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Aging and N-methyl-D-aspartate receptors

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Title:
Aging and N-methyl-D-aspartate receptors
Creator:
Mitchell, Josephine Jean, 1964-
Publication Date:
Language:
English
Physical Description:
x, 136 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Age groups ( jstor )
Brain ( jstor )
Entorhinal cortex ( jstor )
Hippocampus ( jstor )
Memory ( jstor )
Messenger RNA ( jstor )
N methyl D aspartate receptors ( jstor )
Rats ( jstor )
Receptors ( jstor )
Septum of brain ( jstor )
Age Factors ( mesh )
Aging ( mesh )
Brain -- physiology ( mesh )
Brain Chemistry ( mesh )
Cell Aging ( mesh )
Department of Neuroscience thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Neuroscience -- UF ( mesh )
Glutamic Acid -- physiology ( mesh )
Neuronal Plasticity ( mesh )
Rats, Inbred F344 ( mesh )
Receptors, N-Methyl-D-Aspartate -- physiology ( mesh )
Receptors, N-Methyl-D-Aspartate -- ultrastructure ( mesh )
Research ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1995.
Bibliography:
Bibliography: leaves 124-135.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Josephine Jean Mitchell.

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University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
002298782 ( ALEPH )
49818746 ( OCLC )
ALQ2044 ( NOTIS )

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Full Text












AGING AND N-METHYL-D-ASPARTATE RECEPTORS


By

JOSEPHINE JEAN MITCHELL











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


1995




AGING AND N-METHYL-D-ASPARTATE RECEPTORS
By
JOSEPHINE JEAN MITCHELL
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
1995




ACKNOWLEDGMENTS
I would like to thank my advisor, Dr. Kevin J. Anderson,
for sharing his extensive knowledge and for his support
throughout my doctoral studies. My research was supported by
a National Institute of Aging grant (NIA #AG08843). I would
also like to recognize my other committee members: Dr. A.
John MacLennan, Dr. Joanna Peris, Dr. Tom Vickroy and Dr. Don
Walker for their constructive criticisms and suggestions. I
am also very grateful to Dr. Janet Zengel's for her support
and friendship through the years.
Michael S. Sapper deserves a special acknowledgement for
his technical assistance as well as his friendship from the
very beginning. Special thanks go out to Tanya McGraw for
her friendship and support as well as to many of my other
fellow graduate students.
I wish to especially mention my father, Ignaceous J.
Maddalena, who unfortunately passed away just before I began
my doctoral studies. He was and always will be the person
who fueled my love for academics and taught me the importance
of hard work and perseverance in the pursuance of knowledge.
My mother, Helen, has been a loving and supportive figure
throughout my life and I wish to thank her with all of my
heart as well as the rest of my family and friends.
in


Finally, and most importantly, I wish to acknowledge my
husband, Thomas Mitchell (Mitch), who has given me invaluable
support throughout my academic pursuits as the 'one and only
love of my life' and father to our two beautiful sons Kyle
Ryan and Ty Joseph. I could never have achieved all I have
worked for without the constant love and support of my family
and friends.
IV


TABLE OF CONTENTS
ACKNOWLEDGMENTS iii-iv
ABSTRACT vii-ix
CHAPTERS
1 INTRODUCTION 1
Background 1
Previous Studies Examining Age-Related Changes in
NMDA Receptors 4
Molecular Cloning and Characterization of the NMDA
Receptor/Channel Complex 8
The Use of NMDARl and NMDAR2A/B Antisera in the
Analysis of NMDA Receptor/Channel Protein
Density and Distribution in the Rat CNS 17
Cooperative Modulation of [3H]MK-801 Binding to the
NMDA Receptor-ion/Channel Complex by L-
Glutamate, Glycine and Polyamines 19
Specific Aims 22
2 GENERAL METHODS 23
Animal Model 23
[3H]MK-801 Autoradiography 24
Tissue Preparation 24
[3H JMK-801 Binding Assay 25
Image Analysis 26
Statistical Analysis 27
Stereological Determination of Neuronal Density 27
Tissue Preparation 27
Data Analysis 28
Determination of Neuronal Density in the Lateral
Striatum, Entorhinal Cortex and Inner Frontal
Cortex 30
L-Glutamate Stimulation of [3H]MK-801 Binding to
NMDA Receptors 31
Tissue Preparation 31
[3H]MK-801 Binding Assay 31
Image Analysis 32
Data Analysis 33
In situ Hybridization 33
Probe Labeling 33
Tissue Preparation 34
v


Fixation 35
Hybridization 35
Autoradiography 36
Data Analysis 37
Immunocytochemistry 37
Tissue Preparation 37
Antibody Specificity 37
Immunocytochemical Procedure 38
3 AGE-RELATED CHANGES IN [3H]MK-801 BINDING IN F-344
RATS 40
Introduction 40
Methods 42
Statistical Analysis for [3H]MK-801 Binding 42
Results 44
[3H]MK-801 Binding 44
Discussion 51
4 EFFECT OF AGE ON L-GLUTAMATE STIMULATION OF [3H]MK-
801 BINDING IN F-344 RAT BRAIN 59
Introduction 59
Methods 61
Tissue Preparation 61
[3H]MK-801 Binding Assay 61
Image Analysis 62
Data Analysis 62
Binding Isotherm Plots 63
Statistical Analysis 63
Results 64
Discussion 70
5 AGE-RELATED CHANGES IN THE LEVELS OF mRNA CODING
FOR SPECIFIC NMDA RECEPTOR SUBUNITS IN THE CNS OF F-
344 RATS 77
Introduction 77
Methods 78
In Situ Hybridization 78
Data Analysis 79
Statistical Analysis 79
Results 80
Discussion 97
6 AGE-RELATED EFFECTS ON NMDARl AND NMDAR2A/B PROTEIN
LEVELS IN F-344 RAT BRAIN UTILIZING
IMMUNOHISTOCHEMISTRY 102
Introduction 102
Methods 105
Tissue Preparation 105
vi


Antibody Specificity 105
Immunocytochemical Procedure 106
Data Analysis 106
Statistical Analysis 107
Results 108
Discussion 113
7 SUMMARY AND DISCUSSION 117
Research Summary 117
Discussion 121
LIST OF REFERENCES 124
BIOGRAPHICAL SKETCH 136
vil


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
AGING AND THE N-METHYL-D-ASPARTATE RECEPTOR
By
Josephine Jean Mitchell
December, 1995
Chairman: Kevin J. Anderson
Major Department: Neuroscience
Aging is associated with a reduction in cognition and
memory in humans and other animals. Excitatory amino acids
(EAA) play pivotal roles in learning and memory. L-Glutamate
is thought to be the major EAA transmitter in mammalian
brain. L-Glutamate transmitter systems may therefore be
involved in age-related deficits. The N-methyl-D-aspartate
(NMDA) ionotropic glutamate receptor subtype appears
important in learning and memory. It is critical to long
term potentiation and NMDA antagonists impair performance of
rats in spatial and reference memory tasks. Antagonism of
NMDA receptor-mediated neurotransmission produces behavioral
deficits strikingly similar to those detected in aged
animals.
Vlll


IX
Thus deficits in NMDA receptor neurotransmission may
underlie age-related changes in neuronal plasticity. One
possible way NMDA neurotransmission could be reduced in aged
animals is by alterations in NMDA receptor/channel complexes.
The central hypothesis tested in this study was that
there are measurable, anatomically specific age-related
changes in the NMDA receptor and its individual subunits. An
initial examination of age-related differences in NMDA
receptor density in brains of 6-, 12- and 24-month-old F344
rats was performed with [3h]MK-801 in vitro autoradiography.
An age-related decrease was found in the entorhinal and inner
frontal cortices and the lateral striatum.
An analysis of L-glutamate's ability to enhance [3h]MK-
801 binding to NMDA receptor channels was performed by
varying L-glutamate concentration. Emax and EC50 values were
obtained for several brain regions showing an age-dependent
decrease in Emax values without changes in EC50.
The possibility that receptor binding changes were due
to decreases in specific NMDA receptor subunits was analyzed
using in situ hybridization. A significant difference was
seen in NMDARl subunit mRNA in all brain regions. Splice
variants NRloxxr NRlixx and NRlxlx changed in fewer regions
while NMDAR2 subunits did not change with age.
No age-related differences in NMDARl and NMDAR2A/B
protein density levels were found using immunocytochemistry.
These results indicate the NMDA receptor undergoes
regionally specific changes during aging and these changes


X
may account for some of the cognitive deficits in the aging
population.


