deficits relative to young at long delays. At the completion of behavioral testing, western blotting was used to assess several proteins associated with GABA signaling in homogenates prepared from dissected prefrontal cortex. Immunoblots of PFC homogenates showed that the GABA synthesizing enzyme GAD67 was increased but the transporter important for reuptake of GABA after synaptic release, GAT 1, was decreased in aged PFC. GABA(B) receptor (GABA(B)R) expression was also reduced in aged PFC. Among aged rats, expression of GAD and GAT 1 was not associated with working memory performance. In contra st, GABA(B)R expression was significantly and negatively

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40 associated with working memory such that lower GABA(B)R expression predicted better performance among aged rats. Together, the increase in GAD and decrease in GAT 1 expression suggest that aging is a ccompanied by increased GABA availability and increased tonic inhibition within the prefrontal cortex. Furthermore, expression of GABA(B) receptors was significantly related to working memory ability in aged rats such that those aged rats that performed w ell on the working memory task had the lowest levels of GABA(B) receptor expression. This inverse relation between working memory performance and GABA(B) receptor expression suggests that low levels of GABA(B) receptors may be a successful compensation fo r increased extracellular GABA availability in the aged PFC and that blocking GABA(B) receptor activation may improve working memory performa nce in aged rats. To test this we evaluated whether pharmacological blockade of GABA(B)Rs could improve working m emory performance in aged rats. One cohort of young and aged rats received intraperitoneal injections of the GABA(B)R antagonist CGP55845 (0.1 mg/kg or .01 mg/kg) or vehicle prior to test sessions, using a within subjects design such that each rat received both drug conditions. Performance of aged rats was significantly improved by CGP55845. To determine whether the behavioral improvements observed with systemic CGP55845 were mediated by drug actions in the prefrontal cortex, an additional experiment was c onducted in which young and aged rats received microinjections of CGP55845 or vehicle directly into medial prefrontal cortex prior to testing. These intra cerebral microinjections restored performance of aged rats to a level comparable to young adults. Re sults from these experiments demonstrate that targeting GABA(B)Rs may provide therapeutic benefit for age related impairments in executive functions.

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41 Together, these data suggest that age related dysregulation of GABAergic signaling in prefrontal cortex m ay play a causal role in impaired working memory and that targeting GABA(B)Rs may provide therapeutic benefit for age related impairments in executive functions

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42 Figure 1 1 Morris water maze performance in young rats across training a nd probe trials. A) Illustration of the Morris Water Maze used in the experiments described in this dissertation. B) Young rats reliably learn the location of the escape platform across training. C) This is observed during probe trials, as well.

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43 Figure 1 2 Spatial memory performance of young and aged rats. A) Both young and aged rats learn the location of the hidden platform over time, however, aged rats are less proficient at learning the platform location compared to young rats. B) Aged rats, reliably, show a tremendous variability in spatial memory.

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44 Figure 1 3 Delayed response task performance in young and aged rats. A ) shows a schematic of the delayed response task used to asses working memory ability. There are thre e phases to this task. During the sample phase rats are presented with either a left or right lever. After the rat presses the extended lever, the delay phase begins, during which both levers are retracted for a variable time period ranging from 0 to 24 s econds, during which the rat must nosepoke into the food trough to initiate the choice phase. During the choice phase, both levers are presented and the rat must choose the lever presented in the sample phase in order to obtain a food reward. B ) shows youn g and aged performance on the delayed response task. Aged rats displayed significantly less accurate performance relative to young and were disproportionately impaired at lo ng delays C) shows individual young and aged rats plotted by Mean Long Delay (ave rage of choice accuracy at 18 24 s) on the delayed response task. This measure was used as an index of individual working memory ability.

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45 Figure 1 4 Schematic of GABAergic synapse in the hippocampus and prefrontal cortex. Modified with per mission from Haley Carpenter.

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46 Figure 1 5 Basal forebrain projections to the hippocampus and prefrontal cortex.

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47 CHAPTER 2 AGE RELATED CHANGES IN SEPTOHIPPOCAMPAL GABAERGIC SYSTEMS: RELATIONSHIP WITH SPATIAL IMPAIRMENT Introduction Explicit an d spatial memory in humans is dependent upon the hippocampus and medial temporal lobe system ; the function of which can decline precipitously with advanced age (Della Maggiore et al., 2002; Squire, 2004; Wilson et al., 2004; Burke and Barnes, 2006) While aged individuals can exhibit learning and memory dysfunction similar to individuals with direct hippocampal damage (Gallagher and Rapp, 1997) neuron number in medial temporal lobe structures is stable across species in normal aging, even in subjects with profound mnemonic impairment s (Rapp and Gallagher, 1996; Rasmussen et al., 1996; Calhoun et al., 1998; Rapp et al., 2002; West et al., 2004; Shamy et al., 2006) Despite the absence of frank neuron loss in the hippocampus, there is clear evidence that agin g can negatively impact the processing of hippocampal dependent spatial information (Barnes et al., 1997; Shen et al., 1997; Tanila et al., 1997) and that altered integrity of both cholinergic and GABAergic basal forebrain afferents to hippocampus may cont ribute to this functional decline (Gage et al., 1984; Gallagher and Nicolle, 1993; Smith and Pang, 2005; Ypsilanti et al., 2008) Given their vulnerability to degeneration in basal for ebrain cholinergic neurons have been studied extensively within the context of aging and cognitive decline. Some studies have reported that cholinergic basal forebrain neurons decline with age, supporting the notion that their degeneration mediates age related spatial learning impairments (Bartus et al., 1982; Fischer et al., 1989; Altavista et al., 1990; Durkin, 1992; Fischer et al., 1992; Armstrong et al., 1993; De Lacalle et al., 1996; Gustilo et al., 1999; Fadda et al., 2000; Fragkouli et al., 2005) Notably, however,

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48 others have found no relation ship between decline in cholinergic cell number and loss of cognitive abilities, and several recent studies have reported that cholinergic neuron number remains relatively stable at advanced ages (Lee et al., 1994; Ypsilanti et al., 2008; McQuail et al., 2 011) The latter findings are consistent with those from studies showing that hippocampal dependent spatial memory is largely spared following selective neurotoxic ablation of cholinergic neurons in rodents (Baxter et al., 1995) It is becoming increasing ly clear that corticopetal basal forebrain GABAergic neurons influence hippocampal physiology and hippocampal supported cognition (Freund and Antal, 1988; Kiss et al., 1990; Pang et al., 2001) and that the combined influence of cholinergic and GABAergic af ferents is important for optimal hippocampal function. For example, septohippocampal connectivity is critical for the generation of h ippocampal theta rhythms, 3 12 Hz oscillations which are strongly implicated in successful spatial cognition, memory proces ses and sensorimotor integration (Winson, 1978; Rawlins et al., 1979; Bland and Colom, 1993; Bland and Oddie, 2001; Buzsaki, 2002; Colom, 2006) Lesion studies have shown that both cholinergic and GABAergic afferents from rostral basal forebrain neurons ar e critically important for generating these oscillations (Yoder and Pang, 2005) In addition, pronounced spatial learning impairments are produced by disruption of both cholinergic and GABAergic input to the hippocampus but not by the disruption of either projection system in isolation (Becker et al., 1980; McDonald and White, 1994; Baxter et al., 1995; Everitt and Robbins, 1997; Pang et al., 2001; Parent and Baxter, 2004) Alterations in inhibitory circuitry occur in a variety of neurodegenerative and neu ropsychiatric diseases such as epilepsy, depression, schizophrenia, and autism,

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49 many of which are associated with abnormal cognitive function (Lewis et al., 2005; Briggs and Galanopoulou, 2011; Gonzalez Burgos et al., 2011; Pizzarelli and Cherubini, 2011) Indeed, GABA mediated transmission appears crucial for processing information both within and between brain regions essential for mediating a variety of neurocognitive processes (Volk and Lewis, 2002; Bartos et al., 2007) While inhibitory circuitry is increasingly the focus of mechanistic studies associated with neurodegenerative diseases, considerably less attention has been paid to the anatomical integrity of the GABAergic inhibitory circuits in normal aging (McKinney, 2005) Nevertheless, there is em erging evidence that GABAergic indices change in normal aging. For example, interneurons in both prefrontal cortex and hippocamp us of aged rats degenerate or cease to express glutamic acid decarboxylase (GAD) 67, the GABA synthesizing enzyme (Shetty and Tu rner, 1998; Stanley and Shetty, 2004; Stranahan et al., 2012) Moreover, normal aging produces regionally specific changes in GABA(B) receptor expression and function (McQuail et al., 2012) and attenuated GABA(A) receptor activity and expression (Yu et al. 2006) Evoked GABA release is also reportedly decreased in the CA1 subregion of the aged rat hippocampus (Stanley et al., 2012) Finally, drugs targeting GABAergic signaling can improve cognitive functioning in both young and aged rats (Mondadori et al., 1996a; Mondadori et al., 1996b; Getova and Bowery, 1998; Getova and Bowery, 2001; Helm et al., 2005; Lasarge et al., 2009) This study was designed to first assess GABAergic signaling proteins in the hippocampus and then to determine if changes in the i ntegrity of hippocampal targeting

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50 basal forebrain cholinergic and GABAergic neurons are associated with loss of spatial learning abilities in aging. Western blotting was used to quantify the expression of GABAergic signaling proteins in the hippocampus of young and aged rats which were first characterized on a spatial learning task. Confocal and stereological methods were employed to determine cholinergic (ChAT immunopositive), GABAergic projection (GAD67 immunopositive) and total neuron (NeuN immunoposit ive) numbers in rostral basal forebrain of second cohort of youn g and aged rats which were also characterized on a spatial learning task. The findings indicate that GAD67 expression is selectively reduced in rats that exhibit spatial impairment and that t he aging process differentially impacts cholinergic and GABAergic projection neurons in rostral basal forebrain suggest ing that alterations in inhibitory networks may be important contributors to age related mnemonic dysfunction. Methods Subjects Young adu lt (6 months) and aged (24 months ) male F344 rats were obtained from the Nation al Institute on Aging colony. Rats used in experiments assessing basal forebrain projection neuron numbers (young n=8; aged n=16) were housed in the vivarium in the Psychology Building at Texas A&M University for two weeks prior to the st art of behavioral testing. Rats used in experiments assessing hippocampal GABAergic signaling protein expression ( young n=7 ; aged n=11) were housed in a vivarium facility in the McKnight Brain Institute at University of Florida two weeks prior to the start of behavioral testing. These AALAC accredited vivarium s were maintained at a consistent 25 C with a 12:12 hour light/dark cycle (lights on at 0800 hours). Rats were maintained under specifi c pathogen free conditions and had free access to food

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51 and water at all times. All rats in the study were screened for health problems including, but not limited to, cataracts, jaundice, food and water intake, and tumors. Sentinel rats, housed alongside the rats in this study, routinely tested negative for a range of pathogens. All animal procedures were conducted in accordance with approved institutional animal care procedures and National Institutes of Health guidelines. A total of 42 rats were used in this study (young n=15, aged n=27). Experiment 1: GABA signaling Protein Expression and Spatial Memory Abilities Water Maze Task Procedures Apparatus Spatial learning abilities were assessed using the Morris water maze task as described previous ly (Bizo n, et al., 2009; LaSarge, 2007) The water maze apparatus consisted of a white circular tank ( 183 cm in diameter with a wall height of 58 cm ) filled with water (27C) made opaque with the addition of non toxic white tempera paint. A retractable white esca pe platform (12 cm diameter, HVS Image, UK) was submerged 2 Black curtains, to which white geometric shapes (extramaze cues) were affixed large, surrounded the maze. Data were acquired via a video camera mounted above the maze which was connected to a DVD recorder and computer with a video tracking system and Water 2020 software (HVS Image, Buckingham, UK). Spatial reference memory (hidden platform) task ning abilities were tested ac cording to methods developed by Gallagher and colleagues (Gallagher et al., 1993) with specific modifications for training F344 rats (Bizon, et al., 2009; LaSarge, 2007) Briefly, rats received three training trials /day with a 30 s inter trial interval over eight consecutive days. On each trial, rats

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52 were placed into the water facing the wall of the maze at one of four equally spaced start positions (north, south, east, or west). The start positions were varied in a pseudo r andom fashion, such that all rats started from each of the locations approximately the same number of times. Rats were allowed to search until they found the hidden platform or until 90 s elapsed, at which time rats were guided to the escape platform by t he experimenter. Rats remained on the platform for 30 s and then were placed in a holding chamber for a 30 s inter trial interval Every sixth trial was a probe trial in which the platform was lowered to the bottom of the maze for the first 30 s of the t rial, after which it was raised to allow the rats to escape. Cued (visible platform) task Following spatial reference memory training, rats were given a single session with six trials of cue training to assess sensorimotor abilities and motivation to esc ape For cue training, rats were trained to escape to a visible platform (painted black and were varied on each trial, making the extra maze cues explicitly irreleva nt to the platform location. On each trial, rats were allowed to search for the platform for 90 s and then were allowed to remain there for 30 s before a 30 s inter trial interval. Statistical analyses D ata files were created by the Water 2020 software an d exported to SPSS (v. 16.0 ; Cary, NC) for analysis. Accuracy of performance on training and probe trials was assessed using a search error measure originally described by Gallagher et al. (1993). tform location was sampled 10 times/s and these distances were averaged into 1 s bins. For training trials, cumulative search error was derived by summing these 1 s averages and then subtracting the

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53 optimal path between the start location and the platform location. For probe trials, a mean search error measure was derived by dividing cumulative search error by the 30 s duration of the probe trials. Training trial data were averaged into four blocks consisting of the five trials preceding each probe trial. Comparisons between groups on training trials were conducted using two factor repeated measures ANOVA (age X training In all statistical comparisons, p va 0 .05 were considered significant. probe trials as described previously (Bizon, et al., 2009; Gallagher, et al., 1993) Mean search error on probe trials was weighted and summed to provide the spatial learning index (Bizon, et al., 2009) For some comparisons of cell number, aged rats were subgrouped on the basis of their spatial learn ing index. This classification approach has been successfully used in prior studies to identify and investigate structural and signaling alterations in the hippocampus and related circuitry that are relevant to decline of spatial learning abilities in age d rats (Bizon, et al., 2001; Bizon, et al., 2004; Colombo, et al., 1997; Foster and Kumar, 2007; Nicolle, et al., 1999; Rapp and Gallagher, 1996) Aged rats that fell more than two times the standard deviation outside of the mean spatial learning index cal Western Blotting of GABAergi c Signaling Proteins in the Hippocampus Approximately 2 weeks after completion of behavioral testing, rats were decapitated and brains were removed from the skull, cooled on ice, and sliced into 2

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54 hemispheres on an ice cold plate. The hippocampus wa s dissected from each hemisphere changes in protein expression (Davis et al., 1996; Wass et al., 2013) this two week post training interval was selected to evaluate baseline rather than behaviorally stimulated prote in levels. Protein expression of the GABA(B) receptor R1a, R1b, and R2 subunits, the primarily neuronal GABA transporter GAT 1, the GABA synthesizing enzyme GAD67, and the vesicular GABA transporter VGAT was assessed in the hippocampus of young and aged F3 44 rats Sample Preparation Frozen tissue was weighed, thawed, and homogenized in 10 volumes of an ice cold buffer (50 mM 4 (2 Hydroxyethyl)piperazine 1 ethanesulfonic acid, N (2 Hydroxyethyl)piperazine (2 ethanesulfonic acid) (HEPES), pH 7.4, 1 mM ethylenediaminetetraacetic acid and 1 mM ethylene glycol bis(2 aminoethylether) N,N,N tetraacetic acid and protease inhibitors; Roche, Mannheim, Germany) using a glass Teflon Dounce homogenizer. Homogenates were centrifuged at 14,000 rpm for 20 minutes at 4 C. The supernatant was collected and stored in aliquots for western blotting a ssays of non membrane bound proteins. The pellet was resuspended in 20 mL of the same buffer without protease inhibitors and incubated on ice for 30 minutes followed by centrifugation at 16,500 rpm for 15 minutes at 4 C. This pellet was resuspended in 10 C until used for Western blotting assays. Protein concentration was determined using the Pierce BCA Kit according to the manufacturer's protocol (Rockford, IL, USA).