CHAPTER 1
INTRODUCTION
Background
Ramon y Cajal, as early as the turn of the century,
proposed the connective foundation of neural memory (Cajal,
1911). Many years later, Hebb (1949) formulated his
principles of memory formation which was based upon
facilitation of contacts between neurons. Both Cajal and
Hebb focused on the synapse as the location of plastic change
in the formation of memory. They also theorized that the
processes of learning and memory result from changes in the
strength of synaptic transmission (Cajal, 1911; Hebb, 1949).
Bliss and Lomo (1973) found that in the hippocampus, a
brain region known to be important for learning (Nicoll et
al., 1988), brief repetitive activation of excitatory
pathways resulted in a substantial increase in synaptic
strength that lasted for many hours and, in vivo, even for
weeks. Since its initial discovery, this synaptic
enhancement, referred to as long-term potentiation (LTP), has
provided a model in vertebrate brain for a cellular mechanism
of learning and memory (Bliss and Lomo, 1973). LTP is
defined as an electrophysiological phenomenon of persistent
change in synaptic strength or efficacy as a result of
1


2
impulse transmission across synapses. These activity-induced
changes in the efficacy of existing synapses are thought to
be mediated by excitatory synaptic transmission throughout
the central nervous system (Bliss and Lomo, 1973).
Excitatory glutamatergic synapses are abundant
throughout the central nervous system (CNS), especially in
the hippocampus and cerebral cortex (Westbrook and Jahr,
1989). There are at least three types of ionotropic
glutamate receptors classified by the potent, selective
agonists N-methyl-D-aspartate (NMDA), kainate (KA) and a-
amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)
(Foster and Fagg, 1984). During low-frequency synaptic
transmission, glutamate is released from the presynaptic
terminal and acts on both the NMDA receptor subtypes as well
as the non-NMDA receptor subtypes (KA and AMPA). AMPA and KA
receptors are permeable to K+ and Na+ and provide a voltage-
independent means of depolarizing the postsynaptic neuron.
However, the NMDA receptor has unique biophysical
characteristics making it highly relevant to neural
plasticity and memory formation (Collingridge, 1987; Cotman
and Iversen, 1987; Harris et al., 1984). During low
frequency stimulation, ion flux through the NMDA channel is
blocked by Mg+2 in a voltage-dependent manner. Mg+2 ions are
expelled from membrane channels after the postsynaptic
membrane has reached a certain level of depolarization.
Following relief of the Mg+2 block, the receptor channel is
permeable to Na+ and Ca+2. Furthermore, NMDA receptor-


3
mediated current increases as a function of the degree of
depolarization are longer in duration than those from non-
NMDA receptors. The necessity for both presynaptic release
of transmitter to bind to the NMDA receptor as well as
sufficient postsynaptic depolarization to relieve the
voltage-dependent channel block by Mg+2 give this receptor
Hebb-like properties.
It is now thought that the activation of NMDA receptors
is responsible for the induction of LTP in the hippocampus
(see Nicoll et al., 1988 for review). NMDA antagonists have
been shown to block the development of LTP in the CA1 region
of the hippocampus during high-frequency tetanic stimulation
of the Schaffer commissural pathway (Collingridge et al.,
1983) as well as LTP in the dentate gyrus after perforant
path stimulation (Errington et al., 1987). In addition, a
variety of NMDA antagonists, both competitive (e.g., 2-amino-
5-phosphonovalerate [AP5], CGS 19755) and noncompetitive
(e.g., MK-801, phencyclidine [PCP]), have been shown to
impair performance of rats in the acquisition of spatial
working and reference memory tasks (Shapiro and Caramanos,
1990). NMDA receptor channel antagonism by MK-801 also
impairs performance of rats in aversively motivated complex
maze tasks (Spangler et al., 1991). These results provide
evidence for the NMDA receptor playing a role in memory and
spatial learning and LTP (see also Danysz et al., 1988;
Morris et al., 1986).


4
Aging is associated with a reduction in cognition and
memory in humans as well as other animals (Barnes, 1979;
Barnes and McNaughton, 1985). Senescent rats are slower than
young rats in learning various spatial tasks (Barnes, 1979;
DeToledo-Morrell et al., 1984) which may be due to age-
related changes in NMDA receptor-mediated neurotransmission.
In support of this, it has been shown that antagonism of NMDA
receptor-mediated neurotransmission produces behavioral
deficits strikingly similar to those detected in aged animals
(Bonhaus et al., 1990).
Taken together, the above evidence suggests that a
reduction in NMDA receptor-mediated neurotransmission may
underlie age-related changes in neuronal plasticity. One
means by which NMDA neurotransmission could be reduced in
aged animals is by an alteration in the NMDA receptor/channel
complex itself.
Previous Studies Examining Age-Related Changes in NMDA
Receptors
Previous studies have examined the effects of aging on
the density of central excitatory amino acid (EAA) receptors
in brain membrane preparations. For example, Tamaru et al.
(1991) reported a reduction in the number of NMDA receptors
in the cerebral cortex and hippocampus of aged rats.
Specifically, they found a significant decrease in
[3H]glutamate binding displaceable by NMDA, in strychnine-
insensitive [3H]glycine binding and in [3H]MK-801 binding in


5
both brain regions. Scatchard analysis revealed that the
reduction was due to a decrease in the number of binding
sites, not to an alteration in affinity. The reduction in
the number of receptors was apparently not due to a relative
increase of insoluble protein in membrane fractions since the
protein concentrations and ratio of protein concentration to
tissue wet weight were not significantly different between
young and aged animals. Similar results were obtained by
Ingram et al. (1992), who observed a marked (>50%) age-
related reduction in NMDA receptor binding in rat hippocampal
brain homogenates, and by Peterson and Cotman (1989) who
found reductions in NMDA-displaceable [^HjL-glutamate binding
density in two strains of aged mice.
The effect of increasing age on the binding of [3H]L-
glutamate to NMDA receptors in the brain of the BALB/c and
C57B1 mouse strains was also determined using in vitro
guantitative autoradiographic analysis (Magnusson and Cotman,
1993). In this study, a significant decrease in binding to
NMDA receptors occurred with increasing age (ranging from 3
through 30 months). NMDA receptors, as opposed to non-NMDA
receptors, were selectively affected by age-related changes
in the majority (17 of 20) of the cortical, subcortical and
hippocampal regions assayed in both strains of mice.
[3H]Kainate and [3H]AMPA (non-NMDA) binding, on the other
hand, was decreased in only 7 of 21 and 4 of 21 regions,
respectively. They concluded that the NMDA receptor is


6
selectively vulnerable to the aging process throughout most
cerebral cortical, subcortical and hippocampal regions.
A similar autoradiographic study has also demonstrated
that specific brain regions in aged animals exhibit a decline
in NMDA receptor density (Anderson et al., 1989). The
density of NMDA receptors labeled with [3H]L-glutamate in
young-adult (4-month-old) Fischer 344 rats was compared to
aged (24- to 26-month-old) rats. The areas that showed a
greater than 30% decrease in aged rats included the lateral
striatum, inner layers of the entorhinal cortex and the
lateral septal nucleus. The receptor density in inner
parietal cortex and anterior cingulate cortex was decreased
by 10% in aged rats when compared to young-adults. Within a
given brain region there was no significant difference in the
affinity (Kd) of the NMDA receptor for [^H]L-glutamate.
However, a significant decrease in the Bmax was seen within
the brain regions most affected by increasing age which
indicated that there was a decrease in the total number of
receptors (Anderson et al., 1989). This group also utilized
[]glycine binding to probe the associated allosteric
activating site on the NMDA receptor. Areas that showed the
greatest degree of loss of [^H]glycine binding included the
lateral striatum, parietal cortex and the entorhinal cortex.
There was no significant decrease in the lateral septal
nucleus and the loss of glycine binding sites in aged rats
was more profound in the parietal cortex and outer portions


7
of the entorhinal cortex when compared to [3H]L-glutamate
binding.
Age-related changes in the glutamatergic neuro
transmitter systems in rats and rhesus monkeys has been
examined utilizing NMDA-displaceable [3H]L-glutamate binding
to brain homogenates (Wenk et al., 1991). [3H]L-Glutamate
binding density was decreased in many brain regions in aged
rats (24 month-old) when compared to young (5 month-old)
rats. Specifically, the sensory-motor cortex, the parietal-
occipital cortex, the hippocampus and the caudate nucleus all
showed a significant decrease in binding density. In monkey
brains, NMDA-displaceable [3H]L-glutamate binding was
decreased in most brain regions analyzed and particularly
noticeable in the frontal and temporal lobes of the aged
monkeys (29-34 years) when compared to young (4-9 years)
monkeys.
Senescence-accelerated mice (SAM-P/8) have been used as
a murine model of aging and memory dysfunction (Kitamura et
al., 1992). This strain of mice shows an age-related
deterioration of learning and memory at an earlier age when
compared with control mice. In brain homogenates of
hippocampus and cerebral cortex from SAM-P/8 mice there was a
significant increase in the content of glutamate and
glutamine when compared to controls. Potassium-evoked
endogenous glutamate release from the brain slices of SAM-P/8
mice was increased in comparison with the control strains at
9 and 11 months. Additionally, the Bmax of [3H]dizocilpine


8
(MK-801) binding in the cerebral cortex was decreased in SAM
P/8 but not in controls. This suggests that synaptic
dysfunctions in the glutamatergic system occur in the CNS of
the SAM-P/8 mouse strain (Kitamura et al., 1992).
Molecular Cloning and Characterization of the NMDA
Receptor/Channel Complex
A functional cDNA clone for the rat NMDA receptor
(NMDARl) was first isolated in 1991 (Moriyoshi et al., 1991)
The single protein encoded by the cDNA was shown to form a
functional receptor/ion-channel complex with
electrophysiological and pharmacological properties
characteristic of the NMDA receptor; i.e. agonist and
antagonist selectivity, modulation by glycine, Ca2+
permeability, a voltage-dependent channel block by Mg2+, and
Zn2+ inhibition (Moriyoshi et al., 1991).
Three cDNAs encoding different NMDA receptor subunits
were isolated by polymerase chain reaction (PCR) (Monyer et
al., 1992). Two degenerate oligonucleotide primers were
designed after largely conserved peptide sequences in
ionotropic EAA receptor subunits, with which NMDARl shares
several small sequence islands around putative transmembrane
(TM) segments. These primers were used to PCR amplify
homologous sequences from rat brain cDNA. Three full-length
cDNAg, having sequences identified from the PCR products,
were named NMDAR2A (NR2A), NR2B and NR2C. The predicted
proteins were between 55% (NR2A and NR2C) and 70% (NR2A and


9
NR2B) identical to each other, but were only about 20%
identical to homologous AMPA-selective glutamate receptor
subunits (GluRs) and NMDARl (Monyer et al., 1992).
The functional properties of the expressed NR2 subunit
were examined using a Xenopus oocyte expression system. No
detectable calcium currents were recorded after bath
application of glutamate or NMDA to oocytes expressing one or
two NR2 subunits, which indicated that NR2 subunits may not
form functional homomeric or heteromeric channels. However,
large currents were measured in oocytes coexpressing NR1 and
any one o the NR2 subunits. On average, the NMDA-induced
currents in oocytes expressing NRl and NR2A, NR2B or NR2C
were 100 times larger than they were in oocytes expressing
homomeric NRl channels. These currents also more closely
resemble native NMDA receptors. This indicated that
heteromeric configurations are likely to form from NRl
subunits and members of the NR2 subunit family (Monyer et
al., 1992).
NMDARl and NMDAR2 subunits carry an asparagine residue
in the putative channel forming region TMII, whereas a
glutamine or arginine residue resides in the homologous
position of the AMPA-selective glutamate receptor subunits
(Nakanishi et al., 1992). The importance of the asparagine
residue in the regulation of Ca+2 permeability and channel
blockade was shown by electrophysiological characterization
of receptors in which the asparagine was replaced with either
glutamine or arginine (Nakanishi et al., 1992). These


10
substitutions reduced or abolished Ca+2 permeability and
inhibition by Mg+2, Zn+2, and MK-801. Thus, this particular
asparagine residue may constitute a distinctive functional
determinant in subunits belonging to the NMDA receptor
(Monyer et al., 1992).
Previous in situ hybridization studies have revealed
that NMDAR1 messenger RNA (mRNA) in adult rat brain is
expressed in almost all neuronal cells throughout the brain
(Moriyoshi et al., 1991). This group observed prominent
expression of NMDAR1 mRNA in the cerebellum, hippocampus,
cerebral cortex and olfactory bulb. High expression was also
seen in the granular layer of the cerebellum, in granule
cells of the hippocampal dentate gyrus and in pyramidal cells
throughout hippocampal areas CA1-CA4.
The anatomical distribution of mRNA for NMDAR2 subunits
has been examined with in situ hybridization (Buller et al.,
1993; Kutsuwada et al., 1992; Meguro et al., 1992; Monaghan
et al., 1993; Monyer et al., 1992; Moriyoshi et al., 1991;
Nakanishi, 1992). NMDAR2A mRNA is widely expressed in many
brain regions, and this expression is prominent in the
cerebral cortex, hippocampus, olfactory bulb, some thalamic
nuclei, pontine nuclei, inferior olivary nuclei and
cerebellar cortex. The NMDAR2B mRNA expression is prominent
in most of the telencephalic and thalamic regions but
relatively low in the hypothalamus, cerebellum and lower
brain stem. The distribution of the NMDAR2C mRNA is more
discrete; its expression is extremely high only in the


11
granular layer of the cerebellum. NMDAR2D mRNA is mainly
expressed in the diencephalic and lower brain stem regions.
Recently, the distribution of NMDA receptor subtypes, as
identified with autoradiography, have been shown to
correspond to specific receptor subunits (Monaghan et al.,
1993). Early studies had indicated that there were two
populations of NMDA receptors; agonist-preferring (striatal-
type) and antagonist-preferring (thalamic-type) (Monaghan et
al., 1988). These populations were classified by whether
they displayed a higher affinity for agonist or for
antagonist. Monaghan (1991) showed that low concentrations
of L-glutamate selectively promoted the binding of [3h]MK-801
to striatal-type NMDA sites. He concluded that low
concentrations of L-glutamate appeared to preferentially
activate striatal NMDA receptors without activating thalamic
and cortical NMDA receptors. Conversely, with higher
concentrations of L-glutamate, [3H]MK-801 will also label
NMDA receptors of the lateral thalamic nuclei and cerebral
cortex (Monaghan and Anderson, 1991).
More recently, two other pharmacologically-distinct
populations of NMDA receptors have been defined by
autoradiography. These receptors were identified in the
midline thalamic nuclei and in the cerebellum (Monaghan et
al., 1993). It has been shown that the anatomical
distribution of each of these four native receptor
populations corresponds to a specific NMDAR2 subunit
(Monaghan et al., 1993). The NMDAR2A transcript has a


12
distribution very similar to the "antagonist-preferring" NMDA
receptor subtype. The NMDAR2B subunit mRNA is the only
NMDAR2 species found in regions enriched in "agonist-
preferring" sites. NMDAR2C subunits are largely restricted
to the cerebellum which contains a pharmacologically-distinct
receptor subtype and the NMDAR2D subunits are restricted to
the midline-thalamic nuclei NMDA receptor subtype (Monaghan
et al., 1993). These data suggest that NMDAR2 subunits may
contribute to the pharmacological diversity of native NMDA
receptors.
Alternative splicing has been shown to generate several
functionally distinct NMDARl subunits (Anatharam et al.,
1992; Durand et al., 1992; Nakanishi et al., 1992; Sugihara
et al., 1992). Structures and properties of seven isoforms
of the NMDARl receptor are differentiated from each other by
an insertion at the extracellular amino-terminal regions or
deletions at two different carboxy-terminal regions, or by
combinations of the insertion and deletions. All of these
isoforms have been shown in the Xenopus oocyte expression
system to induce electrophysiological responses to NMDA and
respond to various antagonists selective to the NMDA receptor
(Sugihara et al., 1992). The nomenclature of Durand et al.
(1993) denotes each NMDARl splice variant by the presence or
absence of the three alternatively spliced exons in the 5' to
3' direction. A subscripted o denotes exclusion of an exon
while a subscripted i denotes its inclusion. For example,


13
NRlin has all three exons, NRIqoo has none, and NRlioo has
only the N-terminal insert (Figure 1-1).
insert! insert2 insert3
coding
non-coding
j coding when insert3 is absent
Figure 1-1. Proposed gene structure of the NMDARl receptor
(Durand et al., 1993). Three putative inserts can be spliced
in or out to form the mature mRNA. The NMDARl splice
variants are denoted by subscripts indicating the presence or
absence (1 or 0) of the three inserts in the 5 to 3'
direction.
A recent study by Buller et al. (1994) described the
anatomical distributions of NMDARlixx (the NMDARl splice
variant containing insert 1) and NMDARloxx (the NMDARl splice
variant lacking insert 1). They found NMDARlixx mRNA density
varied across cortical regions with the parietal, temporal,
and superficial entorhinal cortices displaying threefold
higher levels of NMDARlixx mRNA than the anterior cingulate,
perirhinal, and insular cortices. In contrast, higher levels
of NMDARl oxx niRNA were found in the anterior cingulate,
perirhinal, and insular cortices. They concluded that the
localization of NMDARlixx and NMDARloxx mRNA between cortical
regions paralleled the distribution of antagonist-preferring
and agonist-preferring NMDA receptors, respectively.