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55 Immunoblotting P roteins w mercaptoethanol (Fisher, Pittsburgh, PA, USA) and heated at 95 C for 5 minutes. Initial experiments focused on evaluating expression of PFC GABA(B) receptor R1 and R2 subunits a s well as GAT 1 T en micrograms of protein per lane were electrophoretically separated on a 4% 15% Tris HCl gel at 200 V for 3 5 minutes then transferred to nitrocel lulose membranes using a wet transfer apparatus for 90 minutes at 0.35A Blots were washed 3 times with tris buffered saline (TBS; pH 7.4) then blocked for 1 hour in blocking buffer (Rockland, Gilbertsville, PA, USA). Blots were then incubated overnigh t at 4 C with antibodies [anti GAD67 (Millipore Temecula, CA ), a nti GAT 1 (Millipore), anti VGAT (Mi llipore) anti GABA(B)R1 (Cell Signaling Technology, Beverly, MA, USA) anti GABA(B)R2 (Cell Signal ing Technology ) diluted 1:1000 in blocking buffer (Rockland, Gilbertsville, PA, USA) with 0.1% Tween 20 (Bio Rad Hercules, CA, USA). Blots were then washed 3 times with 0.1 M Tris Buffer Solution (TBS) and incubated with the appropriate AlexaFluor 680 conjugated anti IgG (Invitrogen, Carlsbad, CA, USA) diluted 1:20,000 in TBS with 0.1% Tween 20 (Bio Rad) for 1 h. Following 3 additional TBS washes, blots were scanned on an Odyssey imaging system (LI COR Biosciences, Lincoln, NE, USA). Statistical Analysis I ntegrated protein density was measured for each band and the individual values of both young and aged samples were normalized to the mean expression of youn g samples run on the same gel. Age comparisons of protein expression were conducted using i ndependent t tests and one way ANOVAs were used to compare cognitive group protein expression. Note that each blot was probed separately for g lyceraldehyde 3

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56 phospha te dehydrogenase ( GAPDH ) expression to confirm that age effects were not a result of non specific effects of loading or quantification of protein content. In no cases were age comparisons performed on GAPDH measures significant and as such, these data are not reported individually To directly test relationships between changes in protein expression and spatial memory abilities among aged rats, spatial learning index was calculated for each subject as described above. The r elationships between expression of each protein of interest with whose age or cognitive group comparisons yielded a significant difference and spatial learning index were tested using correlations. For this and all subsequent experiments, data are presented as the mean standard error of the mean. All statistical analyses were conducted using SPSS 21.0 (Cary, NC, USA) and GraphPadPrism (La Jolla, California). For all statistical comparisons, values of p < 0.05 were considered significant. Experiment 2: Stereological Quantifi cation of Rostral Basal Forebrain Cholinergic, GABAergic and Total Neurons Immunofluorescent Labeling of Cholinergic, GABAergic Projection and Total Rostral Basal Forebrain N eurons Rats were trained on the water maze task a s described for Experiment 1. One week after completion of behavioral testing, rats were rapidly euthanized with an overdose of pentobarbital and perfused transcardially with ice cold 0.9% saline followed by 4% paraformaldehyde. Brains were removed from the skull, postfixed for 24h in perfusate and then cryoprotected in 20% sucrose in 0.1M phosphate buffer. Systematic uniform random sampling was achieved by exhaustively sectioning brains coronally on the full rostro caudal extent of the medial septum and vertical limb of the diagonal band of Broca (beginning just caudal to the olfactory bulbs and ending caudal to the crossing of the anterior

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57 commissure). A registered 1 in 4 series of sections was obta ined for each animal and separate series (spaced at 140 m intervals) were randomly assigned for processing to detect rostral basal forebrain cholinergic (ChAT), GABAergic projection (GAD67) or total (NeuN) immunopositive neurons (Gundersen and Jensen, 1987) Sections were collected into cold 0.1M PBS and store d at 4 o C until stained immunohistochemically. For immunohistochemistry, free floating sections were washed several times in 0.1M Tris buffered saline (TBS; 100 mM Tris HCL, 150 mM NaCl, pH 7.5), pre incubated in a blocking solution containing 3% normal do nkey serum (NDS) and 0.3% Triton X 100 in 0.1M TBS for 1 hour at room temperature and then incubated in blocking solution that contained rabbit anti GAD67 (Bioworld, 1:500); goat anti ChAT (Millipore AB144P, 1:1000) or mouse anti NeuN (Millipore MAB377, 1: 500) for 72 hours at 4 o C. After primary incubation, sections were washed in 0.1M TBS, and incubated in 0.1M TBS containing 2% NDS and the appropriate Alexa 488 conjugated secondary antibodies (Molecular Probes, 1:300) for 2 hours at room temperature in the dark. The sections were washed in 0.1M TBS, and mounted onto Superfrost++ slides (Fisher Scientific). The sections were then coverslipped under ProLong Gold (Invitrogen), sealed with clear fingernail polish, and stored at 4C until analysis. Stereologica l Counts of ChAT, GAD67, and NeuN Immunopositive N eurons Delineation of the rostral basal forebrain The basal forebrain neurons that innervate the hippocampal formation are primarily localized within the medial septum (MS) and the vertical limb of the di agonal band of Broca (vDB; Dutar, et al., 1995; Lewis and Shute, 1967; McKinney, et al., 1983; Meibach and Siegel, 1977; Segal and Landis, 1974; Swanson and Cowan, 1979) As shown in Figure. 2 1 these are contiguous nuclei that emerge along the midline ju st

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58 caudal to the joining of the corpus callosum. The vDB is positioned ventral to the MS and these nuclei are bordered laterally by the lateral septum and the medial edge of the nucleus accumbens shell. The vDB extends ventrally to the medial intersection of the two hemispheres on the ventral edge of the tissue section. In more caudal planes, the rostral most portion of the horizontal limb of the diagonal band of Broca (hDB) emerges. Contiguous with the vDB, the hDB extends laterally along the medial edge o f the nucleus accumbens shell and is bordered ventrally by the ventral pallidum and olfactory tubercle. The caudal most edge of the vDB is rostral to the joining of the anterior commissure. After the crossing of the anterior commissure, the hDB (sometimes also referred to as the magnocellular preoptic area in this plane) is a clearly defined nucleus located in the ventral and lateral basal forebrain. As such, the crossing of the anterior commissure is often used as a boundary between rostral basal forebrai n and more caudal neocortical innervating basal forebrain nuclei (Colom, et al., 2005; McQuail, et al., 2011b; Peterson, et al., 1999; Ypsilanti, et al., 2008) In the current study, counts were obtained from equally spaced (140 m apart) sections througho ut the entire rostro caudal extent of the medial septum and vDB. Because there are not clear boundaries that allow the hDB to be reliably distinguished in the rostral basal forebrain, neurons within the hDB rostral to the crossing of the anterior commissur e were also included in the population estimates. The rostral basal forebrain nuclei as a whole can be readily distinguished from surrounding structures (described above) within ChAT, GAD67 and NeuN immun olabeled material (See Figures. 2 2 through 2 4 ).

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59 Estimation of neuron number using the optical fractionator Starting at a randomly selected level within the first sampling interval, the optical fractionator method (Gundersen, 1986; Peterson, 1999; West, et al., 1991) was implemented using an Olympus Fluo view 300 confocal microscope, equipped with the appropriate filter sets, a CCD camera, and a computer driven x, y, and z Ludl motorized stage controlled with StereoInvestigator software (MBF Bioscience, Williston, VT). Regional boundaries for the rostral basal forebrain nuclei (shown in Figure 2 1) were delineated at low power magnification (4X) in an evenly spaced series of immunolabled sections. The series spanned the rostro caudal extent of the MS and vDB, which are the basal forebrain nuclei that inne rvate the hippocampal formation (Dutar, et al., 1995; Lewis and Shute, 1967; McKinney, et al., 1983; Meibach and Siegel, 1977; Segal and Landis, 1974; Swanson and Cowan, 1979) This design yielded 7 8 sections for quantification (per 1 in 4 series) from ea ch brain. The motorized stage of the microscope was moved in evenly spaced x y intervals under the computer control, surveying the regions of interest in each section according to a systematic random sampling scheme (see Table 2 1 for sampling details). U sing a 60X oil immersion objective (with 1.4 numerical aperture), section thickness was measured at each sampling site and z slices) were acquired through the full section thickness at the appropriate emission wavelengths. The mean section thickness was measured at 30.62 m (CV =0.13), indicating an approximate 12.5% tissue shrinkage in the z plane. This degree of shrinkage is significantly less than that observed from immunohistological procedures that require dehydration and i s consistent with other reports of immunofluorescent tissue processing (Hart and

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60 Terenghi, 2004; Prasad and Richfield, 2010) Quantification was performed offline on the acquired z stacks and was confined to an optical disector 25 m in height which was po sitioned 3 m below the surface of the tissue. The top most nucleus associated with an immunopositive neuron was counted only when it first came into focus within the optical disector, provided it did not encroach on the exclusion lines of the counting fr ame (Gundersen, 1986; Sterio, 1984) The sufficiency of the guard zone was confirmed by plotting the distribution of the cells counted for each marker in the z axis (Andersen and Gundersen, 1999; Dorph Petersen, et al., 2009; Dorph Petersen, et al., 2001). The number of cells counted was highly consistent across the disector height, indicating that the guard zone of 3 m was sufficient to minimize the effects of superficial damage associated with tissue sectioning (i.e., to avoid lost caps (Gundersen, 1986) Figure 2 5 ). Moreover, the distribution of cells along the z axis did not differ between age or cognitive grou ps (Gardella, et al., 2003; Figure 2 5 ), confirming good antibody penetration throughout the full section thickness (see representative examples of cell positions within the disector in the orthogonal wind ows shown in Figure 2 5 ). The total number of ChAT GAD67 and NeuN immunopositive cells in rostral basal forebrain was estimated using the optical fractionator method (West, et al., 1991) in which the product of the cells counted in a known, uniformly random sample of the region of interest is multiplied by the reciprocal of the sampling fraction. Additional details, including stereological sampling parameters are provided in Table 2 1. The precision of the stereological estimates was determined by estimating the coefficients of error (CE) using methods d escribed by Gunderson (1999) Equations

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61 m =1, as the areas defined for the cell counts changed smoothly from the rostral entrance of the MS to the caudal conclusion of the vDB. The se CEs (ranging from 0.04 to 0.06) were less than one half of the observed variation across subjects (CVs ranging from 0.16 to 0.2, Table 2 1), indicating that the sampling and counting parameters should be sufficiently precise to detect frank biologically driven differences in neuronal population estimates among experimental groups (Boyce, et al., 2010; Dorph Petersen, et al., 2001; Gundersen and Jensen, 1987; Gundersen and Osterby, 1981; West, 1999). Statistical Analysis Data were imported into SPSS (v 19 .0) for statistical analysis. E stimates of cholinergic (ChAT immunopositive), GABAergic (GAD immunopositive) and total (NeuN immunopositive) rostral basal forebrain neuron numbers were compared between age and cognitive group using independent sample t te sts and one factor ANOVA s with post hoc analyses where appropriate To further test relationships between numbers of phenotypically distinct neurons in rostral basal forebrain and cognitive ability, bivariate correlational analyses were performed for each age group, using i ndividual spatial learning index scores and individual cell counts for each immunomarker. In all statistical comparisons, p values .05 were considered significant

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62 Results Experiment 1: GABA signaling Protein Expression and Spatial Memory Abilities Cognitive Performance in Young and Aged Rats To relate changes in GABAergic signaling protein expression to hippocampal dependent cognitive abilities, young and aged rats were characterized on the water maze task that assesses sp atial memory. In the spatial water maze task, a comparison of performance on training trials using the cumulative search error measure ( two factor ANOVA ; age X trial block) indicated that both young and aged rats improved performance over the course of tra ining trials (main effect of training trial block, F (3,48 ) = 17.6 p <0.0001) but that aged rats were significantly impaired in finding the platform in comparison to young (main effect of age, F (1,16 ) =19.1, p<0.0001, Fig ure 2 6 A). Notably, these differences were not present on the very first training trial ( F (1,16 ) =0. 84 n.s) rats. Performance on probe trials as assessed with the mean search error measure (two factor ANOVA ; age X probe trial) revealed results similar to those on training trials, in that search for the platform became more accurate as training progressed (main effect of probe trial, F (3,48 ) = 12.4, p<0.0001) but aged rats were overall less proficient in their search than young rats (main effect of age, F (1,16 ) 11.0, p<0.05;). In contrast to the spatial task, there was no impairment in the ability of aged rats to locate the visible escape platform during cue (visible platform) training (one factor ANOVA, F (1,16 ) =0.431, n.s.), indicating intact sensorimotor and motivational processes. In order to relate population estimates to cognitive abilities, m ean search error during probe trials was used to calculate a Spatial Learning Index (SLI) for each rat as described above (Bizon, et al., 2009; Gallagher, et al., 1993) This measure, specifically

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63 designed to maximize individual differences in water maze performance with in the context of aging, has been shown to correlate with age related changes in numerous neurobiolo gical substrates of spatial memory (Bizon, et al., 2001a; Bizon, et al., 2004; Colombo, et al., 1997; Nicolle, et al., 1999; Smith, et al., 2000) Figure 2 6B shows individual spatial learning indices of young and aged rats Age and C ognitive Comparisons of GAD67, VGAT, GAT 1, GABA(B)R1a, GABA(B)R1b, and GABA(B)R2, expression in the hippocampus In order to assess the age related changes in GABA mediated inhibition in the hippocampus, expression of signaling proteins that are key components of GABAergic in hibitory synapses in the hippocampus were quantified using immunoblots. We first evaluated levels of the GABA synthesizing enzyme, GAD67 as a means to assess GABA differences in GABA production in young and aged hippocampus. As shown in Figure 2 7A there was no significant difference in GAD67 expression in aged hippocampus compared to young (GAD67: t (16 ) =1.25, p=0.23 ). Interestingly, however, there was a significant difference in GAD67 expression was detected with a one way ANOVA comparison of cognitive groups ( GAD67: F (2,19 ) 4.73, p=0.03 ) Post hoc analysis, revealed that GAD67 expression in aged spatially impaired rats was significantly reduced compared to expression in young and aged spatially unimpaired rats p ost hoc: young vs AI, p=.009; Au v s AI, p=.035 We next evaluated expression of the vesicular GABA transporter, VGAT, in the hippocampus to determine if GABA is transported differ ently in young and aged rats Figure 2 7B shows that there are no differences in VGAT expression between you ng and aged rats (VGAT: t (16 ) =1.03, p=0.33; Figure 2G) or among cognitive groups Next, we assessed expression of the primary neuronal GABA transporter, GAT 1 in young and aged hippocampus. GAT

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64 1 is the primary neuronal transporter in the hippocampus and is responsible for the reuptake of GABA at the synapse. GAT 1 was not significantly reduce d in aged compared to young hippocampus (GAT 1: t (16) = 0.70, p=0.50, Figure 2 7C ) nor was there any difference in GAT 1 expression among cognitive groups(GAT 1: F (2 ,19 ) 0.28, p=0.76 ) GAT 1 has been strongly implicated in occupancy of the metabotropic GABA(B)R (Gonzalez Burgos et al., 2009) GABA(B)Rs are located extrasynaptically are well positioned to influence tonic inhibition in the hippocampus. In order to as sess age related changes in GABA(B)R levels, we also used immunoblotting to quantify expression. As GABA(B)Rs are obligate heterodimers and functional receptors require at least one GABA(B)R1 subunit with one GABA(B)R2 subunit (Jones et al., 1998; Kaupman n et al., 1998; White et al., 1998) hippocampal expression of both R1 and R2 subunits was assessed. In addition, note that two distinct bands were detected for GABA(B)R1 (130kD and 95kD), corresponding to the two different isoforms of this subunit: GABA(B )R1a and GABA(B)R1b, respectively (Kaupmann et al., 1997) The GABA(B)R1a isoform contains a pair of short consensus repeats at the N terminal that traffick GABA(B)R complexes containing this isoform to presynaptic terminals where these receptors modulate neurotransmitter release (Biermann et al., 2010) Conversely, GABA(B)R1b lacks this N terminal extension and is primarily localized to dendrites where it mediates postsynaptic inhibition (Vigot et al., 2006) Given the distinct localization and function of these R1 isoforms, R1a and R1b were analyzed separately. In agreement with previous work from our lab (McQuail et al., 2012), there was no significant difference in expression of both R1 isoforms and of the R2 subunit between