14
NMDARlixx mRNA displayed a lateral-to-medial gradient pattern
within the striatum whereas NMDARlqXx was shown to be
moderately higher (15%) in the medial striatum than the
lateral striatum. NMDARlixx was present in low levels
throughout the septum while high levels of NMDARloXx mRNA were
found in this region. NMDARlixx mRNA was present at high
levels throughout the thalamus with low levels of NMDARIqxx*
Overall, this study showed that agonist-preferring NMDA
receptors are found predominately in the subset of brain
regions that contain both NMDAR2B and NMDARl oXx mRNA whereas
the antagonist-preferring NMDA receptors are found
predominately in brain regions containing both NMDAR2A and
NMDARlixx mRNA. Monaghan and Buller (1994) also found that
NMDARl mRNAs that are alternatively spliced at the second and
third inserts have distribution patterns that are dissimilar
to that of previously described NMDA receptor subtypes. No
differences in NMDA receptor pharmacological properties have
been found after studying homomeric receptors of NMDARl mRNA
that contain alternatively spliced exons at the second and
third insert sites (Durand et al., 1993; Nakanishi et al.,
1992). Because of these findings, alternative splicing at
these C-terminal sites is thought to have less of an effect
upon NMDA receptor pharmacology than N-terminal alternative
splicing events.
Expression cloning in Xenopus oocytes isolated two
different cDNAs encoding functional NMDA receptor subunits.
These receptor subunits were termed NMDA-R1A (NRIqh) and -RIB


15
(NRlm) (Durand et al., 1992). The two subunits displayed
different pharmacologic properties as a consequence of
alternative exon addition within the putative ligand-binding
domain. The splicing choice is regulated such that NRlm is
the predominate form of the receptor in the cerebellum,
whereas NRIqh predominates in the cerebral cortex,
hippocampus and olfactory bulb. Durand et al. (1992) showed
that the functional differences between NRlm and NRIqh are
marked and include differences in agonist affinity and
potentiation by spermine. Clearly, alternative splicing
contributes to NMDA receptor diversity. The expression of
distinct NMDA receptors with different electrophysiological
properties and anatomical distribution may be responsible for
the different forms of activity-dependent synaptic plasticity
in the mammalian brain such as the generation of different
forms of LTP (e.g., hippocampal associative LTP and
motorcortical LTP) (Nakanishi et al., 1992).
Recent studies examining the transmembrane topology of a
glutamate receptor GluRl (an AMPA subunit) showed that the N-
terminus is extracellular, whereas the C-terminus is
intracellular (Hollmann et al., 1994). In addition, three
transmembrane domains (TMD), (designated TMD A, TMD B, and
TMD C) corresponded to the previously proposed TMDs I, III,
and IV, respectively. It was found by N-glycosylation
tagging that, contrary to earlier models (Barnard et al.,
1987; Hollmann et al., 1989), the putative channel-lining
hydrophobic domain TMD II does not span the membrane.


16
Instead, this domain was suggested to either lie in close
proximity to the intracellular face of the plasma membrane or
loop into the membrane without traversing it. Furthermore,
the region between TMDs III and IV, in previous models
thought to be intracellular, is an entirely extracellular
domain (Hollmann et al., 1994).
Previously, Durand et al. (1993) showed that the 21-
amino acid insert in the N-terminal domain reduced the
apparent affinity of homomeric NMDARl receptors for NMDA and
nearly abolished potentiation by spermine and glycine. For
both of these properties, the N-terminal insert was the
determining structural feature; the C-terminal domain
produced only a very minor effect. These observations
correlate with a three transmembrane spanning topology where
the N-terminal domain is extracellular and therefore should
affect the ligand binding affinity characteristics of the
NMDA receptor. Therefore, it is not surprising that the C-
terminus would have minimal, if any, effect on ligand binding
characteristics due to its intracellular location. This was
demonstrated by Durand et al. (1993) when they examined the
electrophysiological characteristics of six splice variants
of NMDARl receptors and concluded that variants differing
only in their C-terminal domain showed little change in
agonist affinity or spermine potentiation.


17
The Use of NMDAR1 and NMDAR2A/B Antisera in the Analysis of
NMDA Receptor/Channel Protein Density and Distribution in the
Rat CNS
One of the first studies examining the NMDARl protein in
the rat CNS was provided by Hennegriff et al. (1992).
Polyclonal antibodies were raised against synthetic peptides
corresponding to the carboxyterminal region of the putative
NMDA receptor cloned by Nakanishi (1991). The affinity
purified antibodies to the NMDA receptor subunit labeled
antigens of 75 and 81 kDa. In order to determine the
regional distribution of NMDARl, the 75/81 kDa doublet
protein was quantitated by laser densitometry from Western
blots in homogenate samples prepared from seven brain
regions. They found the doublet to be most abundant in
neocortex, followed by hippocampus > striatum thalamus >
olfactory bulb > cerebellum brain stem. The same regional
distribution was found in synaptosomes using antibodies to
NMDARl and resembled earlier autoradiographic studies
measuring NMDA-sensitive [ 3H]glutamate binding sites
(Monaghan and Cotman, 1985). A more thorough light and
electron microscopic analysis of the distribution of the NMDA
receptor subunit NMDARl in the rat CNS was performed by
Petralia et al. (1994a). This group made a polyclonal
antiserum that recognized four of the seven splice variants
of NMDARl and subsequently utilized this antiserum to perform
a comprehensive immunohistochemical survey of the
distribution of this antigen. They showed that the NMDARl


18
subunit is widespread throughout the rat CNS. The most
densely stained cells included the pyramidal and hilar
neurons of the CA3 region of the hippocampus, Purkinje cells
of the cerebellum and paraventricular neurons of the
hypothalamus. Ultrastructural localization of NMDAR1 antigen
showed labeling present in postsynaptic densities in a
pattern consistent with the synthesis, processing and
transport of this protein. Staining was seen in the
cytoplasm of dendrites and concentrated in patches associated
with groups of microtubules and/or the surface of one pole of
a mitochondrion. In addition, patches of staining in the
cell bodies were found to form similar associations with
microtubules and mitochondria, as well as an association with
rough endoplasmic reticulum, Golgi apparatus, and the nuclear
envelope. No staining was found in the synaptic cleft. The
antiserum did not cross-react with extracts from transfected
cells expressing other glutamate subunits, nor did it label
non-neuronal tissues. The pattern of staining correlated
closely with previous in situ hybridization studies but
differed somewhat from binding studies (Petralia et al.,
1994a).
Another study by this laboratory (Petralia et al.,
1994b) examined the histological and ultrastructural
localization patterns of the NMDA receptor subunits NMDAR2A
and NMDAR2B. They made a polyclonal antiserum to a C-
terminus peptide of NMDAR2A. In analysis of membranes from
transfected cells, this antiserum recognized NMDAR2A and


19
NMDAR2B, and to a slight extent, NMDAR2C and NMDAR2D.
Immunostained sections of rat brain showed significant
labeling throughout the CNS that was similar to that seen
previously with their antiserum to NMDARl. Dense staining
was present in postsynaptic densities in the cerebral cortex
and hippocampus. Since there is physiological evidence that
both NMDARl and NMDAR2 subunits coexist in the native NMDA
receptor, their findings are consistent with this idea
(Petralia et al., 1994b).
Cooperative Modulation of r-^HlMK-801 Binding to the NMDA
Receptor-ion/Channel Complex by L-Glutamate, Glycine and
Polyamines
L-Glutamate binding to the NMDA receptor, in the
presence of glycine, produces channel opening. MK-801 is an
NMDA receptor antagonist that binds with high affinity (in
the low nanomolar range) to a site located within the NMDA
channel. L-Glutamate markedly stimulates [3H]MK-801 binding
by increasing the affinity for the ligand. Other NMDA
receptor agonists (e.g. NMDA, D-aspartate) are capable of
enhancing this binding as well, but the non-NMDA receptor
agonists (AMPA and kainate) have no effect on [3h]MK-801
binding (Foster and Wong, 1987). Because MK-801 binding has
been shown to take place only if the NMDA receptor channel is
in the transmitter-activated state (Huettner and Bean, 1988),
the efficacy with which L-glutamate produces receptor
activation and channel opening can be measured by determining


20
the amount of [3H]MK-801 binding as a function of glutamate
concentration.
Glycine appears to be a constitutive co-agonist for NMDA
receptor activation. Glycine alone is ineffective in opening
NMDA-linked cation channels. However, glycine greatly
potentiates the frequency of channel opening in response to
NMDA receptor agonists (Johnson and Ascher, 1987; Reynolds et
al., 1987). Glycine also potentiates NMDA-stimulated Ca2+
influx in cultured striatal neurons (Reynolds et al., 1987).
An allosteric role for glycine in NMDA receptor function is
supported by autoradiographic studies. These studies showed
that the distribution of strychnine-insensitive [2H]glycine
binding sites in supraspinal brain regions mirrored the
regional distribution of NMDA-sensitive L-glutamate sites
(Monaghan and Cotman, 1985). Since glycine has been shown to
promote channel activation in response to agonist, it can be
predicted to enhance [3H]MK-801 binding since MK-801 prefers
to bind to the activated state of the channel.
Along with the allosteric co-agonist glycine site on the
NMDA receptor/complex, there is also a polyamine recognition
site. The polyamines spermine and spermidine have been shown
to increase both the maximum NMDA response amplitude (Lerma,
1992; McGurk et al., 1990; Ransom and Deschenes, 1990;
Sprosen and Woodruff, 1990) as well as the binding of [3H]MK-
801 to the NMDA receptor channel (Ransom and Stec, 1988).
Because spermine does not activate NMDA receptors in the
absence of glutamate and glycine, it has been suggested to