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65 aged and young hippocampus ( R 1a: t (16 ) = 0.80 p=0 .44, Figure 2 7D; R1b: t (16) =0.78, p=0.45 Figure 2 7E ; and GABA(B) R2: t (16) =0.59, p=0.39, Figure 2 7F ). Similarly, there were no significant differences detected with a one way ANOVA comparing cognitive groups ( R1a: F (2,19 ) 0.39, p=0.6 8; R1b: F (2,19 ) 0.35, p=0.71; R2: F (2,19 ) .038, p=0.96 ) In order to determine whether there was a significant relationship between protein expression and water maze performance, bivariate correlational analyses were performed for each age group, using i nd ividual spatial learning index scores and individual expression for each protein. Notably, no significant relationships were observed between GAD67 expression and spatial learning performance as measured by the spatial learning index (GAD67: r= 0.12, n.s; VGAT: r= 0.25, n.s GAT 1: r=21, n.s; GABA(B)R1a: r=.34, n.s; GABA(B)R1b: r=.27, n.s; GABA(B)R2: r=.14, n.s) Experiment 2: Stereological Quantification of Rostral Basal Forebrain Cholinergic, GABAergic and Total Neurons Cognitive Performance in Young a nd Aged Rats In the spatial water maze task, a comparison of performance on training trials using the cumulative search error measure ( two factor ANOVA ; age X trial block) indicated that both young and aged rats improved performance over the course of tra ining trials (main effect of training trial block, F (3,66 ) = 35.8 p <0.0001) but that aged rats were significantly impaired in finding the platform in comparison to young (main effect of age, F (1,22 ) =9.8, p<0.005, Fig ure 2 8A ). Notably, these differences we re not present on the very first training trial ( F (1,2 2 ) =0. 77 n.s) search strategies were comparable to those of young rats. Performance on probe trials as assessed with the mean search error measure (two factor ANOVA; age X probe trial) revealed results similar to those on training trials, in that search for the platform became

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66 more accurate as training progressed (main effect of probe trial, F (3,66 ) = 13.8, p<0.0001) but aged rats were overall less proficient in their s earch than young rats (main effect of age, F (1,2 2 ) 7.34, p<0.05;). In contrast to the spatial task, there was no impairment in the ability of aged rats to locate the visible escape platform during cue (visible platform) training (one factor ANOVA, F (1,22) = 1.4, n.s.), indicating intact sensorimotor and motivational processes. In order to relate population estimates to cognitive abilities, m ean search error during probe trials was used to calculate a Spatial Learning Index (SLI) for each rat as described abov e (Bizon, et al., 2009; Gallagher, et al., 1993) This measure, specifically designed to maximize individual differences in water maze performance with in the context of aging, has been shown to correlate with age related changes in numerous neurobiologica l substrates of spatial memory (Bizon, et al., 2001a; Bizon, et al., 2004; Colombo, et al., 1997; Nicolle, et al., 1999; Smith, et al., 2000) Figure 2 8B shows individual spatial learning indices of young and aged rats For some analyses, aged rats were subgrouped based on their SLI such that a ged rats performing more than two times the standard deviation of the mean SLI of the young group (SLI > 275 ) were c lassified as and all other aged rats (SLI < 275) were c lassified as trial block X cognitive group) confirmed that cumulative search error across training blocks differed between these subgroups (F (2, 21) = analyses ind icated that aged spatially impaired rats performed significantly worse than both young and aged spatially unimpaired rats (ps<0.05); however, performance of young and aged spatially unimpaired rats did not differ (n.s.).

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67 Distribution of Ch AT GAD67 and Ne uN I mmunopositive N eurons Figures. 2 2 through 2 4 show low power photomicrographs of representati ve immunolabeling for ChAT (Figure 2 2), GAD67 (Figure 2 3) and NeuN (Figure 2 4) in independent series of sections through the rostral basal forebrain of r epresentative young, aged spatially unimpaired and aged spatially impaired rats. Both cholinergic (ChAT im munopositive; Figure 2 2 ) and GABAergic project ion (GAD67 immunopositive, Figure 2 3) neurons were distributed heterogeneously throughout the rostral basal forebrain nuclei and represented clear subpopulations of those neur ons immunolabeled for NeuN (Figure 2 4). For each label and across age and cognitive groups, the rostral basal forebrain nuclei could be readily distinguished from bordering structure s (including the lateral septum, nucleus accumbens, and olfactory tubercle). High magnification immunofluorescent labeling of ChAT, GAD67 and NeuN immunopos itive neurons is shown in Figure 2 9 ChAT immunopositive cells (Fig 2 9A 2 9 C) were robustly lab eled with well defined nuclei and tended to be polygonal and fusiform in shape. Overall, ChAT immunopositive neurons appeared larger but less densely distributed than G AD67 immunopositive cells (Fig 2 9D 2 9 F) in the same region. In agreement with previous reports (Brashear, et al., 1986; Colom, 2006; Gritti, et al., 2006; Gritti, et al., 2003) the GAD67 immunopositive cells exhibited diverse morphologies (oval, fusiform or polygonal cell bodies were observed) and sizes (ranging from small oval cells to la rge multipolar cells). Labeling of ChAT and GAD67 was consistent with numerous neuroanatomical studies that have demonstrated that ChAT and GAD immunopositive neurons in the rostral basal forebrain are distinct nonoverlapping neuronal populations (Brashear et al., 1986; Formaggio, et al., 2011; Gritti, et al., 1993; Kohler, et al., 1984; Semba, 2000) While the ChAT immunopositive

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68 cells were primarily clustered within the medial septum and along the ventral edge of the hDB, the GAD67 immunopositive cells w ere distributed more homogenously throughout the rostral basal forebrain subfields. As expected, NeuN immunopositive cells (which include both ChAT and GAD67 immunopositive neurons as well as a variety of other projection and interneurons) had discernible nuclei, were located throughout the rostral basal forebrain, and exhibited diver se sizes and morphologies (Figure 2 9G 2 9 I). No obvious morphological differences in ChAT, GAD67 or NeuN immunopositive cells were detected at either low or high magnification in sections obtained from rats that differed in age or cognitive status. Age and Cognitive Comparisons of ChAT, GAD67, and NeuN Immunopositive cell Numbers in the Rostral Basal Forebrain The number of ChAT, GAD67 and NeuN immunopositive neurons were ass essed using confocal stereology. ChAT immunopositive neurons A comparison between young and aged rats yielded a significant but modest decline in the number of ChAT immunopositive cells in aged rats relative to young ( 12.5%; t (22) =2.00, p<0.05; Figure 2 1 0 A). A comparison of ChAT immunopositive cells among young and aged rats sub grouped based upon spatial learning ability indicated a similar trend toward decreased ChAT immunopositive cell number with age (F (2,21) 2.23, p=0.13) but the magnitude of reducti on in aged rats was comparable across aged spatially unimpaired ( 14%) and aged spatially im paired ( 12%) subgroups (Figure 2 10 B). In agreement with the observation that ChAT immunopositive cell number was reduced by a similar magnitude in both aged spati ally unimpaired and aged spatially impaired rats, there was no reliable relationship between ChAT immunopositive cell

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69 number and spatial learning index among aged rats (r= 0.10, n.s.; Figure 7D). Notably, however, a significant correlation between ChAT im munopositive cell number and spatial learning index was evident in young rats, such that higher ChAT immunopositive cell numbers were associated with better spatial learning perf ormance (r= 0.76 p<0.05; Figure 2 10 C). GAD67 immunpositive neurons A very different pattern of results was obtained from estimates of total GABAergic projection (GAD67 immunopositive) neuron numbers in the same subfields. As shown in Figure 2 11A, GAD67 immunopositive cell number was greater in aged relative to young rats, alth ough this difference was not statistically reliable (t (21) = 0.74, n.s.). However, a one factor ANOVA comparing GAD67 immunopositive cell number in young and aged rats sub grouped on the basis of spatial learning ability revealed a highly significant differ ence among cognitive groups (F (2,20) =16.2, p<.0001; Figure 2 11 B). P ost hoc comparisons confirmed a robust statistically significant increase in the number of GAD67 immunopositive cells specifically in aged spatially impaired rats relative to both young (+ 18%; p<0.005) and aged spatially unimpaired (+25%, p<0.0001) rats. The number of GAD67 immunopositive cells did not differ significantly between young and aged spatially unimpaired rats (n.s.). The strong relationship between elevated GAD67 immunopositive cell number and cognitive impairment was supported by a significant correlation among aged rats (r=0.60, p<0.05), such that higher numbers of GAD67 immunopositive cells were associated with higher spatial learning indices (i.e., worse learning; Figure 2 1 1D). Among young rats the relationship between spatial learning ability and GAD67 immunopositive cell number was in the same direction but did not reach statistical significance ( r=0.49, p=0.22;Figure 2 11C ).

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70 NeuN immunopositive neurons To investigate whet her age and cognition related alterations in ChAT immunopositve and GAD67 immunopositive cell estimates contributed to an overall difference in neuron number in rostral basal forebrain, NeuN immunopositive cells were also quantified. As shown in Figure 2 12A, the mean estimated number of NeuN immunopositive cells was numerically lower in aged rats relative to young but this difference was not reliable (t (1,21) = 1.2, n.s.). Likewise, no significant differences were evident using a one factor ANOVA performe d on young and aged rats subgrouped by cognitive ability (F (2,20) =0.78, n.s.; Figure 2 12B). As expected based on these analyses, no reliable correlations were observed between NeuN cell number and spatial learning indices in young (r= 0.1 3 n.s. ; Figure 2 12C ) or aged rats (r= .26 ; n.s., Figure 2 12D). Discussion This aim of this study was to assess age related GABAergic changes in hippocampal systems in relation to spatial memory performance in young and aged rats. Experiment 1 investigated inhibitory s ystems in the hippocampus by evaluating expression of GABAergic signaling proteins that are crucial to successful inhibitory synapses in the hippocampus. Interestingly, levels of the GABA synthesizing enzyme, GAD67 were selectively and significantly decre ased in the hippocampus of aged rats that were assessed as spatially impaired. This is in line with previous studies that have reported a decrease in GAD67 immunopositive neurons (Shetty and Turner, 1998; Stanley and Shetty, 2004) This reported decrease i n immunopositve GAD67+ cells occurs in the absence of frank neuronal loss in the aged hippocampus (West et al., 1993) Experiment 2 in this study was designed to test the hypothesis that coordinated age related alterations in both rostral basal forebrain cholinergic and GABAergic

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71 neurons that project to the hippocampus contribute to hippocampal dependent spatial learning abilities in aged rats. Indeed, age dependent changes were evident in both cholinergic and GABAergic neuronal populations, but the direct ion of these changes and their relationship to cognitive abilities were distinct. A modest but significant decline in cholinergic neuron number occurred with age, although this reduction was not related to spatial learning ability in aged rats. H owever, t here was a strong relationship between cholinergic cell number and spatial learning abilities in young rats. In contrast to findings with cholinergic neurons, a robust increase in the number of GABAergic projection (GAD67 immunopositive) neurons was observ ed selectively in the rostral basal forebrain of aged rats with impaired spatial abilities. These phenotypically specific alterations were not reflected in the total number of rostral basal forebrain neurons, as NeuN immunopositive cell number was stable w ith age and across cognitive groups. In addition, GAD67 expression was selectively reduced in aged spatially impaired rats compared to rats that were young and aged spatially unimpaired. Cholinergic (ChAT immunopositive) neurons Cholinergic signaling in t he hippocampus and neocortex has been heavily implicated in cognitive functioning, including learning, memory, and attention (Deutsch, 1971,Dunnett and Fibiger, 1993,Everitt and Robbins, 1997,Fragkouli, et al., 2005, McKinney, 2005,Ormerod and Beninger, 20 02,Sarter and Bruno, 1997,Sarter, et al., 2003,Schliebs and Arendt, 2006,Schliebs and Arendt, 2011,Woolf, 1997) A number of cholinergic indices decline with age, and the cholinergic system remains a primary target of drugs currently used to treat age rel ated cognitive impairment (Giacobini, 2004,Lane, et al., 2004,Lane, et al., 2006,Nordberg, 2006) Indeed, degeneration of cholinergic neurons in the nucleus basalis of Meynert and concomitant depletion of

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72 acetylcholine in cortical targets is a hallmark of (Whitehouse, et al., 1982) and has suggested that degeneration of cholinergic neurons may be an important contributor to cognitive deficiencies observed in normal aging. However, across species, findings regarding cholinergic neuronal integrity at advanced ages have been mixed. A number of studies, employing a variety of counting techniques, report a decline in cholinergic neuron number in aging (Altavista, et al., 1990,Armstrong, et al., 1993,Bartus, et al., 1982,Baskerville, et al., 2006,Fischer, et al., 1992,Fischer, 1989,Lee, et al., 1994,Stroessner Johnson, et al., 1992) although others report no or only a modest decline (Bigl, et al., 1987,McQuail, et al., 2011b,Ypsilanti, et al., 2008) While current findings provide support for a significant decline in rostral basal forebrain cholinergic neurons in normal aging, it should be noted that this reduction was modest, even with the relatively large sample size used. Indeed, some of the reported differences in findings across studies c omparing cholinergic neuron number in normal aged rats may be attributable to the numbers of subjects employed relative to the numbers necessary to achieve sufficient statistical power for detecting small magnitude group differences. In addition to th e modest but significant decline in cholinergic neuron number with advancing age, there is considerable evidence that age related decline in cholinergic neuronal function and signaling contributes to cognitive impairment. For example, age associated dysreg ulation in calcium signaling in cholinergic neurons could affect a range of neuronal functions that include neurotransmitter release and synaptic plasticity (Disterhoft, et al., 1996,Foster, et al., 2001,Kumar, et al., 2009) Specifically, Murchison and G riffith (1998) reported increased intracellular calcium buffering in

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73 cholinergic basal forebrain neurons in aged rats, and m ore recently, that age related calcium dysregulation in basal forebrain cholinergic neurons strongly predict s impaired hippocampal d ependent cognition using the same rat model employed here (Murchison, et al., 2009) Other evidence indicates that aging may disrupt cholinergic signaling at the receptor level. Specifically, muscarinic signaling in the hippocampus, a principal target o f rostral basal forebrain projecting neurons, is significantly altered in aged rats. While muscarinic receptor mediated phosphoinositide turnover is blunted in hippocampal CA1, CA3 and subiculum across aged rats (Nicolle, et al., 2001) G protein coupling to muscarinic receptors in hippocampus is specifically associated with compromised hippocampal dependent spatial learning (Zhang, et al., 2007) Together with the current data, these findings suggest that age related alterations in cholinergic signaling a re not predominantly the result of loss of acetylcholine synthesizing neurons in basal forebrain but rather reflect suboptimal functioning of these neurons and/or postsynaptic signaling associated with the cholinergic receptors in hippocampus and other cor tical targets. This interpretation is consistent with the finding that cholinergic cell number strongly correlated with cognitive abilities in young but not aged rats (see Figure 2 5). In young rats the cellular machinery and environment is largely intact and functioning optimally, and thus the overall number of cholinergic neurons might provide an accurate index of acetylcholine signaling in target fields In aged rats disruption in cholinergic signaling at multiple levels might result in a functional decoupling such that cell number is a less accurate predictor of overall cholinergic function. GABAergic projection (GAD67 immunopositive) neurons A major finding from the current work is the selective and significant increase in GABAergic projection neu rons (indicated by greater numbers of GAD67 immunopositive

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74 cells) in aged spatially impaired rats. Notably, the 67 isoform of GAD is preferentially expressed by GABAergic neurons in rostral basal forebrain that project to cortical targets, whereas the 65 i soform is expressed by rostral basal forebrain interneurons (Castaneda, et al., 2005) Very few prior studies have quantified rostral basal forebrain GABAergic projection neurons in aging, and to our knowledge, no other studies have quantified this specif ic GAD67 immunopositive neuronal population. In a study investigating GABAergic cell number, Smith and Booze (1995) reported no age related change in GAD immunopositive rostral basal forebrain neurons in 3 and 27 month F344 rats using a non isoform specif ic GAD antibody (Smith and Booze, 1995) The current findings are consistent with this previous report in that no significant difference in GABAergic neuron number was evident in rostral basal forebrain when age groups were compared in the absence of cogni tive subgrouping. Together, these data indicate that GABAergic neurons do not degenerate with advancing age, an important finding for interpretation of other phenotypic and functional age related alterations that have been associated with this population o f rostral basal forebrain GABAergic neurons. For example, the calcium binding protein parvalbumin is highly co expressed in GABAergic projection neurons and is often used as a marker for these neurons as it is not expressed by GABAergic interneurons (Grit ti, et al., 1993,Gritti, et al., 2003,Kiss, et al., 1990b,Krzywkowski, et al., 1995,Miettinen, et al., 2002) The present findings would suggest that previously reported reductions in parvalbumin immunopositive cell number reflect dysregulated calcium sign aling which parallels that described above for cholinergic neurons, in the absence of overt GABAergic neuronal loss. Additional work

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75 to characterize the age related changes in the signaling properties and function of these GABAergic neurons represents an important direction of future work. Importantly, the increase in GAD67 immunopositive cell number observed in aged spatially impaired rats did not appear to be due to an overall increase in total neuron number in rostral basal forebrain, as stereological estimates of NeuN immunopositive cells in adjacent sections did not differ with age or as a function of cognitive ability. Although the specific mechanisms responsible for the elevation in GABAergic cell number observed in spatially impaired aged rats rem ains an outstanding question, it seems plausible that this change might reflect an age related upregulation of GAD67 protein expression in a subset of rostral basal forebrain neurons which might otherwise express only nominal levels of GAD67. Such an upreg ulation would increase the detectability of these neurons, which in turn would be reflected as an increase in the total number of GAD67 immunopositive neurons Indeed, GAD67 expression can be regulated by a number of factors including activity, stress, oes trous cycle, and caloric restriction (Carta, et al., 2008,Cashion, et al., 2004,Cheng, et al., 2004,Liang, et al., 1996) Moreover, altered GAD expression has been reported in a number of brain disorders ophrenia (Briggs and Galanopoulou, 2011,Lanoue, et al., 2010,Lewis, et al., 2005) Another interesting finding was the significant reduction of hippocampal GAD67 expression in the in aged spatially impaired rats compared to young and aged spatially impai red counterparts, the same cognitive group that, based on the results of this study, received increased GABAergic input from the basal forebrain. GABAergic interneurons in the hippocampus are thought to be the target of basal forebrain GABAergic projectio n

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7 6 neurons and certain interneuronal phenotypes are known to decline in aging in the absence of frank neuronal loss (Shetty and Turner, 1998; Stanley and Shetty, 2004). Another possible explanation for the increase in basal forebrain GABAergic projection n eurons in aged spatially impaired rats is that it is a compensatory mechanism for decreased GABAergic tone in the hippocampus as indicated by a significant reduction in GAD67 expression in the hippocampus of aged spatially impaired rats. Future studies ar e needed to investigate this possibility. Alternatively, it is important to consider that the neurons in rostral basal forebrain are quite heterogeneous and the full extent of distinct neuronal subtypes in these fields remains undetermined and was not exh austively evaluated in the current study. In addition to cholinergic and GABAergic projection neurons, other cell types include GABAergic interneurons and a recently identified subpopulation of hippocampal projecting glutamatergic neurons (Colom, et al., 2005; Manseau, et al., 2005) As such it is possible that the failure to detect differences in numbers of NeuN immunopositive cells between age and cognitive groups reflects differential effects of age on multiple phenotypically distinct neuronal populati ons in this region. Notably, the rostral basal forebrain glutamatergic neurons share size and certain neurochemical characteristics with GABAergic projection neurons (Colom, et al., 2005; Manseau, et al., 2005) and some basal forebrain neurons have been i dentified that have the capacity to synthesize both GAD67 and phosphate activated glutaminase (PAG), a mitochondrial enzyme used in the production of glutamate (Gritti, et al., 2006) In addition, RT PCR studies have shown that mRNA for the glutamate vesic ular transporter 2 (VGLUT2) may be coexpressed with ChAT and GAD67 mRNA in young and adult rats (Danik, et al., 2005)

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77 These findings make it intriguing to speculate that the present results represent a functional shift from excitatory to inhibitory signal ing in a subpopulation of hippocampal projecting neurons as has been shown in kindling models in which stimulation induces the production of GAD67 in hippocampal granule cells (Gomez Lira, et al., 2005,Sloviter, et al., 1996) It will be important in futu re studies to determine the effects of age on glutamatergic projections as well as on GABAergic interneuronal populations, to better understand how age related changes in basal forebrain dynamics contribute to a loss of hippocampal dependent learning and m emory. It is becoming increas ingly clear that corticopetal basal forebrain GABAergic neurons influence neural transmission and cognitive functions linked to their terminal fields (Freund and Antal, 1988,Kiss, et al., 1990b,Pang, et al., 2001) GABAergic ne urons are well positioned to modulate cortical circuitry, both through their direct input to cortical structures and via the highly interconnected cholinergic and GABAergic neur on al network s within the rostral basal forebrain itself (Freund and Antal, 1988 ,Freund and Buzsaki, 1996) Neuronal tracing studies indicate that GABAergic hippocampal interneurons are a primary target of GABAergic afferents from rostral basal forebrain (Freund and Antal, 1988,Gulyas, et al., 1991) As such an increase in the inhibi tory influence from rostral basal forebrain would result in enhanced excitability of the hippocampal principal neurons. Indeed, many studies have reported hyperexcitability in the hippocampus with advanced age, and such alterations have been linked to hip pocampal dysfunction and a decline in hippocampal supported cognition (Dickerson, et al., 2005; Gallagh er and Koh, 2011;Wilson, et al., 2005; Yassa, et al., 2010) The decrease in GAD67 expression observed in Experiment 1 of this

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78 study may be an underlyin g mechanism of hippocampal hyperexcitability. Less GABA synthesis by hippocampal interneurons could result in less inhibitory tone on pyramidal cells and, thus, increased excitability of pyramidal cells. The present findings support the hypothesis that a ge related alterations in inhibitory signaling within septohippocampal circuitry might contribute to this reported shift in the inhibitory excitatory dynamics of the aged hippocampal formation. Overall, the findings from the current work add to the evide nce that rostral basal forebrain systems are significantly altered in aging. In particular, these studies support a growing literature which indicates that inhibitory networks are particularly vulnerable to dysfunction in aging and that such dysfunction ca n profoundly impact cognition. Future work in which the present findings are extended to determine the innervation patterns of both inhibitory and excitatory afferents from rostral basal forebrain and that better characterize the intrinsic dynamics within basal forebrain of aged behaviorally characterized rats will help to further elucidate the role of this system in age related cognitive decline.

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79 Figure 2 1. Rostral basal forebrain boundaries used for cell count estimations. Schematic illustrations modified from Paxinos and Watson (2007) showing the rostro caudal boundaries (extending from 1.7 mm anterior to 0.26 mm posterior of Bregma) and the delineation of rostral basal forebrain nuclei (red outline) used in the current study. Abbreviations: ac; anterior commissure; AcbC, nucleus accumbens core; AcbS, nucleus accumbens shell; cc, corpus callossum CPu; caudate putamen, hDB, horizontal limb of the diagonal band of Broca; LS, lateral septum; LV, lateral ventricle; MS, medial septum; MCPO magnocellula r preoptic area; Tu, olfactory tubercle, vDB, vertical limb of the diagonal band of Broca; 2n, optic nerve.

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80 Figure 2 2 Distribution of ChAT immunopositive cells in rostral basal forebrain. Low magnification photomicrographs of ChAT immunolabeling in coronal sections taken through the rostral basal forebrain (approximately 0.5 mm anterior to bregma) of representative young (A), aged spatially unimpaired (B) and aged spatially impaired (C) rats. Staining was robust in individual neurons localized to ros tral basal forebrain nuclei including the medial septum (MS), vertical limb of the diagonal band of Broca (vDB) and horizontal limb of the diagonal band of Broca (hDB), and was also evident in scattered interneurons distributed throughout the caudate putam en (CPu), nucleus accumbens (Acb) and olfactory tubercle (Tu). The dense immunolabeling of the ChAT immunopositive neurons within the MS, vDB and hDB made these nuclei readily distinguishable from neighboring structures (including lateral septum, the media l wall of the nucleus accumbens shell and the olfactory tubercle) across age and cognitive groups. For orientation, white matter regions (i.e., corpus callosum and anterior commissure) and the lateral ventricle (LV) are labeled. Abbreviations: ac; anterio r commissure; AcbS, nucleus accumbens shell; cc, corpus callossum CPu; caudate putamen; hDB, horizontal limb of the diagonal band of Broca; LS, lateral septum; LV, lateral ventricle; MS, medial septum; Tu, olfactory tubercle, vDB, vertical limb of the dia gonal band of Broca. Scale bar = 200 m.

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81 Figure 2 3 Distribution of GAD67 immunopositive cells in rostral basal forebrain. Low magnification photomicrographs of GAD67 immunolabeling in coronal sections taken through the rostral basal forebrain (approximately 0.5 mm anterior to bregma) of representative young (A), aged spatially unimpaired (B) and aged spatially impaired (C) rats. GAD67 labeling was robust throughout rostral basal forebrain nuclei, including medial septum and the vertical and hor izontal limbs of diagonal band of Broca (vDB and hDB, respectively). Somewhat diffuse GAD67 immunolabeling was also observed in lateral septum (LS) and in nucleus accumbens (Acb), whereas scattered individual GAD67 immunopositive cells were observed in the caudate putamen (CPu) and olfactory tubercle (Tu). Across age and cognitive groups, dense labeling within basal forebrain nuclei allowed these regions to be readily delineated from surrounding nuclei. For orientation, white matter regions (i.e., corpus c allosum and anterior commissure) and the lateral ventricle (LV) are labeled. Abbreviations: ac; anterior commissure; AcbS, nucleus accumbens shell; cc, corpus callossum; CPu; caudate putamen; hDB, horizontal limb of the diagonal band of Broca; LS, lateral septum; LV, lateral ventricle; MS, medial septum; Tu, olfactory tubercle, vDB, vertical limb of the diagonal band of Broca. Scale bar = 200 m

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82 Figure 2 4 Distribution of NeuN immunopositive cells in rostral basal forebrain. Low magnification phot omicrographs of NeuN immunolabeling in coronal sections taken through the rostral basal forebrain (approximately 0.5 mm anterior to bregma) of representative young (A), aged spatially unimpaired (B) and aged spatially impaired (C) rats. NeuN expression is prominent in most neurons through rat forebrain, although differences in density and intensity of expression allow discrete nuclei to be clearly distinguished. NeuN immunolabeling is robust throughout the medial septum (MS) and the vertical and horizontal limbs of the diagonal band of Broca (vDB and hDB, respectively). It is also particularly prominent in lateral septum (LS) and the nucleus accumbens shell (AcbS), as well as in olfactory tubercle (Tu), allowing the boundaries of basal forebrain nuclei to b e clearly delineated across age and cognitive groups. For orientation, white matter regions (i.e., corpus callosum and anterior commissure) and the lateral ventricle (LV) are labeled. Abbreviations: ac; anterior commissure; AcbC, nucleus accumbens core; A cbS, nucleus accumbens shell; cc, corpus callossum CPu; caudate putamen, hDB, horizontal limb of the diagonal band of Broca; LS, lateral septum; LV, lateral ventricle; MS, medial septum; MCPO magnocellular preoptic area; Tu, olfactory tubercle, vDB, verti cal limb of the diagonal band of Broca; 2n, optic nerve. Scale bar = 200 m.

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83 Figure 2 5 Antibody penetration throughout the tissue slice. Left panels show representative examples of immunolabeling for ChAT A), GAD67 B), or NeuN C). The orthog onal views (showing xz, and yz planes) illustrate good antibody penetration and robust immunolabeling throughout the entire z plane for each label (shown in a 25 m z slice corresponding to the disector height). Right panels show plots of the proportion of the localized in the z axis. As shown in Table 2 1, cells were counted using a disector height of 25 m with a top guard zone of 3 m. Gray bars show that the proportion of total cel ls counted across all animals (N= 24 for ChAT and N=23 for both NeuN and GAD67) did not appreci ably differ across the first 5 m in the z axis (distance shown is from the top of the disector). These data indicate that the 3 m guard zone was sufficient to minimize any effect of cells lost to sectioning or the non systematic inclusion of caps. The proportion of cells counted in the remaining 20 m is shown in 5 m bins, plotted by cognitive group to illustrate that, for each label, the proportion of cells sa mpled was distributed evenly throughout the z axis for both age and cognitive groups. Scale bar =50 m

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84 Figure 2 6 Spatial learning in young and aged rats in Experiment 1. (A) Young and aged rats did not differ on the first training trial, and bot h groups improved over the course of training. As a group, aged rats were significantly impaired relative to young in learning to swim to a hidden (submerged) platform within the water maze. (B) Spatial learning index (SLI) scores were calculated from prob e trial performance to provide an overall index of spatial learning ability for each subject. Note that there was considerable variability among aged rats, with many performing within the range of young (aged spat ially unimpaired rats ) and others performin g more than two times the standard deviation of mean young rat performance, demonstrating impairment (aged spatially impaired rats ). See text for statistical analysis.

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85 Figure 2 7. Age related changes in GABAergic signaling pr otein expression and relationship to working memory ability.

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86 Figure 2 8 Spatial learning in young and aged rats in Experiment 2 (A) Young and aged rats did not differ on the first training trial, and both groups improved over the course of training. As a group, aged rats were significantly impaired relative to young in learning to swim to a hidden (submerged) platform within the water maze. (B) Spatial learning index (SLI) scores were calculated from probe trial performance to provide an ov erall index of spatial learning ability for each subject. Note that there was considerable variability among aged rats, with many performing within the range of young (aged spatially unimpaired rats; SLI < 275) and others performing more than two times the standard deviation of mean young rat performance, demonstrating impairment (aged spatially impaired rats, SLI>275). See text for statistical analysis.

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87 Figure 2 9 High magnification immunofluorescent labeling of ChAT, GAD67 and NeuN immunopositive ne urons. Representative ChAT (A C), GAD67 (D F), and NeuN (G I) immunopositive cells in young (left), aged spatially unimpaired (middle) and aged spatially impaired rats (right). Across ChAT, GAD67, and NeuN material, immunopositive cells were robustly labe led and contained a well defined nucleus. Overall, ChAT immunopositive cells tended to be polygonal and fusiform shaped whereas GAD67 immunopositive cells exhibited diverse morphologies and sizes (ranging from small oval cells to large multipolar cells). T he NeuN immunopositive cells (which would include both ChAT and GAD67 immunopositive neurons as well as a variety of other projection and interneurons) had discernable nuclei and exhibited diverse sizes and morphologies. No obvious morphological difference s between ChAT, GAD67 or NeuN immunopositive cells were detected from rats that differed in age or c ognitive status. Scale bar = 20 m

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88 Figure 2 10 Cholinergic (ChAT immunopositive) cell number in the rostral basal forebrain of young and aged rats. A) T he number of ChAT immunopositive cells was modestly but significantly decreased in aged relative to young rats. B) The reduction in ChAT immunopositive cells was of a similar magnitude (approximately 12.5%) in both aged spatially unimpaired and aged spati ally impaired rats. C) Scatter plot illustrating a strong relationship between ChAT immunopositive cell number and spatial le arning index among young rats. D) This relationship was not present among aged rats. See text for statistical analyses. *p<0.05; Y = young; AU= aged spatially unimpaired; AI= aged spatially impaired.