21
act at an allosteric site independent from the glycine site
(Ransom and Stec, 1988).
The mechanism by which L-glutamate and these other
agonists affect [3H]MK-801 binding affinity has not been
critically addressed. In response to varying agonist
concentrations, [3H]MK-801 binding sites may undergo a
continuum of conformational changes expressing a smooth range
of different affinities for [3H]MK-801. However, it seems
more probable that [3H]MK-801 binding is an all (open
channel) or nothing (closed channel) phenomenon since most
ion channels have not been shown to exhibit broad conductance
ranges (i.e. they are either open or closed [Ransom and Stec,
1988]). This would be more consistent with the
electrophysiological characterization of MK-801 as a use-
dependent, open channel blocker (Foster and Wong, 1987). A
more likely explanation for the observed changes in Kp for
[3H]MK-801 is that they result from changes in the length of
time a channel spends in the activated state (Ransom and
Stec, 1988). At high agonist concentrations, channels are
opened for a proportionately greater amount of time and the
affinity for [3H]MK-801 is high. At low agonist
concentrations, the freguency with which the channel opens is
comparatively low. The result is an apparent reduction in
[3H]MK-801 binding affinity since there is a much lower
probability, due to decreased access, that an infreguently
opened channel will bind radioligand (Ransom and Stec, 1988).


Specific Aims
The central hypothesis tested in this dissertation
project was that there are measurable, anatomically specific
aging-related changes in the NMDA receptor/channel complex
and its individual subunits. Four specific hypotheses were
addressed within the framework of the central hypothesis.
The first hypothesis was that there are age-related
differences in the density of the NMDA receptor in F-344 rat
brain as a function of age. This hypothesis was addressed
using in vitro quantitative [3H]MK-801 binding analyses
(Chapter 3). The second hypothesis was that there are age-
related differences in the ability of L-glutamate to enhance
[3h]MK-801 binding to NMDA receptors. [3H]MK-801 binding as
a function of L-glutamate concentration was performed to test
this hypothesis (Chapter 4). The third hypothesis was that
there are selective changes in mRNA coding for subunits of
the NMDA receptor as a function of age. In situ
hybridization analyses were performed with cDNA probes
specific to NMDA receptor subunits (Chapter 5). The fourth
hypothesis was that there are specific age-related changes in
NMDARl and NMDAR2 protein density in the rat CNS.
Immunocytochemistry was performed with antisera specific to
the NMDA receptor subunit protein (Chapter 6).


CHAPTER 2
GENERAL METHODS
A description of the general methods that were used
throughout this dissertation will follow and will be referred
to when describing individual experiments.
Animal Model
Fischer 344 (F-344) male rats 6-, 12-, and 24-months-of-
age were used for these experiments. These animals were
obtained from the National Institute on Aging (NIA) breeding
colony (Harlan). In 1981, the Committee on Animal Models for
Research on Aging described the life span characteristics of
two rat strains, the F-344 and the Brown Norway (BN) rat, and
recommended their use as models for aging research. Among
the reasons for choosing F-344 and BN rat strains over other
rat strains was that these rats displayed delayed onset of
kidney problems (nephropathy) as well as certain tumors.
Currently, the NIA recommends and maintains a total of three
rat strains for aging studies, the F-344, the BN, and the Fi
F-344/BN hybrid. The F-344 was chosen for these studies
because it has long been a standard model for aging research
and thus has been extremely well characterized. Survival
curves for the F-344 rat show that 24-month-old males have a
23


24
40% probability of survival and the probability of survival
curve drops rapidly to only 10% by 27-months-of-age.
Therefore, 24-month-old animals represent advanced stages of
aging in this rat strain and 12-month-old animals can be
classified as middle-aged. 6-month-old animals represent
young-adults and are used throughout these studies for
comparisons to middle-aged and aged rats.
r^HlMK-801 Autoradiography
Tissue Preparation
Male F-344 rats 6-, 12- and 24-months-of-age (n=6 per
age group) were decapitated, their brains rapidly removed and
frozen with powdered dry ice. Brains were stored at -80 C
until used. Sections (6pm thick) were cut on a cryostat and
representative sections from each age group were thaw-mounted
onto chromic acid washed and gelatin-subbed slides. A rat
brain atlas (Paxinos and Watson, 1982) was referenced in
order to delineate the brain regions for analyses. The
brains were sectioned in the horizontal plane. The bregma
coordinate corresponded to the dorsoventral distance of the
sections from the horizontal plane passing through bregma and
lambda on the surface of the skull. The start and stop
bregma coordinates were -4.1 mm and -6.1 mm, respectively.
This anatomical range was used to thoroughly analyze brain
regions of interest that were present on each sectional
profile. Every fifth serial section was taken and a total of


25
fifty sections were cut from each rat brain. One section
from each of the three age groups was mounted onto each slide
which represented one block of animals. Ten slides were used
per block of animals for each [3H]MK-801 binding assay. Two
slides per assay were used to determine non-specific binding
and these values were subtracted from each total binding
value in order to obtain specific binding. These tissue
sections were used immediately or stored for no longer than
24 hours at -20 C prior to the [3H]MK-801 binding assay.
r3H1MK-801 Binding Assay
Slides were thawed and preincubated at room temperature
for 10 minutes in 50 mM Tris acetate with 1.0 mM EDTA and
0.1% saponin (pH 7.7). Sections were then rinsed at 30 C
for 60 minutes in 50 mM Tris-acetate buffer (pH 7.7). This
treatment removes endogenous glutamate, glycine and various
ions. Sections were then incubated for 60 minutes at room
temperature in 10nM [3H]MK-801 (30 Ci/mmol;New England
Nuclear, Boston,MA,U.S.A.) in 50 mM Tris-acetate buffer
containing 20 pM D-2-amino-5-phosphonopentanoic acid (D-
AP5), 250 jjM spermine, 25 ¡u glycine and 20 pM L-glutamate.
D-AP5, a competetive NMDA antagonist, was added to all
[3H]MK-801 incubations to give lower and more consistent
basal binding levels (Monaghan 1991). The addition of
spermine, glycine and L-glutamate ensures a maximal degree of
stimulation of the NMDA receptor/ channel complex and
therefore optimal [3H]MK-801 binding. Sections were then


26
washed for 60 minutes in ice-cold Tris-acetate buffer
containing 20 nM D-AP5. Nonspecific binding was defined in
sections treated identically in the presence of 50 jjM. MK-801.
Following the rinse, the sections were dried under an air
stream. The sections were then placed in x-ray cassettes and
apposed to tritium-sensitive film (Hyperfilm, Amersham).
Tritium standards calibrated against brain paste were
included in the cassettes (Microscales, Amersham). The film
was exposed for six weeks and then developed using Kodak D-19
developer.
Receptor densities (expressed in pmol/mg protein) from
the 12- and 24-month rat brains were normalized against
values derived from 6-month-old animals. Data from the 12-
and 24-month-old rats were presented as a percentage of the
receptor density values from the 6-month-old animals.
Image Analysis
Each slide contained a representative horizontal section
from each age group. Ten slides were analyzed per block of
animals, with each block consisting of an animal from each
age group. Autoradiograms were analyzed, with the
investigator blind to age, by computer assisted densitometry
with a Microcomputer Imaging Device (MCID, Imaging Research,
Inc., St. Catherines, Ont.). Densitometric measurements were
converted on line to pmol/mg protein binding.


27
Statistical Analysis
Six blocks of animals, each block consisting of an
animal from each age group, were assessed. The experimental
design consisted of a within-subject factor with eight levels
(brain region), a between-subject fixed factor with three
levels (age), and each of six levels (experimental day).
Repeated measures analysis of variance (ANOVA) with between-
subject randomized blocks and age considered as a linear
covariate were used to model the amount of receptor density
as a function of age within each brain region and to
determine if this pattern differed significantly among brain
regions.
The brain regions that were analyzed included the outer
and inner frontal cortices (OFCTX and IFCTX); the entorhinal
cortex (ERC); the molecular layer of the dentate gyrus (DG);
hippocampal area CAl stratum radiatum (CA1); the lateral
septum (LSEP); the lateral striatum (LSTR); and the lateral
thalamus (LTHAL).
Stereoloqical Determination of Neuronal Density
Tissue Preparation
Thirty micron sections of brains from 6-, 12-, and 24-
month-old F-344 rats (n=4 per age) were cut on a cryostat.
One section from each age group was thaw-mounted on each
slide with a total of fifty slides for each block of animals.