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89 Figure 2 11 GABAergic (GAD67 immunopositive) cell number in the rostral basal forebrain of young and aged rats. A) GAD67 immunopositive cell number was numerically but not significant ly greater in age d rats in comparison to young. B) A significant difference in GAD67 immunopositive cell number was evident between cognitive age groups, such that aged spatially impaired rats exhibited a marked, reliable elevation in GAD67 immunopositive cells in comparison to both young and a ged spatially unimpaired rats. C) Scatter plot shows a trending though nonsignificant relationship between GAD67 immunopositive cell number and spatial learning index in young rats such that higher numbers were a ssoci ated with worse learning. D) Scatterplot shows that the same relationship was observed among aged rats, but in this case, greater GAD67 immunopositive cell number was significantly associated with worse spatial learning ability. See text for statistical an alyses. *p<0.05 relative to young and aged spatially unimpaired; Y= young; AU= aged spatially unimpaired; AI= aged spatially impaired.

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90 Figure 2 12 Total (NeuN immunopositive) cell number in the rostral basal forebrain of young and aged rats. NeuN immu nopositive cell number in the rostral basal forebrain did n ot differ as a function of age A) or cognitive ability B). Moreover, the total number of NeuN immunopositive cells did not predict spatial le arning ability in either young C) or aged D) rats. See t ext for statistical analyses. Y= young; AU= aged spatially unimpaired; AI= aged spatially impaired.

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91 Table 2 1 Sampling parameters used for estimating total number of rostral basal forebrain neurons. Object Samplin g Grid ( m X m) Countin g F rame ( m X m) Disector height ( m) Average Object Counted SD Average CE CV ChAT+ cells 336X448 140X140 25 292 42.89 0.05 0.16 GAD67+ cells 448X448 110X100 25 679 101.14 0.06 0.20 NeuN+ cells 400X400 89X89 25 781 175.33 0.04 0.16

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92 Table 2 2. Estimates of ChAT, GAD67 and NeuN immunopositive cells in rostral basal forebrain of young and aged behaviorally characterized rats. Young Aged Total Aged SU Aged SI ChAT+ cells (SD, n) 21003.47 (3284.54, n=8) 18383.71 (2626.83, n=16) 18113.89 (2515.56, n=8) 18653.53 (2879.54, n=8) GAD+ cells (SD, n) 48586.17 (5564.82, n=8) 51118.00 (8721.43, n=15) 44459.45 (4408.62, n=8) 58727.78** (5317.29, n=7) NeuN+ cells (SD, n) 140007.13 (19561.76, n=8) 128798.47 (2 1035.71, n=15) 124938.86 (22388.96, n=7) 132175.63 (20674.01, n=8)

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93 CHAPTER 3 AGE RELATED CHANGES IN PREFRONTAL CORTICAL GABAERGIC SYSTEMS: RELATIONSHIP TO WORKING MEMORY IMPAIRMENT Introduction With advancing age, many individuals will experience a si gnificant decline of cognitive capacities supported by the prefrontal cortex (PFC), resulting in deficits across a wide range of adaptive behaviors that are essential for maintaining independence and quality of life (Robbins et al., 1998; Salthouse et al., 2003; Glisky, 2007; Bizon et al., 2012) One fundamental aspect of PFC function is w orking memory which involves the ability to maintain a mental of information that is no longer present in the environment and to use this repre sentational information to guide future action (Goldman Rakic, 1996) Within the PFC, maintenance of information in mind following the removal of a sensory stimulus is associated with persistent excitation of pyramidal neurons, providing a possible neuroph ysiological basis for working memory ( Goldman Rakic, 1996 ; Wang et al., 2013 ) In addition, input from a diverse group of GABAergic interneurons onto somatodendritic compartments of pyramidal cells refines s patial and temporal specificity in this system (G oldman Rakic, 1995; Zaitsev et al., 2009) Altered excitability of PFC pyramidal neurons is a central feature of cognitive disorders associated with a wide range o f pathological conditions such as Do schizophrenia (Kleschevnikov et al., 20 04; Gonzalez Burgos and Lewis, 2008) and experimentally induced disruption of PFC GABAergic circuits can produce profound impairments in working memory (Kleschevnikov et al., 2004; Enomoto et al., 2011; Murray et al., 2011) A large body of work has impli cated changes in PFC monoamine signaling in age related working memory decline (Goldman

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94 Rakic and Brown, 1981; Arnsten et al., 1994; Moore et al., 2005) ; however less attention has been paid to whether disruptions in inhibitory synaptic substrates also con tribute to such decline. GABA(B)Rs are G protein coupled receptors localized to perisynaptic sites on GABAergic and glutamatergic terminals where they regulate neurotransmitter release as well as on dendritic spines and shafts where they are localized ext rasynaptically and contribute to tonic inhibition of pyramidal neurons (Gonzalez Burgos, 2010; Pinard et al., 2010) Recently, McQuail et al. (2012) reported a marked and regionally specific reduction of GABA(B) receptor (GABA(B)R) proteins in aged PFC. Th e impact on cognition of this age related decline in PFC GABA(B)Rs is not yet known. Notably, however, other evidence from electrophysiological studies conducted in both rodents and nonhuman primates indicates that PFC pyramidal neurons may be subject to i ncreased inhibitory input in advanced aging (Luebke et al., 2004, Bories et al., 2013). The first goal of the current study was to investigate expression of GABA(B)Rs and other GABAergic signaling proteins in the young and aged medial prefrontal cortex (mP FC) the rodent homologue of the primate dorsolateral prefrontal cortex (Uylings et al., 2003) in relation to performance on a mPFC dependent delayed response working memory task. The second goal of this study was to determine the effects of pharmacologic al manipulations of GABA(B)R signaling on working memory performance and, specifically, to determine whether modulation of GABAergic signaling in the mPFC can attenuate working memory deficits that accompany normal aging.

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95 Methods Subjects Young (6 months old) and aged (22 months old) male Fischer 344 rats were obtained from the National Institute on Aging colony (Taconic Farms, Hudson, NY) and housed in the AAALAC accredited vivarium facility in the McKnight Brain Institute at University of Florida in acc ordance with the rules and regulations of the University of Florida Institutional Animal Care and Use Committee and NIH guidelines. The facility was maintained at a consistent 25C with a 12 h light/dark cycle (lights on at 0800 hours). Rats were maintaine d under specific pathogen free (SPF) conditions and had free access to food and water at all times except as noted below. A total of 59 rats (young n=24, aged n=35) were used in this study. Numbers of rats in each experiment were as follows: Experiment 1: young n= 6 and aged n=12; Experiment 2: young n=10 and aged n=13; Experim ent 3: young n=8 and aged n=10. Experiment 1: GABA signaling Protein Expression and Working Memory Abilities Delayed Response Task Procedures Apparatus. Testing in the delayed respo nse task was conducted in eight identical standard rat behavioral test chambers (30.5 X 25.4 X 30.5 cm, Coulbourn Instruments, Whitehall, PA) with metal front and back walls, transparent Plexiglas side walls, and a floor composed of steel rods (0.4 cm in d iameter) spaced 1.1 cm apart. Each test chamber was housed in a sound attenuating cubicle, and was equipped with a recessed food pellet delivery trough located 2 cm above the floor in the center of the front wall. The trough was fitted with a photobeam to detect head entries and a 1.12 W lamp for illumination. Food rewards consisted

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96 of deliveries of a single 45 mg grain based food pellet for each correct response (PJAI, Test Diet, Richmond, IN). Two retractable levers were located to the left and right of t he food trough (11 cm above the floor). An additional 1.12 W house light was mounted near the top of the rear wall of the sound attenuating cubicle. A computer interfaced with the behavioral test chambers and equipped with Graphic State 3.01 software (Coul bourn Instruments) was used to control experiments and collect data. Shaping. Prior to the start of behavioral testing, rats were reduced to 85% of their free feeding weights over the course of five days and maintained at this weight for the duration of b ehavioral testing. Rats progressed through three stages of shaping prior to the onset of the delayed response task, with a new stage beginning on the day immediately following completion of the previous stage. On the day prior to Shaping Stage 1, each rat was given five 45 mg food pellets in its home cage to reduce neophobia to the food reward used in the task. Shaping Stage 1 consisted of a 64 min session of magazine training, involving 38 deliveries of a single food pellet with an inter trial interval (IT I) of 100 40s. Shaping Stage 2 consisted of lever press training, in which a single lever (left or right, counterbalanced across age groups) was extended and a press resulted in delivery of a single food pellet. After reaching a criterion of 50 lever pre sses in 30 min, rats were then trained on the opposite lever using the same procedures. During Shaping Stage 3, either the left or right lever (counterbalanced across trials in this Stage of testing) was extended and a press resulted in a single food pelle t delivery. Rats were trained in Shaping Stage 3 until achieving 80 lever presses in a 30 min session.

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97 Working Memory Assessment The working memory assessment was based on Sloan et al. (2006), and was used previously to demonstrate age related impairment s in Fischer 344 rats (Beas et al., 2013) Each session was 40 min in duration, and the house light was illuminated throughout the entire session except during timeout periods (see below). Rats received only a single test session/day. A trial began with th e ever) into the chamber (Figure 3 1A ). The left/right position of this lever was randomly selected within each pair of trials, and a lever press caused it to retract and started the delay period timer. During the delay, rats were required to nosepoke into the food trough in order to timer expired caused both levers to extend. During this choice phase, a response on the same lever pres sed during the sample phase (a correct response) resulted in both levers being retracted and delivery of a single food pellet. Entry into the food trough to collect the food pellet initiated a 5 s inter trial interval, after which the next trial was initia ted. A response on the opposite lever from that chosen during the sample phase (an incorrect response) resulted in both levers light was extinguished, followed immediately by t he start of the next trial. During initial sessions in this task, there were no delays between the sample and choice phases, and a correction procedure was employed such that the sample lever was repeated on the same side following an incorrect response to prevent development of side biases. Once rats reached a criterion of 80% correct choices across a session for two consecutive sessions, this correction

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98 procedure was discontinued and a set of seven delays was introduced. The presentation of delay duration s was randomized within each block of seven trials, such that each delay was presented once within a block. Upon establishing greater than 80% correct performance across two consecutive sessions in a Set, rats were progressed to the next Set (Set 1: 0, 1, 2, 3, 4, 5, 6 s; Set 2: 0, 2, 4, 8, 12, 16 s; Set 3: 0, 2, 4, 8, 12, 18, and 24 s). Rats were tested for 5 consecutive sessions on the delays in Set 3 to acquire baseline performance data. Approximately 2 weeks after completion of behavioral testing and r eturn to ad libitum food, rats were decapitated and brains were removed from the skull, cooled on ice, and sliced in 1 mm coronal sections using a rat brain matrix. Coronal sections were placed on an ice cold plate, and the mPFC was dissected and stored at Watson (AP: +3.7 through +2.2), the boundaries of the mPFC were delineated dorsally and medially by the emergence of the white matter tracts surrounding the striatum, and ventrally by the ve ntral tip of the corpus collosum. As cognitive task performance can elicit changes in protein expression (Davis et al., 1996; Wass et al., 2013) this two week post training interval was selected to evaluate baseline rather than behaviorally stimulated pro tein levels. Western blot analysis performed on homogenates generated from these dissected tissue samples was used to evaluate the influence of age and cognitive performance on the following GABAergic signaling proteins: the GABA(B) receptor R1a, R1b, and R2 subunits, the primarily neuronal GABA transporter GAT 1, the GABA synthesizing enzyme GAD67, and the vesicular GABA transporter VGAT.

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99 Western Blotting Procedures Sample Preparation. Frozen tissue was weighed, thawed, and homogenized in 10 volumes of an ice cold buffer (50 mM 4 (2 Hydroxyethyl)piperazine 1 ethanesulfonic acid, N (2 Hydroxyet hyl)piperazine (2 ethanesulfonic acid) (HEPES), pH 7.4, 1 mM ethylenediaminetetraacetic acid and 1 mM ethylene glycol bis(2 aminoethylether) tetraacetic acid and protease inhibitors; Roche, Mannheim, Germany) using a glass Teflon Dounce homoge nizer. Homogenates were centrifuged at 14,000 rpm for 20 minutes at 4 C. The supernatant was collected and stored in aliquots for western blotting assays of non membrane bound proteins. The pellet was resuspended in 20 mL of the same buffer without protea se inhibitors and incubated on ice for 30 minutes followed by centrifugation at 16,500 rpm for 15 minutes at 4 C. This pellet was resuspended in 10 volumes of 50 mM HEPES, pH 7.4, and aliquots were stored Protein concentration was determined using the Pierce BCA Kit according to the manufacturer's protocol (Rockford, IL, USA). Immunoblotting. Proteins were denatured and reduced in Laemmli sample buffer mercaptoethanol (Fisher, Pittsburgh PA, USA) and heated at 95 C for 5 minutes. Initial experiments focused on evaluating expression of PFC GABA(B) receptor R1 and R2 subunits as well as GAT 1 Subsequently, and to the extent possible, expression of GAD67 and VGAT were assessed in this sa me cohort. The GABA(B)R1 and GABA(B)R2, GAT 1, and VGAT assays were conducted using the membrane preparation whereas the GAD67 assay was conducted using the supernatant fraction collected as described above. Note that

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100 the supernatant was not available for all subjects and therefore only a subset of young and aged rats were included in the GAD67 study (n= 6 young and n= 7 aged). Similarly, for some animals, there was no remaining membrane homogenate available for use in the VGAT study, resulting in fewer ani mals included in this study (n= 6 young and n= 8 aged). In all western blot experiments, ten micrograms of protein per lane were electrophoretically separated on a 4% 15% Tris HCl gel at 200 V for 35 minutes then transferred to nitrocellulose membranes us ing a wet transfer apparatus for 90 minutes at 0.35A. Blots were washed 3 times with tris buffered saline (TBS; pH 7.4) then blocked for 1 hour in blocking buffer (Rockland, Gilbertsville, PA, USA). Blots were then incubated overnight at 4 C with antibodi es [anti GAD67 (Millipore, Temecula, CA), anti GAT 1 (Millipore), anti VGAT (Millipore), anti GABA(B)R1 (Cell Signaling Technology, Beverly, MA, USA), anti GABA(B)R2 (Cell Signaling Technology), diluted 1:1000 in blocking buffer (Rockland, Gilbertsville, P A, USA) with 0.1% Tween 20 (Bio Rad, Hercules, CA, USA). Blots were then washed 3 times with 0.1 M Tris Buffer Solution (TBS) and incubated with the appropriate AlexaFluor 680 conjugated anti IgG (Invitrogen, Carlsbad, CA, USA) diluted 1:20,000 in TBS with 0.1% Tween 20 (Bio Rad) for 1 h. Following 3 additional TBS washes, blots were scanned on an Odyssey imaging system (LI COR Biosciences, Lincoln, NE, USA). Statistical analyses Raw data files generated from delayed response test sessions were exported from Graphic State software and compiled using a custom macro written for Microsoft Excel (Dr. Jonathan Lifshitz, University of Kentucky). To assess

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101 working memory abilities m ean accuracy (percent correct) at each delay across 5 test sessions performed at the final set of delay s (Set 3) was calculated and age comparisons were conducted using two factor repeated measure ANOVA (Age X Delay). For western blot analyses, integrated protein density was measured for each band and the individual values of both you ng and aged samples were normalized to the mean expression of young samples run on the same gel. Age comparisons of protein expression were conducted using i ndependent t tests Note that each blot was probed separately for g lyceraldehyde 3 phosphate dehydr ogenase ( GAPDH ) expression to confirm that age effects were not a result of non specific effects of loading or quantification of protein content. In no cases were age comparisons performed on GAPDH measures significant ( R1a and R1b: t (16 ) =0.34, p=0.74; GAB A(B ) R2: t (16 ) = 0.98, p=0,35; GAT 1 : t (16 ) =0.49, p=0,63; GAD67 : t (11 ) = 0.10, p=0,34 ) and as such, these data are not reported individually To directly test relationships between changes in protein expression and working memory abilities among aged rats, me an percent accuracy averaged across the two longest delays (18 and 24 s) was calculated for each subject and measure was used as an index of working memory ability Relationships between expression of each protein of inte rest and Mean Long Delay performance were tested using correlations. For this and all subsequent experiments, data are presented as the mean standard error of the mean. All statistical analyses were conducted using SPSS 21.0 (Cary, NC, USA) an d GraphPadPrism (La Jolla, California). For all statistical comparisons, values of p < 0.05 were considered significant.

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102 Experiment 2: Systemic Administration of the GABA(B) Receptor Antagonist CGP55845 This experiment was designed to test the hypothe sis that blockade of GABA(B) receptors via systemic administration of the selective antagonist CGP55845 can improve working memory performance in aged rats. Drug Administration Procedures Rats were trained on the delayed response task as described above for Experiment 1. Pharmacological testing began on the day after establishing baseline performance measures (i.e., after completing 5 sessions of testing on the longest set (Set 3) of delays). Rats received intraperitoneal (i.p., 1.0 ml/kg) injections of the selective GABA(B)R antagonist CGP55845 (0.01 or 0.1 mg/kg; Tocris, Ballwin, MO) or 0.9% saline vehicle 40 minutes prior to testing. The choice of this drug and dose regimen was based on several previous studies in which systemic administration of CGP55 845 was shown to exert behavioral effects in rodents. Froestl et al. (1996), showed that CGP55845 reversed baclofen induced hypothermia, supporting in vivo actions at GABA(B) receptors. Moreover, previous work from our lab and others has shown that i.p. a dministration of CGP55845 can exert cognitive benefit in a rodent model of Kleschevnikov et al., 2012, LaSarge et al., 2009) Drugs were administered in a randomized, counterbalanced order such that each rat was tes ted under each drug condition. A 48 h washout period was interposed between injections, during which delayed response testing was conducted in the absence of drug administration.

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103 Data Analysis Analysis of baseline performance in the delayed response task was calculated as described above in Experiment 1. The effects of GABA(B)R antagonist administration were assessed using a three factor repeated measures ANOVA, wit h both Drug Dose and Delay as repeated measures variables and Age as a between subjects vari able Post hoc comparisons within each age group were performed using two factor repeated ANOVAs (Drug Dose X Delay). When justified, additional two factor ANOVAs were used to compare individual drug doses against vehicle conditions within each age group to determine those doses that sig nificantly altered performance. P roteins were denatured and reduced in Laemmli sample buffer with 5% mercaptoethanol (Fisher, Pittsburgh, PA, USA) and heated at 95 C for 5 minutes. GAT 1, VGAT, GABA(B)R 1 and G ABA(B)R 2 assays were conducted using m embrane preparation while GAD67 assays were conducted using the supernatant fraction collected as described above. Ten micrograms of protein per lane were electrophoretically separated on a 4% 15% Tris HCl gel at 200 V for 3 5 minutes then transferred to nitrocel lulose membranes using a wet transfer apparatus for 90 minutes at .35A Blots were washed 3 times with tris buffered saline (TBS; pH 7.4) then blocked for 1 hour in blocking buffer (Rockland, Gilbertsville, PA, U SA). Blots were then incubated overnigh t at 4 C with antibodies [anti GAD67 (Millipore Temecula, CA ), a nti GAT 1 (Millipore), anti VGAT (Millipore) anti GABA(B)R1 (Cell Signaling Technology, Beverly, MA, USA) anti GABA(B)R2 (Cell Signal ing Technology ) diluted 1:1000 in blocking buffer (Rockland, Gilbertsville, PA, USA) with 0.1% Tween 20 (Bio Rad

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104 Hercules, CA, USA ). Blots were then washed 3 times with 0.1 M T ris B uffer S olution (TBS) and incubated with the appropriate AlexaFluor 680 conjugated anti Ig G (Invitrogen Carlsbad, CA, USA ) diluted 1:20,000 in TBS with 0.1% Tween 20 (Bio Rad) for 1 hour. Following 3 additional TBS washes, blots were scanned on an Odyssey imaging system (LI COR Biosciences, Lincoln, NE, USA). Experiment 3: Intracerebral Microi njections of the GABA(B) Receptor Antagonist CGP55845 This experiment was designed to test the hypothesis that the mPFC is a critical site of action for the cognitively enhancing effects of the selective GABA(B)R antagonist CGP55845. Cannulation Surgery Y oung and aged rats were anesthetized with isofluorane gas and fixed into a stereotaxic frame (Kopf Instruments, Tojunga, CA) fitted with atraumatic earbars. The incisor bar was set at 3.3 mm relative to the interaural line to provide a flat skull position B ilateral guide cannula e, which consisted of a plastic body holding two 22 gauge stainless steel cannulae 1.5 mm apart (Plastics One, Roanoke, VA), were implanted to target mPFC (AP : +1.7 relative to bregma, ML: 0.7, DV: 3 .8 relative to skull ). Cannula e were secured to the skull with dental acry lic and stainless steel screws, and wire stylets were used to occlude the guide cannulae to prevent infection. Intracerebral Microinjections of the GABA(B) Receptor Antagonist CGP55845 Following a two week reco very period, rats were trained in the delayed response task until they completed 5 sessions at the longest set of delays as in

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105 Experiments 1 and 2. On the next day, rats received a dummy injection during which the stylets were removed from the guide cannul ae and injectors (28 ga. needles which extended 1.0 mm beyond the end of the guide cannulae; Plastics One) were lowered into the mPFC. This dummy injection (in which no drug was injected) served to acclimate the rat to the handling necessary to administer the drug. In subsequent test sessions, rats received bilateral microinjections of the GABA(B)R antagonist CGP55845 (0.2 mol, 0.6 mol, 2.0 mol) or vehicle (artificial cerebral spinal fluid, Harvard Apparatus, Holliston, MA) using a randomized, counterbal anced design such that each rat was tested in each drug condition. Microinjections (0.5 l per hemisphere over 60 s) were administered 5 minutes prior to the start of delayed response test sessions. Microinjections were delivered via 10 l syringes connect ed to the injection needles by a length of PE 20 tubing. These parameters were chosen based on previous work showing that drug microinjections of this volume into mPFC produce reliable and specific effects on mPFC dependent cognitive tasks including workin g memory, with minimal diffusion to the midline or ventricles (Taylor et al., 1999; Ragozzino, 2002; Stefani and Moghaddam, 2005; Allen et al., 2008; St. Onge et al., 2011). The syringes were mounted on a syringe pump (Pump 11 Elite, Harvard Apparatus) an d needles were left in place for one min after injections to allow for drug diffusion. A 48 h washout period was interposed between drug injection sessions, during which delayed response testing was conducted in the absence of drug. Performance on these no n drug, washout days was monitored for residual effects of the microinjec tions.

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106 Histological Assessment of Cannulae Placement Following completion of testing, rats were killed with 100 mg/kg sodium pentobarbital, then perfused with 0.1M phosphate buffer s olution (PBS) followed by 4% paraformaldehyde in 0.1 M PBS. Brains were removed and postfixed in 4% paraformaldehyde overnight and then cryoprotected in 20% sucrose in PBS. Brains were then flash frozen and sliced coronally at 50 microns on a cryostat (Lei ca Jung Frigocut 2800E, Richmond, IL). Every other tissue slice was thionin stained and visualized using a microscope under conventional bright field illumination. Cannula tip placements were verified and mapped onto standardized coronal sections of the rat brain (Paxinos and Watson, 2005) Data Analyses Baseline working memory performance of young and aged rats in this cohort was assessed using a two factor repeated measure ANOVA (Age X Delay) as in Experiments 1 and 2 The effects of drug manipulation s were assessed using three factor repeated measures ANOVA as in Experiment 2, wit h both Drug Dose and Delay as repeated measures variables and Age as a between subjects variable Post hoc comparisons within each age group were performed using two factor r epeated measures ANOVAs (Drug Dose X Delay). When justified, additional two factor ANOVAs were used to compare individual drug doses against vehicle within each age group to determine those doses that significantly affected performance.

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107 Results Experimen t 1: GABA Signaling Protein Expression and Working Memory Abilities Working Memory Performance is Impaired in Aged F344 Rats In order to relate age related changes in GABAergic signaling protein expression to PFC dependent cognitive abilities, young and ag ed rats were characterized on a delayed response task that a ssesses working memory (task schematic shown in Figure 3 1A). Previous work has shown that performance on this task critically depends upon the mPFC and that aged rats are impaired relative to you ng cohorts (Sloan et al., 2006; Beas et al., 2013) Delayed response p erformance of youn g and aged rats is shown in Figure 1B. Consistent with previous findings, a two factor repeated measures ANOVA ( A ge X D elay) indicated that both young and aged rats sho wed reduced accuracy as delays increased (main effect of D elay: F (6, 96 ) =65.69 p< 0 .001 ). Notably, however, aged rats performed significantly worse than young cohorts (main effect of A ge: F (1, 16 ) =7.07 p =.02 ) and this impairment was disproportionately evi dent at long delays (interaction between D elay and A ge: F (6,96 ) =3.14 p=.0 1). Given that the greatest magnitude age related deficits occurred at the longest two delays tested (i.e., 18 and 24 s), performance at these delays was averaged for each subject an d this value (Mean Long Delay) was used as an index of individual working memory ability. Figure 3 1C shows the Mean Long Delay performance for individual young and aged rats.

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108 Age Related Alterations in GABAergic Signaling Protein Expression in Aged mPFC Figure 2A shows representative immunoreactive bands from young and aged mPFC samples when incubated with antibodies to GABA(B) receptor R1 or R2 subunits, GAT 1, GAD67, or VGAT. As GABA(B)Rs are obligate heterodimers and functional receptors require at lea st one GABA(B)R1 subunit with one GABA(B)R2 subunit (Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998) mPFC expression of both R1 and R2 subunits was assessed. In addition, note that two distinct bands were detected for GABA(B)R1 (130kD and 95 kD), corresponding to the two different isoforms of this subunit: GABA(B)R1a and GABA(B)R1b, respectively (Kaupmann et al., 1997) The GABA(B)R1a isoform contains a pair of short consensus repeats at the N terminal that traffick GABA(B)R complexes containin g this isoform to presynaptic terminals where these receptors modulate neurotransmitter release (Biermann et al., 2010) Conversely, GABA(B)R1b lacks this N terminal extension and is primarily localized to dendrites where it mediates postsynaptic inhibitio n (Vigot et al., 2006) Given the distinct localization and function of these R1 isoforms, R1a and R1b were analyzed separately. In agreement with previous work from our lab (McQuail et al., 2012), expression of both R1 isoforms and of the R2 subunit were robustly and significantly reduced in aged compared to young mPFC ( R1a: 29%, t (16) =2.80, p=0.01, Figure 3 2B; R1b: 42% t (16) =3.42, p=0.003 Figure 3 2C; and GABA(B) R2: 28%, t (16) =2.67, p=0.02, Figure 3 2D ). In addition, bivariate correlation analyses w ere performed on individual data from aged rats in order to compare the expression of each GABA(B)R isoform and subunit to individual performance on the delayed response task. As shown in Figure 3 2B, expression

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109 of the GABA(B)R1a isoform in aged rats was n ot significantly associated with Mean Long Delay performance (R1a: r= .48, p=.12; Figure 2B). In contrast to R1a, expression of both R1b and R2 in aged rats was significantly and negatively associated with Mean Long Delay performance ( R1b: r= 0.66, p=0.02, Figure 3 2C; R2: r= 0.75, p=.01, Figure 3 2D), such that lower expression was associated with better performance on the working memory task. Bivariate correlation analyses were also performed separately on data from young rats and in no cases were signifi cant relationships observed between individual GABA(B)R subunit expression and Mean Long Delay performance (R1a: r= 0.12, p=0.82; R1b: r=0.28, p=0.60; R2: r= 0.03, p=0.96). The primary neuronal GABA transporter, GAT 1 has been strongly implicated in GABA(B )R occupancy (Gonzalez Burgos et al., 2009) therefore expression of GAT 1 was also evaluated in the same membrane homogenates. Similar to GABA(B)R expression, GAT 1 was significantly reduced in aged compared to young mPFC ( 31%, t (16) =3.82, p=.002, Figure 3 2E). Notably, however, no significant relationship was observed between GAT 1 expression and Mean Long Delay performance in aged rats (GAT 1: r= 0.30, p=0.34). Likewise, there was no significant relationship between GAT 1 expression and working memory a bilities among young rats (GAT 1: r= 0.42, p=0.15). Given the marked reduction of GABA(B)Rs and GAT 1 expression in aged mPFC described above, subsequent experiments were performed to provide additional information regarding how aging influences inhibitor y synapses in this brain region. Specifically, expression of the GABA synthesizing enzyme, GAD67, and of the vesicular transporter for GABA, VGAT, was evaluated in the mPFC of

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110 young and aged rats. As shown in Figure 3 2F, GAD67 expression was robustly and significantly elevated in aged mPFC compared to young (GAD67: +80%, t (11) = 2.80, p=.02). In contrast, VGAT expression in mPFC did not differ between young and aged rats (VGAT: t (12) =1.03, p=0.33; Figure 3 2G). As described in the methods section, tissue from only a subset of the rats was available for GAD67 and VGAT immunoblots. Analysis of delayed response performance of the rats included in these assays revealed the same working memory impairment in aged rats compared to young that was observed in the larger cohort [(GAD67 main effect of Delay: F (6,66) =68.31, p<0.001; main effect of Age: F (1,11) =7.27, p=0.02; interaction between Delay and Age: F (6,66) =4.68, p=0.001) (VGAT main effect of Delay: F (6,72) =99.51, p<0.001; main effect of Age: F (1,12) =7.08, p=0.02; interaction between Delay and Age: F (6,66) =5.08, p<0.001)]. Notably, no significant relationships were observed between GAD67 or VGAT expression in mPFC and Mean Long Delay performance in the working memory assessment among aged (VGAT 1: r= 0.34, p =0.40; GAD67: r=0.22, p=0.64) or young (VGAT: r= 0.30, p=0.47 ; GAD67: r= 0.30, p=0.56) rats. Experiment 2: Systemic Administration of the Selective GABA)B)R Antagonist CGP55845 Restores Working Memory Performance in Aged Rats Data from Experiment 1 show t hat expression of GABAergic signaling proteins is altered in aged mPFC in a manner that is consistent with increased inhibition of cortical pyramidal neurons (Luebke et al., 2004; Bories et al., 2013) Moreover, reduced expression of postsynaptic GABA(B)Rs (GABA(B)R1b subunit) is significantly associated with better performance on the delayed response task. Together, these data support the hypothesis that reducing

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111 GABA(B)R activity should improve working memory performance in aged rats. To test this hypothe sis, performance of young and aged rats was evaluated in the delayed response task following systemic administration of the selective GABA(B)R antagonist CGP55845 or vehicle. Baseline performance prior to dr ug administration, shown in Figure 3 3A, was simi lar to that observed in Experiment 1. A two factor ANOVA (Age X Delay) indicated that both young and aged rats performed less accurately at longer delays (main effect of Delay: F (6,126) =110.72, p<.001) and that aged rats performed significantly worse than young rats (main effect of Age: F (1,21) =9.23, p=0.006: Figure 3 3A), particularly at longer delays (interaction between Delay and Age: F (6,126) =3.94, p=0.04). With respect to the effects of GABA(B)R blockade, a three factor ANOVA (Age X Delay X Drug Dose) revealed main effects of Delay (F (6,252) =89.53, p<.001) and Drug Dose (F (2,42) = 3.64, p=0.04) as well as a significant interaction between Age and Drug Dose (F (2,42) = 7.42, p =.002). No other main effects or interactions were statistically significant. To better understand the nature of the interaction between age and the effects of the GABA(B)R antagonist on delayed response performance, two factor ANOVAs (Drug Dose X Delay) were performed separately in young and aged rats. Among aged rats, there were significant main effects of Delay (F (6,72) = 98.46, p<.001 ) and Drug Dose (F (2,24) = 7.81, p=0.002 ) but no interaction between these two variables. Given the main effect of Drug Dose, subsequent post hoc analyses were performed in order to individually compare the two doses of GABA(B)R antagonist to vehicle and to each other. These comparisons showed that in aged rats, 0.1 mg/kg (F (1,12) = 16.89, p=0.001 ), but not 0.01 mg/kg CGP55845 (F (1,12) =0 .93, p=0.35 ), significantly enhanced performance