28
The brain regions of interest in these studies were those
that showed an age-related decrease in [3h]MK-801 binding as
determined by the first experiment in Chapter 3. These
regions were the lateral striatum (LSTR), the inner frontal
cortex (IFCTX) and the entorhinal cortex (ERC). Sections
were stained with Cresyl violet in order to view neuronal
cell bodies.
A rat brain atlas (Paxinos and Watson, 1982) was
referenced in order to delineate the stereotaxic coordinates
of brain regions for analyses. Brains were sectioned in the
horizontal plane. Bregma coordinates correspond to the
dorsoventral distance of the sections from the horizontal
plane passing through bregma and lambda on the surface of the
skull. The start and stop bregma coordinates were -3.1 mm
and -7.9 mm, respectively. This broader anatomical range was
chosen to thoroughly analyze the dorsal to ventral extent of
the lateral striatum as well as to view the entorhinal and
inner frontal cortices. Two serial sections were taken every
120pm throughout the stereotaxic range.
Data Analysis
Five blocks of animals were used for these analyses with
each block containing one 6-month-, one 12-month- and one 24-
month-old rat. An initial analysis was performed to
ascertain whether the lateral striatum (LSTR) underwent
volumetric changes by sampling this brain region through its
dorsoventral extent. The volume of this structure could be


29
readily determined because its entire anatomical distribution
could be viewed throughout the series of sections. The LSTR
was sampled at five separate anatomical levels (2 serial
slides per level) with computer assisted image analysis
(Microcomputer Imaging Device, Imaging Research, Inc.)* A
determination was then made as to whether there was a
significant age-related change in the overall volume (mm3) of
this structure. In addition, a quantitative analysis of the
laminar thickness of the inner frontal cortex (IFCTX) and the
entorhinal cortex (ERC) in 6-, 12- and 24-month-old animals
was performed. This study was performed to determine whether
there were changes in laminar thickness in these cortical
structures in the aged brain when compared to the young
animals. Calculations of laminar thickness were used because
both the entorhinal cortex and inner frontal cortex have
highly organized laminar structures. There were no
significant age-related differences in the volume of the LSTR
or the laminar thickness of the ERC or the IFCTX.
Neuronal packing density units are in neurons/mm2. No
significant age-related differences in the volume of the LSTR
or in the laminar thickness of the ERC or IFCTX were found,
therefore packing density should not vary as a function of
age. Therefore, any change seen in neuronal packing density
would be attributable to differences in the number of neurons
per mm2. In support of this, Coleman et al. (1987) examined
the volumes of the components of the hippocampus in the aging
F-344 rat and found that there was no change in volume


30
between 12 and 37 months. They suggested that neuronal
packing density (expressed as the number of neurons per mm2)
could be compared in aged animals independent of a volumetric
effect.
Determination of Neuronal Density in the Lateral Striatum,
Entorhinal Cortex and Inner Frontal Cortex
An MCID impage analysis system was used to determine
neuronal packing density in three brain regions. A fully
automated sampling scheme was utilized where the general
target characteristics were defined. The two target
definitions that were used were target density and target
size. For the first criteria of target density, MCID was
given upper and lower density thresholds (segmentation
ranges) and all pixels darker or lighter than the threshold
values were ignored. Pixels that were lying within the
segmentation range were valid targets. The second criteria
for target acceptance was defined as a range of target
lengths where the target neurons smaller or larger than the
defined size range ( >5pm and <20pm) would be ignored with
those targets fulfilling the length criteria being counted.
After the system found the defined targets, post-scan editing
of the digitized image was performed using editing tools.
This enabled the investigator to separate any neuronal
targets that may have been overlapping.
The area searched for targets (scan area) was also
guantified, thus neuronal packing density (number of


31
neurons/mm^) was determined for each brain region. Ten
serial sections were analyzed from each age group with the
investigator blind to age. Neuronal density from 6-month-old
animals was used to normalize counts from 12-, and 24-month-
old animals. Results were then statistically compared
between age groups (one-way repeated measures ANOVA, paired
t-tests).
L-Glutamate Stimulation of r3HlMK-801 Binding to NMDA
Receptors
Tissue Preparation
Male F-344 rats 6-, 12- and 24-months-of-age (n=6 per
age) were decapitated and their brains were rapidly removed
and frozen with powdered dry ice. The brains were stored at
-80 C until used. Fifty 6¡um sections were cut on a cryostat
and representative sections from each age group were thaw-
mounted onto acid washed and gelatin-subbed slides. These
tissue sections were used immediately or stored for no longer
than 24 hours at -20 C prior to the [3H]MK-801 binding
assay.
r 3H1MK-801 Binding Assay
The [3H]MK-801 binding assay was the same as described
previously with the exception that varying concentrations of
L-glutamate were added to the incubation step in order to
examine glutamate's ability to stimulate [3H]MK-801 binding.


32
The following concentrations (in micromolar) of L-glutamate
were used: 0.0, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5, 10 and 20.
Non-specific binding was defined by the addition of 50 pM
unlabeled MK-801 with 20pM L-glutamate. Each slide contained
one section from each of the three age groups. A total of
four slides, from each block of animals, were analyzed per
assay (n=6) for each L-glutamate concentration.
Receptor density (expressed in pmol/mg protein) from 12-
and 24-month rat brains was normalized against 6-month-old
rats. Data from 12- and 24-month-old animals were
statistically compared to receptor density values from 6-
month-old animals.
Image Analysis
Each slide consisted of a horizontal section from each
age group taken from between bregma coordinates -4.1 mm and
-6.1 mm (the dorsoventral distance of the sections from the
horizontal plane passing through bregma and lambda on the rat
skull). This stereotaxic range was used for a thorough
analysis of the brain regions of interest that were present
on each sectional profile. A total of fifty sections were
cut for each block of animals, with every fifth section being
taken throughout the stereotaxic range. Autoradiograms were
analyzed, blind to age, by computer assisted densitometry
with an MCID system. Four sections from each block of
animals, for each concentration of L-glutamate, were
analyzed. Two slides per assay were analyzed for non-


33
specific binding and these values were subtracted from each
total binding value in order to obtain specific binding.
Densitometric measurements obtained were converted on line to
pmol/mg protein binding.
Data Analysis
The brain regions that were analyzed included the OFCTX
and IFCTX; the ERC; the molecular layer of the DG;
hippocampal area CAl (stratum radiatum); the LSEP; the LSTR;
and the LTHAL.
INPLOT (GraphPad Software, San Diego, CA) was used to
generate rectangular hyperbola plots or binding isotherms.
These binding isotherm plots yielded EC50 (effective
concentration at 50%) and Emax (maximal effect) values. EC50
values and Emax values were obtained for each age group and
each brain region. Statistical analyses were then performed
to ascertain significant age-related differences in these
parameters (repeated measures ANOVA; F tests).
In situ Hybridization
Probe Labeling
Oligonucleotide probes for this study were graciously
supplied by Dr. Daniel T. Monaghan, University of Nebraska
Medical Center, Omaha NE. These probes are 45 nucleotides in
length (45 mer) and are specific to NMDARl, four
alternatively spliced versions of NMDARl (NRloxx NRlixx/


34
NRlxix / NRlxxi) and the members of NMDAR2 subunit family
(NMDAR2A, NMDAR2B, NMDAR2C, NMDAR2D). The specificity of
these probes was previously confirmed by Buller and Monaghan
et al. (1994) by incubation of the radiolabeled
oligonucleotides in the presence of excess (100 nM )
unlabeled probe. In this study, only oligonucleotides with
the same sequence inhibited probe hybridization.
The oligonucleotide probes were labeled using a New
England Nuclear (NEN) labeling kit and following the methods
described by Wisden et al. (1991). The probes were labeled
at the 3'- end using [35S]dATP (1000-1500 Ci/mmol, NEN) and 3'
terminal deoxynucleotidyl transferase (NEN). After labeling,
the probes were used immediately or stored for a limited time
at -70 C.
Tissue Preparation
Frozen brains from 6-, 12- and 24-month-old rats (n=6
per age) were cut at 12 pm, in the horizontal plane, on a
cryostat and thaw-mounted on Fisher Superfrost Plus glass
slides. The stereotaxic bregma coordinates were from -4.1 mm
to -6.1 mm. Sections were refrozen in the cryostat. One
section from each age group was mounted per slide for a total
of three sections per slide. A total of fifty slides was
prepared per block of animals. Four slides per probe, each
containing a section from each age group, were used. Gloves
were worn at all times during sectioning of the brains and


35
handling of the slides in order to avoid contamination with
ribonucleases.
Fixation
The sections were fixed immediately after cutting by
placing the slides in 4% paraformaldehyde at 4C for 5-15
minutes. Slides were washed in 0.1M phosphate-buffered
saline (PBS) for 1 minute and 70% EtOH (RNase-free) for
several minutes. Slides were then stored in 95% EtOH in a
cold room (4C) until needed. All solutions were made with
diethylprocarbonate (DEPC)-treated water.
Hybridization
Slides were removed from storage and air dried at room
temperature. Each labeled probe was dissolved in appropriate
amounts of hybridization buffer (NEN, Boston, MA), containing
0.2 M dithiothreitol to achieve a final concentration of 2000
cpm/pl. 100 pi of this solution was applied to the slide and
covered with a glass coverslip. Slides were incubated at 42
C overnight with a parafilm wrap. The following day,
parafilm and coverslips were removed and the slides were
placed in lx SSC (saline sodium citrate buffer) at 60 C for
20 minutes. Finally, the sections were rinsed again in lx
SSC at 60 C for 5 seconds and then rapidly dried under an
air stream.
In situ hybridization is an experimental method that can
be used to determine the relative density of individual mRNA