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112 relativ e to the vehic le condition (Figure 3 3B). The 0.1 mg/kg dose also significantly enhanced performance relative to the 0.01 mg/kg dose (F (1,12) =6.76 p=0.02). In contrast to the enhancing effects of GABA(B)R antagonist administration in aged rats, systemic administration of CGP5545 impaired working memory performance in young rats. A two factor ANOVA (Drug Dose X Delay) revealed main effects of Delay (F (6,54) = 41.48, p<0.001 ) and Drug Dose (F (2,18) = 3.93, p=0.04 ), but no interaction between these two variables. Post hoc com parisons showed that the 0.01 mg/kg (F (1,9) = 7.17, p=0.025 ) but not the 0.1 mg/kg dose (F (1,9) = 3.06, p=0.11 ) significantly impaired working memory performance of young rats compared to vehicle conditions. There was no significant difference in performance b etween the 0.1 mg/kg and the 0.01 mg/kg doses (F (1,9) = 1.25, p=0.29 ). Experiment 3: Intra mPFC Infusions of the Selective GABA(B) Receptor Antagonist CGP55845 Restores Working Memory Performance in Aged Rats Data from Experiment 2 demonstrate that blocking GABA(B)Rs via systemic administration of CGP55845 significantly improves working memory performance in aged rats. To determine whether these effects were mediated by actions in mPFC, young and aged rats were implanted with guide cannulae targeting mPFC. F igure 3 4A shows the location of cannula placements for rats used in this study. Figure 3 4B shows pre drug baseline performance of these young and aged rats prior to drug microinjections. A two factor ANOVA (Age X Delay) indicated that both young and aged rats performed less accurately at longer delays (main effect of delay: F (6,96) =76.84, p<0.001) and that aged rats performed

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113 significantly worse than young rats (main effect of Age: F (1,16) =7.66, p=0.02: Figure 3 3B). With respect to the effects of mPFC G ABA(B)R blockade, a three factor ANOVA (Age X Delay X Drug Dose) revealed a main effect of Delay (F (6,96) =104.94, p<.001) and significant interactions between Age and Delay (F (6,96) = 3.15, p =0.007 ) and Age X Drug Dose (F (3,48) = 2.92, p =0.04). No other main efects or interactions were observed. To better understand the nature of the interaction between age and the effects of mPFC GABA(B)R blockade on delayed response performance, two factor ANOVAs (Drug Dose X Delay) were performed separately in young an d aged rats. Among aged rats, there were main effects of Drug Dose (F (3,27) =3.16, p =.04) and Delay (F (6,54) =75.52, p<.001) but no interaction between these variables. Post hoc ANOVAs comparing each dose with vehicle revealed and each other that the 0.6 m ol dose of CGP55845 (F (1,9) =6.25, p=0.03), but not the 0.2 mol (F (1,9) =0.29, p=0.61) or 2.0 mol (F (1,9) =.81, p=.37) doses, significantly improved working memory performance relative to vehicle conditions (Figure 3 3C). Furthermore, performance at the 0.6 mol dose of CGP55845 was significantly better compared to the 0.2 mol dose (F (1,9) =6.83, p=0.03), whereas no other dose comparisons yielded significant differences in performance (0.2 mol vs 2.0 mol: F (1,9) =4.07, p=0.07; 0.6 mol vs 2.0 mol: F (1,9) = 61.45, p=0.26). Performance on the non drug days that intervened between drug microinjection days was also monitored to assess non specific effects on performance of repeated mPFC microinjections. A two factor ta from these intervening days indicated the expected main effect of Delay (F (6,54) =58.52, p<.001), but no main

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114 effect of Day (F (4,36) =1.44, p=0.24) nor a Day X Delay interaction (F (24,216) =0.99, p=0.48), indicating that there were no adverse effects on pe rformance of repeated mPFC microinjections. In contrast to aged rats, a two factor (Delay X Drug Dose) ANOVA performed on data from young rats revealed a main effect of Delay (F (6,42) =1.31, p=0.18) but no main effect or i nteraction involving Drug Dose. Discussion Working memory, or the ability to hold task relevant information in mind, is a foundational aspect of cognition that is vulnerable to decline with normal aging. Persistent activity of PFC pyramidal neurons is a central feature of most neural mod els of working memory, and input to these neurons from diverse classes of GABAergic interneurons is considered essential for providing spatial and temporal specificity in the encoding and maintenance of to be remembered information (Goldman Rakic, 1995; Ar nsten, 2013) Data presented in the current study demonstrate that mPFC GABAergic signaling proteins become dysregulated in normal aging, and suggest that these changes contribute to increased pyramidal neuronal inhibition and a reduced ability to maintain information in working memory stores. In direct support of this idea, we demonstrate that reducing inhibition via GABA(B)R antagonist administration both systemically and directly within mPFC can significantly enhance working memory performance in aged ra ts to a level on par with young adult cohorts. Increased Inhibition in Aged Prefrontal Cortex Electrophysiological recordings from PFC in both rodents and nonhuman primates indicate that inhibitory input onto PFC pyramidal neurons can increase

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115 with aging (Luebke et al., 2004; Bories et al., 2013) One previous study described a reduction in excitatory postsynaptic spontaneous currents (PSC) coupled with an increase in inhibitory PSCs onto layer II/III pyramidal neurons in aged non human primate PFC compare d to young (Luebke et al., 2004). More recently, Bories et al. (2013) reported that miniature inhibitory postsynaptic potentials onto mPFC pyramidal neurons were increased in aged rats that were impaired on a corner exploration behavioral task. The biochem ical data presented in the current study provide further evidence for an age related increase in the inhibition of mPFC pyramidal neurons. First, the increase in GAD67, the rate limiting enzyme for GABA synthesis, suggests that there is elevated GABA prod uction in aged mPFC. Second, the R1a isoform of the GABA(B)R, which is localized to presynaptic terminals and negatively regulates neurotransmitter release, was significantly reduced in aged PFC. Loss of the GABA(B)R on inhibitory terminals would be expec ted to attenuate autoinhibition of GABA release. Finally, GAT 1 expression was significantly reduced in aged mPFC. GAT 1 plays an integral role in clearing GABA from the synapse by translocating GABA through the neuronal membrane, and several studies have localized GAT 1 extrasynaptically on presynaptic terminals (Minelli et al., 1995; Conti et al., 2004) where it has been suggested to play an important role in preventing synaptic GABA spillover and maintaining GABA homeostasis. Electrophysiological data from studies in which GAT 1 is selectively blocked or genetically deleted clearly indicate that reduced GAT 1 expression results in an increase in extracellular GABA concentrations (Frahm et al., 2001; Jensen et al., 2003; Chiu et al., 2005; Gonzalez Burgo s et al., 2009) Together, this

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116 constellation of biochemical alterations would be expected to produce increased GABAergic inhibition of pyramidal cells in aged PFC. Against the background of these changes, postsynaptic GABA(B)R subunit expression was robus tly attenuated in aged PFC. This finding agrees with a previous study from our laboratory in which expression of both R1 and R2 subunits of the GABA(B)R was reduced in aged PFC (McQuail et al., 2012) While rats were not characterized on a PFC dependent be havioral task in that prior study, findings herein show that attenuated GABA(B)R expression is significantly and inversely related to mPFC dependent working memory. Notably, this relationship was only observed with the GABA(B)R2 subunit and the postsynapti c GABA(B)R1b isoform of the R1 subunit, but not with the presynaptic R1a isoform of the R1 subunit. Postsynaptic GABA(B) receptors are largely located extrasynaptically and are sensitive to alterations in tonic GABA and GABA spillover from active synapses (Kulik et al., 2003; Kulik et al., 2006; Glykys and Mody, 2007) Indeed, previous work by Wang et al. (2010) has indicated that GABA(B) receptors mediate a small tonic inhibitory current in layer 2/3 mPFC pyramidal neurons in young rats. The data present ed here support a model in which interneuron dysfunction in aging contributes to elevated GABA availability and increased GABA(B) mediated tonic activity in aging. In this context, the downregulation of GABA(B)Rs may reflect an effective compensatory mecha nism for preserving optimal neuronal excitability and working memory abilities.

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117 GABA(B) Receptor Antagonist Administration Improves Working Memory in Aging Consistent with the relationship between lower endogenous GABA(B)R expression and better working me mory in aging, both systemic and intra mPFC administration of a GABA(B)R antagonist (CGP55845) significantly improved working memory performance of aged rats. Indeed, with both routes of administration, doses of CGP55845 were identified which completely re stored working memory performance in aged rats to a level on par with young performance. These cognitively enhancing effects are consistent with prior reports that have primarily focused on the efficacy of this class of GABA(B)R antagonists in improving hi ppocampal mediated cognition in young and aged rats (Mondadori et al., 1993; Froestl et al., 2004; Helm et al., 2005; Lasarge et al., 2009; Froestl, 2010; Kleschevnikov et al., 2012) Interestingly, systemic injections of the same doses of CGP55845 impaire d young rat performance in the delayed response task. One interpretation of these differential effects in young and aged rats is that GABA(B)R blockade in young rats opposes GABAergic transmission that is optimal for working memory. Blocking GABA(B)R activ ity in young rats may shift the mPFC toward a hyper excitable state which could ultimately be as deleterious to working memory as the excessive inhibition that may accompany advanced age. Indeed, in schizophrenia, interneuron dysfunction and PFC hyperexcit ability are thought to contribute to impaired working memory, a hallmark feature of this disorder (Lewis et al., 2005; Yizhar et al., 2011) Alternatively, it is possible that the impairing effects of GABA(B)R blockade in young animals have a site of acti on that is distinct from the enhancing effects of this drug at advanced ages. Data to date support a model in which GABA(B)

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118 blockade in aged mPFC serves to attenuate the effects of postsynaptic and/or extrasynaptic GABA ( B ) receptors expressed by mPFC pyram idal neurons. However, GABA(B)Rs are also localized presynaptically where their blockade could contribute to excess GABA release and increased pyramidal neuron inhibition. It is interesting to speculate that in young animals, GABA(B) antagonists might serv e to disinhibit GABA terminals themselves, ultimately resulting in greater pyramidal neuron inhibition, an effect which could confer impaired working memory. It is notable that while a similar pattern of impaired performance in young rats was observed foll owing both systemic and intra mPFC administration of the GABA(B)R antagonist, the effects did not reach statistical significance when the drug was delivered intracerebrally. As such, it is important to consider the possibility that critical site of action for the impairing effects of GABA(B)R antagonists may be outside of the mPFC. Overall, a better understanding of the synaptic dynamics of GABA(B)R signaling in both the young and aged brain will be essential for clarifying the role of these receptors in n ormal cognition and in age related cognitive disorders. The current biochemical and pharmacological findings as well as several prior electrophysiological studies suggest that pyramidal neurons may be subject to greater inhibition in aged rats. However, it is also important to consider that numbers of both interneurons and inhibitory, i.e.symmetrical, synapses can decline with age (de Brabander et al., 1998; Wong et al., 2000; Jacobs et al., 2001; Poe et al., 2001; Uylings and de Brabander, 2002; Duan et al., 2003; Stranahan et al., 2012) In the current study, expression of VGAT, which is critical for packaging GABA into synaptic vesicles and thus should be present in

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119 all inhibitory terminals, was stable across ages. While the VGAT data support a relative preservation of inhibitory terminals in this model, the failure of this measure to recapitulate the previous anatomical reports of reduced inhibitory synapse number may reflect insufficient sensitivity of the methodologies employed here to detect inhibito ry terminal loss in cortical microcircuits. Alternatively, it is certainly possible given the other dynamic changes in GABA signaling proteins reported here, that while overall synapse number is reduced, there is an upregulation of VGAT expression with ag e in remaining inhibitory synapses. Given the diversity of interneurons and evidence that interneuron subclasses can be differentially sensitive to changes with age (Potier et al., 1994; Shetty and Turner, 1998; Vela et al., 2003) it will be important in future work to determine whether the findings described here are a pervasive feature of aged PFC interneurons, or rather if these biochemical alterations reflect very robust but anatomically restricted effects in specific interneuron subclasses. The prese nt study provides evidence that GABAergic substrates in mPFC are dysregulated during normal aging. The pattern of changes in protein expression suggest that mPFC pyramidal neurons are subject to greater inhibition with advanced aging and that downregulatio n of GABA(B)R expression in these neurons reflects an effective compensatory mechanism for preserving working memory function. GABA(B)R expression was strongly and inversely related to working memory abilities such that lower expression was associated wit h better preservation of working memory function in aged rats, and GABA(B)R blockade effectively reversed age related impairments in working memory. These findings highlight that altered excitatory inhibitory dynamics within the aged PFC

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120 may contribute to working memory decline in aging, and that targeting GABA(B)Rs may provide therapeutic benefits for improving cognitive functions supported by this brain region.

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121 Figure 3 1. Wor king m emory is i mpaired in a ged F344 r ats. A ) shows a schema tic of the delayed response task used to asses working memory ability. There are three phases to this task. During the sample phase rats are presented with either a left or right lever. After the rat presses the extended lever, the delay phase begins, dur ing which both levers are retracted for a variable time period ranging from 0 to 24 seconds, during which the rat must nosepoke into the food trough to initiate the choice phase. During the choice phase, both levers are presented and the rat must choose th e lever presented in the sample phase in order to obtain a food reward B ) shows young and aged performance on the delayed response task. Aged rats displayed significantly less accurate performance relative to young and were disproportionately impaired at long delays (young n=6, aged n=12 ). C) shows individual young and aged rats plotted by Mean Long Delay (average of choice accuracy at 18 24 s) on the delayed response task. This measure was used as an index of individual working memory ability. See text f or statistical analysis.

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122 Figure 3 2. Age related changes in GABAergic signaling protein expression and relationship to working memory ability. A ) shows representative immunoreactive bands from young and aged mPFC homogenates following incubation wit h antibodies to the GABA(B)R subunits R1 and R2, the GABA transporter GAT 1, the GABA synthesizing enzyme GAD67, the vesicular GABA transporter VGAT and loading control GAPDH. B ) In the aged mPFC, GABA(B)R1a expression was significantly reduced compared t o young; however, as shown in the scatter plot of individual aged rats, there was no significant relationship between GABA(B)R1a expression and delayed response performance (young n=6, aged n=12). C) Expression of GABA(B)R1b was significantly reduced in ag ed mPFC compared to young. The scatter plot of individual aged rats shows that, in contrast to GABA(B)R1a, GABA(B) R1b expression was significantly and inversely associated with delayed response performance such that lower expression was associated with be tter working memory ability (young n=6, aged n=12). D) GABA(B)R2 expression was also significantly reduced in aged mPFC compared to young (young n=6, aged n=12). Like the R1b isoform, the scatter plot of protein expression of individual aged rats shows tha t GABA(B)R2 expression in the mPFC is significantly and inversely related to working memory performance such that those aged rats with the lowest levels of GABA(B)R2 exhibited better working memory. E ) Expression of GAT 1 was significantly decreased in age d mPFC compared to young (young n=6, aged n=12). F ) In contrast to all other signaling proteins examined, expression of GAD67, was significantly elevated in aged mPFC compared to young (young n=6, aged n=7). Neither GAT 1, nor GAD67, expression in mPFC wa s reliably related to working memory performance. G) Notably, expression of VGAT, did not differ between young and aged mPFC, supporting that aging does not produce a robust loss of inhibitory terminals in this brain region (young n=6, aged n=8). See text for statistical analyses. Asterisks indicates significant differences (p<0.05).

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123 Figure 3 3. Systemic GABA(B) receptor antagonist administration significantly improves working memory performance in aged rats. A) shows baseline performance on the delayed response task of young (n=10) and aged (n=13) rats prior to drug administration Aged rats displayed significantly less accurate performance relative to young and were disproportionately impaired at long delays. B ) Sys temic injection of the selective GABA(B)R antagonist CGP55845 (shaded circles) significantly improved performance of aged rats in the delayed response task over vehicle conditions (open circles). Note that at the highest dose administered (black circles), accuracy of aged rats was restored to a level on a par with young baseline performance (open triangles in panel A). C ) In contrast to drug effects observed in aged rats, systemic injection of CGP55845 (shaded circles) significantly impaired working memory performance in young rats compared to vehicle conditions (open circles). See text for statistical analyses.