36
species. Each 45 mer probe was constructed to be highly
specific and hybridizes to its particular NMDA subunit mRNA.
One potential drawback is that only the relative density of
mRNA can be obtained from this method. Therefore, only semi-
quantitative analyses were performed. Another drawback is
that each probe corresponding to specific NMDA receptor
subunits could only be compared across each age group. No
quantitative comparisons could be made between various
probes. In other words, the relative density of the NMDAR1
mRNA could only be compared between 6-, 12- and 24-month-old
animals from the same assay. This information could not be
compared to the data obtained for any of the other mRNAs
(i.e. NMDAR2A-D and the four alternatively spliced versions
of NMDAR1). Another drawback is that the analyses gave an
assessment of the relative density of mRNA while not all mRNA
is necessarily translated into protein.
Autoradiography
Dried slides were placed in an x-ray cassette and
apposed to B-max film (Hyperfilm, Amersham) for two weeks,
developed in D-19 (Kodak) developer and fixed with Rapid Fix
(Kodak). Autoradiograms were analyzed by computer assisted
densitometry with a Microcomputer Imaging Device, Imaging
Research, Inc.(MCID) image processing system.


37
Data Analysis
The brain regions analyzed included the OFCTX, IFCTX,
LSEP, LSTR, LTHAL, ERC, DG and hippocampal areas CAl and CA3.
Brain regions were compared between the three age groups and
the relative density of each subunit mRNA was normalized
against the values obtained for young animals. Probes were
labeled simultaneously and labeling experiments were grouped
such that variability was kept to a minimum. A semi-
quantitative measurement of mRNA levels and the distribution
of the different subunits was compared between ages.
Immunocytochemistry
Tissue Preparation
Brains from 6-, 12- and 24-month rats (n=5) were
serially sectioned at 30 pm on a cryostat and thaw-mounted
onto subbed microscope slides and then refrozen in the
cryostat. Every fifth section was taken for a total of fifty
slides. One brain section from each block of animals was
mounted per slide n=5.
Antibody Specificity
AB1516 (Chemicon Inti. Inc., Temecula, CA), an antiserum
raised against a synthetic peptide corresponding to the C-
terminus of rat NMDA receptor subunit (NMDAR1), is selective
for splice variants NRloil, NRlm, NRlooi/ NRlioi* These


38
appear to be the major splice variants expressed in rat brain
(Hollmann et al., 1993). It has been shown previously that
there is no cross reactivity of this antiserum with other
glutamate receptor subunits (Petralia et al.f 1994a). AB1548
(Chemicon Inti. Inc., Temecula, CA), an antiserum raised
against a synthetic peptide corresponding to the C-terminus
of rat NMDAR2A receptor subunit, recognizes both NMDAR2A and
NMDAR2B subunits equally. This antiserum shows no cross
reactivity with NMDARl or other glutamate receptor subunits
(Petralia et al., 1994b).
Immunocytochemical Procedure
An optimal antibody concentration was determined
empirically. Previous studies had used an AB1516 antibody
concentration between 2 and 4 pg/ml (Petralia et al., 1994a)
and AB1548 antibody concentration of 0.5-1.5 pg/ml (Petralia
et al., 1994b). In the present experiments, a concentration
of 2.5 pg/ml was found to produce optimal results with both
NMDARl and NMDAR2A/B antibodies. Sections were incubated in
10% normal goat serum in PBS (pH 7.4) for 1 hour and then
kept overnight in primary antisera (AB1516 or AB1548) in PBS.
Sections were then washed and incubated in biotinylated goat
anti-rabbit antisera (1:250 dilution) for 1 hour, washed,
incubated in avidin-horse radish-peroxidase (1:200 dilution)
for 1 hour, washed, treated for 15 minutes with 3',3-
diaminobenzidine tetrahydrochloride (0.5 mg/ml PBS + 5 pl/ml
of 0.6% hydrogen peroxide), and washed. All washes were


39
performed with PBS (2 x 15 min.)* Sections with PBS
substituted for the primary antibody (PBS controls) were also
used in order to correct for nonspecific staining.
Staining density was semi-quantitated by an MCID image
analysis system. Data were presented as raw optical
densities. Raw optical density of NMDARl and NMDAR2A/B
immunoreactivity from 6-month-old animals was used to
normalize data from 12- and 24-month-old animals. Data from
middle-aged and aged animals were presented as a percentage
of the six-month-old animals.


CHAPTER 3
AGE-RELATED CHANGES IN [3H]MK-801 BINDING IN F-344 RATS
Introduction
Aging is associated with a reduction in many physiologic
forms of neuronal plasticity. Recent findings indicate a
direct correlation in age-dependent deficits in spatial
memory and in hippocampal kindling (a measure of neuronal
plasticity) as measured in 26-month-old Fischer 344 rats
(DeToledo-Morrell et al.f 1984). N-Methyl-D-aspartate (NMDA)
receptor-mediated glutamatergic neurotransmission has been
shown to be important for learning and memory in animals and
man (Collingridge, 1987; Cotman and Iversen, 1987; Harris,
1984). Profound learning and memory impairments are seen in
a variety of animal species after NMDA-receptor activation is
blocked by either competitive or non-competitive antagonists
(Morris et al., 1986). There are also some striking
similarities between NMDA antagonist induced memory
impairments and age-related cognitive deficits, suggesting
that age-related alterations in NMDA receptors may be
involved in cognitive decline with increasing age (Bonhaus et
al., 1990; Ingram et al., 1992).
40


41
To test the hypothesis that NMDA receptors decline
during aging, an examination of the density of NMDA receptors
was performed in aged Fischer 344 rats. In this study,
[ ](+)-5-methyl-l0,1l-dihydro-5H-dibenzo(a,d)-cycloheptan-
5,10-iminehydrogen malate (MK-801 or dizocilpine), a highly
specific non-competitive NMDA receptor antagonist was used to
determine the density of NMDA receptors. This antagonist
binds with high affinity (low nanomolar range KD) to a site
located within the NMDA receptor ion channel. Thus, MK-801
binding occurs only if the NMDA receptor is in the
transmitter-activated (open) state (Huettner and Bean, 1988).
Channel activation by an NMDA receptor agonist like L-
glutamate, in the presence of glycine, promotes high-affinity
MK-801 binding. Glycine alone cannot open NMDA receptor
channels, but is a constitutive co-agonist. Glycine, in the
presence of L-glutamate or other NMDA receptor agonists
(i.e., NMDA or D-aspartate) has been shown to greatly
potentiate the frequency of channel opening (Johnson and
Ascher, 1987; Reynolds et al., 1987). In addition, the
polyamines spermine and spermidine increase both the maximum
NMDA response amplitude as well as binding of MK-801 to the
NMDA receptor. Alone, polyamines do not activate the NMDA
receptor, suggesting the presence of an allosteric binding
site independent from the other co-agonist sites (Lerma,
1992; McGurk et al., 1990; Ransom and Deschenes, 1990;
Sprosen and Woodruff, 1990).


42
Because of the high degree of specificity and affinity
of MK-801 for the NMDA receptor channel complex, [3H]MK-801
is an ideal ligand to examine age-related changes in NMDA
receptors in the CNS. Also, because MK-801 only binds to the
activated state of the NMDA receptor, the total number of
available NMDA receptors can be determined by using optimal
agonist and co-agonist concentrations.
Methods
For a detailed description of the experimental tissue
used in these studies, the [3H]MK-801 binding assay
procedure, and image analyses see Chapter 2.
Statistical Analysis forf3H1MK-801 Binding
The data acquired in this study consisted of
quantitative analyses of the average density of [3H]MK-801
binding (pmol/mg protein) obtained from bilateral brain
regions for each of the 6-,12-,and 24-month-old rats (n=6 for
each age). One "group" consisted of one 6-month-, one 12-
month-, and one 24-month-old animal mounted per series of
slides. Ten slides, from each of these groups, were analyzed
per assay. The regions that were analyzed included the outer
frontal cortex (OFCTX), inner frontal cortex (IFCTX),
entorhinal cortex (ERC), dentate gyrus of the hippocampus
(DG), hippocampal area CA1, lateral septum (LSEP), lateral
striatum (LSTR) and the lateral thalamus (LTHAL).