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124 Figure 3 4 Intra mPFC GABA(B) receptor antagonist administration improves working memory performance in aged rats. A) shows bilateral cann ula placements in mPFC for each rat included in this experiment (schematic illustrations modified from Paxinos and Watson, 2007) B) shows baseline delayed response task performance of young (n=8) and aged (n=10) rats prior to drug administration. Aged ra ts displayed significantly less accurate performance relative to young. C) Direct mPFC administration of the selective GABA(B)R antagonist CGP55845 (shaded circles) significantly enhanced working memory performance in aged rats over vehicle conditions (op en circles). Note that administration of the 0.6 mol dose (dark gray circles), improved accuracy of aged rats to a level on par with young baseline performance (open triangles in panel B). D) In contrast to drug effects observed in aged rats, direct mPFC administration of CGP55845 (shaded circles) did not significantly affect working memory performance of young rats compared to vehicle conditions (open circles). See text for statistical analyses.

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125 CHAPTER 4 SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS At the end of Chapter 1, the importance of maintaining homeostatic balance between excitation and inhibition was emphasized as crucial for optimal cognition across the lifespan. To date, the majority of cognitive aging studies have focused on changes in exci tatory networks; however, emerging data suggests that cognitive impairments in aging may also be impacted by age related changes in inhibitory systems (Luebke et al., 2004; Bories et al., 2013) The primary aim of the this dissertation was to investigate h ow age related changes in GABAergic systems contribute to impairments in hippocampal dependent long term memory and prefrontal cortical dependent working memory, two aspects of cognition that are particularly vulnerable to decline in aging. Indeed, stri king GABAergic changes were described in the experiments of this dissertation project. Interestingly, these changes in inhibitory systems were significantly related to performance in behavioral tasks that are critically dependent on the hippocampus and me dial prefrontal cortex. In Chapter 2, y oung and aged rats were behaviorally characterized on a spatial memory task that is critically dependent on basal forebrain neuronal populations that project onto the hippocampus. We first used w estern blotting to qua ntify the expression of GABAergic signalin g proteins in the hippocampus of young and aged rats. Secondly, we conducted a quantitative investigation of the hippocampal projecting neuronal populations of the medial septum/diagonal Band of Broca complex of t he basal forebrain in a natural rodent model of cognitive aging utilizing confocal microscopy and unbiased stereological methods to assess whether neuron numbers change across the life span of F344 rats. In the

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126 hippocampus, we saw a significant decrease i n GAD67 expression in aged animals that displayed spatial learning impairments. Cholinergic MS DBB neurons did significantly, albeit modestly, decrease with age. This decrease in cholinergic neurons was not significantly related to spatial learning perfo rmance among aged rats. The major finding in this study was that GABAergic projection neurons of the MS DBB complex were selectively increased in aged F344 rats that displayed spatial learning impairments. Among aged rats, there was a significant inverse correlation between GABAergic neuronal number and spatial learning ability such that those animals that performed the best in the spatial learning task had the lowest number of GABAergic basal forebrain projecting neurons. Interestingly, an assessment of NeuN positive cells revealed that overall neuron numbers in the MS DBB complex did not change across the lifespan or across cognitive groups. The important role that basal forebrain projection systems play in cognitive processes is well documented (Rawlin s et al., 1979; Sarter and Bruno, 2000; Ryan M. Yoder, 2005; Colom et al., 2006) Basal forebrain nuclei innervate cortical structures and these innervations are crucial for higher order processes such as learning, memory, attention, emotion and motivatio n (Olton et al., 1991) These same basal forebrain GABAergic neurons that are selectively and significantly increased in spatially impaired aged rats project to other areas of the brain that are known to be vulnerable to changes in age. One such target area is the olfactory bulb which receives cholinergic and GABAergic innervations from the medial septum and the diagonal band of Broca (Senut et al., 1989; Schliebs and Arendt, 2011) Sense of smell is integral to maintaining quality of life as we

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127 age. U nfortunately, olfactory dysfunction affects nearly half of all the people 65 80 years old in the United States. Nearly 75% of people in the U.S. over the age of 80 experience some type of olfactory deficit. There is a vast literature that describes h ow cholinergic neurons from the medial septal nuclei of the basal forebrain directly modulate neural activity within the olfactory bulb and the piriform cortex, a cortical region that is important for olfactory information processing (de Almeida et al., 20 13) The majority of studies that have examined this projection system in aging have focused on al terations to the GABAergic component of the basal forebrain olfacotry bulb/piriform cortex projection system. As shown in Chapter 2, these GABAergic projection neurons undergo marked changes in age and these changes are significantly related to spatial me mory performance. This is particularly important in light of the fact that previous work in our lab found that the same aged F344 rats that showed impairment on a simple odor discrimination task were impaired in the water maze task (LaSarge et al., 2007) ( Figure 4 1). Interestingly, systemic injections of CGP55845, the same selective GABA(B) antagonist shown to improve working memory in aged rats in Chapter 3, completely reversed olfactory learning deficits in aged rats (Lasarge et al., 2009) (Figure 4 2). Together, the data in Chapter 2 and from the previous studies in our lab suggest that there is an age related increase in inhibitory projections from the basal forebrain to the olfactory bulb that may influence chemosensory processing in the

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128 bulb and olf actory learning and memory in the piriform cortex. Future studies are needed to explore this possibility. In Chapter 3, we describe d significant changes in GABAergic signaling proteins in the prefrontal cortex of aged rats behaviorally characterized in a delayed response test of working memory. Western blot data indicated that there was increased ambient GABA in the aged prefrontal cortex compared to young due to increased synthesis of GABA and decreased GABA reuptake. Expression of metabotropic GABA (B)Rs which are located extrasynaptically and activated largely by GABA that spills over from the synapse was also reduced in aged rats. This reduction of GABA(B)Rs among aged rats was significantly related to performance on the delayed response task such that those aged rats that had the lowest levels of GABA(B)R exhibited the best working memory ability. This significant relationship suggested that reduction of GABA(B)Rs was a successful compensation for increased ambient GABA in the aged mPFC. It is n ot uncommon for receptors to be downregulated in the presence of excess neurotransmitter (Frechilla et al., 2001) We then tested whether blocking GABA(B)R activation with a selective GABA(B)R antagonist could improve working memory in aged rats. Both sy stemic injections and direct mPFC infusions of the GABA(B)R antagonist CGP55845 improved working memory performance of aged rats. Data from Chapter 3 clearly demonstrates that GABAergic systems in the medial prefrontal cortex are dysregulated in aged rats Important questions regarding age related changes to local inhibitory networks in mPFC still remain unexplored. One interesting question is how interneuron populations of the

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129 mPFC change with age and in relation to working memory ability. While there is evidence that interneurons in the primate prefrontal cortex do decline with age, very few studies have explored whether interneurons in the rat mPFC change with age. Stranahan et al. (2012) reported a significant decrease in principal and GAD67 immunop ositive cells in the dorsal prefrontal cortex of rats behaviorally characterized in the hippocampal dependent water maze task. No correlation between cell number and spatial learning ability was detected. A comprehensive quantification using confocal ster eology of mPFC principal and interneurons in young and aged rats behaviorally characterized in the mPFC dependent delayed response task would be an informative future study. This study could be expanded to include synaptic markers to determine if synaptic integrity remains intact in the aged mPFC. In Chapter 2 of this dissertation, I described an increase in basal forebrain GABAergic neurons that project to the hippocampus selectively in aged rats that exhibit spatial learning impairments. These same neur ons project to the medial prefrontal cortex, the same region of the brain where I described significant changes in GABAergic circuitry in aged rats in Chapter 3. Specifically, GAD67 expression is significantly increased in the prefrontal cortex of aged ra ts compared to young. While it is probable that the increase in GAD67 and other changes in GABAergic signaling protein expression in the prefrontal cortex may be due to intrinsic processes, it is interesting to speculate whether these changes may be influ enced by extrinsic inhibitory influences (Appendix 1 2)(Figure 4 3).

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130 Final Thoughts and Future Directions From the studies conducted in this dissertation, basal forebrain GABAergic projection systems have emerged as important innervations that can hav e far reaching influences on cortically dependent cognition. These projections have historically been understudied in the context of natural aging with much emphasis being placed on age related changes to excitatory neuronal systems. An intriguing future direction would be to explore aged related changes in basal forebrain projection patterns using a combination of antero and retrograde tracers and immunohistochemistry to visualize distinct vesicular transporters. Henny and Jones (2008) used this techni que to characterize innervation patterns of MCPO projecting neurons in the rat prefrontal cortex. This technique could be used to compare basal forebrain innervation patterns of young and aged rats and determine how these innervations change with age. Da ta from the preliminary studies outlined in Appendices A and B clearly demonstrate that basal forebrain coritcopetal GABAergic neurons can directly impact cognition mediated by the prefrontal cortex. This projection system is much less studied than the s eptohippocampal system critical to hippocampal dependent spatial memory and future studies are necessary to explore the impact these projections have on prefrontal cortical mediated working memory and other executive functions that are also known to declin e at advanced ages. More specifically, future studies are necessary to elucidate how GABAergic basal forebrain corticopetal projections change with age and how these changes may be related to prefrontal cortical impairments in aging.

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131 Finally, a future l ine of study must begin to explore the role of basal forebrain glutamatergic projection neurons on hippocampal and prefrontal cortical mediated cognition and how this role changes with age. Historically, basal forebrain projection s were thought to largely consist of Cholinergic and GABAergic neurons (Swanson and Cowan, 1979). Recently, a glutamatergic neuronal population which projects to the medial temporal lobe was described in the basal forebrain (Colom et al 2005). The role that these neurons play i n age related cognitive decline is still unknown. As we begin to appreciate how age related shifts in excitation and inhibition can profoundly influence cognition, the basal forebrain emerges as an important region whose projection systems are well positi oned to modulate homeostasis throughout the cortex. It will be interesting to explore the possibility of modulating basal forebrain projections to restore homeostasis in cortical regions vulnerable to changes in age and critical to cognitive processes.

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132 Figure 4 1. Performanc e of s patially characterized r ats on d iscrimination Learning. A) There was a main effect of spatial performance group (F (2, 13) = 7, p<0.01) such that aged spatially impaired rats took significantly more trials t o reach criterion compared to both young and aged spatially unimpaired rats (p<0.05 in both cases). B) Digging media discrimination performance was comparable among all groups (LaSarge et al 2007).

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133 Figure 4 2 GABA(B) a ntagonist r everse s a ge r elated o dor l earning deficits. Mean odor discrimination performance after administration of the GABA(B) antagonist CGP55845 or saline vehicle in young adult, aged learning unimpaired, and aged learning impaired rats. There was a significant cognitiv e age group by drug condition interaction such that treatment with 0.01 and .1 mg/kg of CGP55845 improved the trials to criterion measure specifically in the aged learning impaired group

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134 Figure 4 3. Basal forebrain projections to the hippocampus and prefrontal cortex.

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135 APPENDIX A BASAL FOREBRAIN MAGN OCELLULAR PREOPTIC A REA AND HORIZONTAL DIAGONAL BAND OF BROCA GABAER GIC NEURONS INNERVATE INTERNEURO NS OF THE MEDIAL PRE FRONTAL CORTEX The basal forebrain sends parallel choliner gic and GABAergic proj ections to the cortex (Colom et al., 2005; Kiss et al., 1990; Gritti et al., 1993; Rye et al., 1984; Saper et al., 1984; Fisher et al., 1988). These projecting neurons originate in the magnocellular preoptic (MCPO) area and the h orizontal diagonal band of Broca. Anterograde and retrograde tracer studies indicate that basal forebrain GABAergic corticopetal projections innervate GABAergic interneurons in the prefrontal corte x (Fr eu nd and Gulyas, 1991; Fr eu nd and Meskanaite, 1992). These cortical GABAergic interneurons project to multiple pyramidal cells in the PFC (Fr eu nd et a l. 1983). A more recent study by Henny and Jones (2008) reports that inhibitory synapses from the basal forebrain are found on pyramidal cells as well as in terneurons, suggesting that the projections form the basal forebrain are not as organized as previously thought. Regardless, these GABAergic projection neurons are well situated to significantly influence prefrontal cortical mediated cognitive tasks. GAB Aergic neurons projecting onto inhibitory interneurons in the prefrontal cortex are thought to decrease interneuron activity and, thus, disinhibit pyramidal cells resulting in increased pyramidal cell activation (Sarter and Bruno, 2002). This increased py ramidal cell activity could significantly affect information processing in the prefrontal cortex. To begin to investigate the role of basal forebrain corticopetal GABAergic projection neurons on prefrontal cortical mediated cognition we first verified th at GABAergic neurons from the magnocellular preoptic (MCPO) area did indeed

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136 project to the medial prefrontal cortex. To do this, we iontophoretically injected the anterograde tracer biotniylated dextram amine (BDA) 10,000 direclty into the MCPO of young rats. Seven days after injection, rats were sacrificed and their tissue was processed for immunohistochemical visualization of BDA, the vesicular GABA transporter VGAT and interneuron markers (GAD67, parvalbumin, and calbindin). Figure A 1 shows a calbin din positive neuron in the medial prefrontal cortex with colocalized BDA and VGAT puncta, indicating GABAergic innervation of medial prefrontal cortical interneurons from the basal forebrain. This preliminary experiment verified that neurons of the MCPO d o, in fact, innervate inhibitory interneurons in the prefrontal cortex.

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137 Figure A 1. Basal forebrain neurons project to the prefrontal cortex.

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138 APPENDIX B DIRECT APPLICATION O F THE SELECTIVE M3 A GONIST CEVEMILINE ON BASAL FOREBRAIN IMPROVES WORKING MEM ORY PERFORMANCE IN YOUNG RATS. A second preliminary study followed investigating whether direct, selective modulation of basal forebrain GABAergic corticopetal neurons influenced performance on the delayed response task, a prefrontal cortical mediated task of working memory. In the basal forebrain, projecting GABAergic neurons are selectively activated by drugs targeting the M3 muscarinic receptor (Alreja et al ., 2000; We et al., 2000; Wu et al., 2003; Liu et al., 1998; Xu e al., 2006). Young rats were surgically implanted with guide cannula directed at the magnocellular preoptic area. After a 2 week recovery period, rats were trained on the delayed response task. Figure B 1 shows baseline performance of young rats after 5 days of stable working memory training at all delays. Using a within subject design, all rats received direct infusions of vehicle and 2 doses of cevimeline, a selective M3 muscarinic receptor agonist, (5mg and 10 mg) over the course of pharmacological testing Microinfusions were administered 5 minutes prior to testing on the delayed response task and a 48 hr washout period was observed between infusions. Interestingly, direct MCPO administration of cevimeline significantly improved working memory of young F 344 rats as assessed by the delayed response task. These data demonstrate that basal forebrain GABAergic neurons projecting to the prefrontal cortex can significantly influence prefrontal cortical mediated cognition.

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139 Figure B 1. Systemic M3 muscarinic receptor agonist administration significantly improves working memory performance in young rats.

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164 BIOGRAPHICAL SKETCH Cristina Bauelos graduated with a Bachelor of Science from Cornell University. She completed a Master of Science in Biological Science at the Univers ity of Texas at Brownsville in the lab of Dr, Luis Colom. As a graduate student in the Colom Lab, She completed a thesis entitled Status Epilepticus on Neuronal Populations in the Septum of Sprague In the fall of 2008, Cristina entered a doctoral program in Behavioral and Cellular Neuroscience in the Department of Psychology at Texas A&M University under the mentorship of Dr. Jennifer Bizon whose research focus is aimed at understanding the neurobiological underpinnings of age related cognitive decline. In the summer of 2010, Dr. Bizon was recruited to the University of Florida, and Cristina moved with the lab transferring into the Neuroscience concentration of the Interdis ciplinary Program in Biomedical Sciences in the UF College of Medicine. Cristina advanced to c andidacy in the fall of 2011 She successful ly defended her dissertation and rece iv ed her PhD in the spring of 2014.