43
Repeated measures analysis of variance (ANOVA) with
between-animal randomized blocks and age considered as a
linear continuous covariate was used to model the pattern of
MK-801 binding as a linear function of age within each brain
region. F-tests were also performed for the presence of a
significant interaction between age and brain region receptor
density effects. In assessing the validity of ANOVA
assumptions, it was noted that experimental day age profiles
of receptor binding were parallel within each brain region
and that between-animal variability as estimated by the
standard deviation was similar among age groups within each
brain region. This indicated that the patterns of [3H]MK-801
binding obtained for each group of 6-, 12-, and 24-month-old
rats were the same, independent of which experimental day
each group was assayed. Within age groups however, brain
region between-animal standard deviations tended to increase
as the mean level of receptor density in those regions
increased. ANOVA models assume that standard deviations do
not vary in this manner. Therefore, receptor binding density
values were transformed logarithmically, prior to analysis,
in order to appropriately model this inherent pattern of
variability within the ANOVA framework. Plots of residuals
versus predicted values, residual histograms, and residual
normal probability plots were examined to assess goodness of
fit in the ANOVA model. The statistical analyses determined
which mean age profiles of MK-801 binding density appeared to


44
be linearly decreasing in specific brain regions and which
profiles had slopes which were significantly less than zero.
Results
r 3H1MK-801 Binding
The IFCTX, ERC and LSTR were the only regions analyzed
that underwent a significant change (decrease) in [3H]MK-801
binding when 24-month-old animals were compared to 6-month-
old animals. The mean age profiles for the IFCTX, ERC and
the IFCTX all linearly decreased and had slopes significantly
less than 0 with p values of .0026, .0151 and .0018
respectively (Figure 3-1). The other brain regions analyzed,
the OFCTX, the LSEP, the LTHAL, the molecular layer of the DG
and hippocampal area CAl, did not change as a function of
increasing age (Figures 3-2 and 3-3). A representative
autoradiograph shows the distribution of [3h]MK-801 binding
in the rat brain (Figure 3-4).


45
1.0 i
0.8-
0.6-
0.4-
0.2-
0.0
6 mo.
E3 12 mo.
24 mo.
* p< .003
** p< .01
***p< .002
IFCTX ERC LSTR
Brain Region
Figure 3-1. Brain regions that underwent a significant age-
related decrease in [3H]MK-801 binding. (IFCTX=inner frontal
cortex; ERC=entorhinal cortex; LSTR=lateral striatum).
Repeated measures ANOVA with between-animal randomized blocks
and age as a linear covariate presented as a function of
increasing age. The p values represent mean age profiles with
slopes significantly less than 0.


46
2 n
OFCTX LSEP LTHAL
Brain Region
Figure 3-2. Brain regions that did not undergo a significant
age-related change in [3H]MK-801 binding. (OFCTX= outer
frontal cortex; LSEP= lateral septum; LTHAL= lateral
thalamus). Repeated measures ANOVA with between subject
randomized blocks and age as a linear covariate presented as
a function of increasing age.


47
O
O
OO O)
£
£ I
DG CAI
6 mo.
12 mo.
24 mo.
Brain Region
Figure 3-3. Brain regions that did not undergo a significant
age-related change in [3H]MK-801 binding. (DG= molecular
layer of the hippocampal dentate gyrus; CA1= hippocampal area
CAl stratum radiatum). Repeated measures ANOVA with between
subject randomized blocks and age as a linear covariate
presented as a function of increasing age.


Figure 3-4. NMDA receptor distribution as determined by
[3H]MK-801 binding in : (A) 6-month-old, (B) 12-month-
old, and (C) 24-month-old rat brain.


49


50
TABLE 3-1. Laminar thickness (mm), volume (mm3), and
neuronal density (neurons/mm2) values from Fischer 344 rat
brain.
6-month-old
12-month-old
24-month-old
Laminar
Thickness
(mm)
Frontal Cortex
Mean= 0.1952
SEM = 0.0013
0.19561
0.00211
0.1945
0.0017
Entorhinal
Cortex
Mean= 0.2988
SEM = 0.0031
0.2940
0.0037
0.2980
0.0026
Volume (mm3)
Lateral
Striatum
Mean=41.8491
SEM = 1.2064
41.8147
1.0065
41.3812
0.5626
Neuronal
Density
(per mm2)
Entorhinal
Cortex
Mean=1343.86
SEM = 36.60
1300.38
127.03
1319.50
41.33
Inner Frontal
Cortex
Mean=1285.50
SEM = 35.17
1279.89
10.86
1285.31
33.81
Lateral
Striatum
Mean=1284.23
SEM = 24.01
1284.71
51.28
1297.88
21.01
n=5 animals
per age qroup
No statistically significant differences found with ANOVA
(multi-comparison significance level at 95%, repeated
measures).


51
Discussion
These studies showed that the number of NMDA receptors,
as determined by [3H]MK-801 binding, is reduced in the ERC,
the LSTR and the IFCTX in aged F-344 rats. However, receptor
density in the OFCTX, the LSEP, the LTHAL, the molecular
layer of the DG as well as hippocampal area CA1 did not
change as a function of increasing age.
It is interesting that the decrease in the density of
[3h]MK-801 binding was limited to only three of the eight CNS
structures analyzed. The ERC was one structure that did
undergo an age-related decrease in MK-801 binding. The ERC
projects to the hippocampal formation, a component of the
limbic system associated with brain mechanisms for memory.
More specifically, the ERC is the major source of extrinsic
afferents to the dentate gyrus granule cells and pyramidal
cells of the hippocampus proper (Amaral and Witter, 1989).
There are decreased numbers of NMDA receptors in the ERC of
aged rats and this may affect the connectivity from this
structure to the hippocampus. This may result in an
alteration in NMDA receptor-mediated neurotransmission and
subsequently alter some aspects of memory (e.g., spatial
information processing) (Barnes and McNaughton, 1985; Morris
et al., 1978). It has been shown that excitotoxic lesions of
the rat ERC impair the retention of reference memory tasks
(Levisohn and Isacson, 1991). Age-dependent deficits in
spatial memory have been shown to be related to impairments


52
in hippocampal "kindling" (used as a measure of neuronal
plasticity) in F-344 rats (DeToledo-Morrell et al., 1984).
The kindling phenomenon is dependent upon sequential synaptic
modification in a cascading neural system (DeToledo-Morrell
et al., 1984), and would be expected to be especially
vulnerable to age-related alterations in postsynaptic
mechanisms such as a decrease in NMDA receptors in the
entorhinal cortex. The ERC, in addition to having
connections with the hippocampus, has reciprocal connections
with a wide range of cortical association areas (Amaral and
Witter, 1989). The decrease in MK-801 binding in the ERC
could therefore also affect cortical circuitry by decreasing
the overall amount of NMDA receptor-mediated
neurotransmission.
The decrease in MK-801 binding observed in the IFCTX of
the aged animals may contribute to some alteration in the
circuitry necessary for learning and memory as well.
Cortical-striatal projections have been shown to utilize NMDA
receptors (Cherubini et al., 1988), and a decreased density
of NMDA receptors in both IFCTX and the LSTR may affect the
NMDA receptor-mediated neurotransmission between these
projections. Piggott et al. (1992) found that [3H]MK-801
binding declined with age in human frontal cortical
membranes. More recently, Serra et al. (1994) found that the
total number of binding sites for [3H]MK-801 was decreased in
the hippocampus, cerebral cortex and striatum of 18- and 24-
month-old rats, relative to 3-month-old animals. Other


53
groups have also shown a significant age-related decrease in
[3H]MK-801 binding in cerebral cortical and hippocampal brain
homogenates (Kitamura et al., 1992; Tamaru et al., 1991). In
another study, the ERC, IFCTX and LSTR were among various
brain regions that were shown to undergo the greatest percent
decline in NMDA-displaceable [3H]L-glutamate binding when 30-
month-old mice were compared to 3-month-olds (Magnusson and
Cotman, 1993). These regions may be some of the most
vulnerable to the effects of aging. Since these regions are
implicated in normal memory function and plasticity of the
CNS (DeToledo-Morrell et al., 1984; Gonzales et al., 1991;
Petit, 1988), an age-related decrease in NMDA receptors in
these regions suggests that some of the cellular mechanisms
encoding memory are subsequently impaired.
It was determined that the age-related decrease seen in
[3h]MK-801 binding was not a result of a decrease in neuronal
density in the ERC, IFCTX or the LSTR. Age comparisons were
made in neuronal packing densities for each of these brain
regions. As West et al. (1993) stated, the volume and/or
potential shrinkage (laminar thickness) of specific brain
regions needs to be determined in order to obtain accurate
measurements of changes that may be occurring in specific
neuronal populations. He stated that only when these
parameters are known can neuron packing density be used,
instead of total neuronal number, in a meaningful discussion
of the functional state of a neural structure (West et al,
1993). In the present study, neuronal packing density was


54
measured as the number of neurons per square millimeter.
Therefore, this measurement would only be valid after
determining that these brain regions did not undergo an age-
related change in overall volume (LSTR) or laminar thickness
(ERC and IFCTX) (i.e., the mm^ denominator value was not
affected by age). No significant age-dependent differences
were found in the volume of the LSTR or in the laminar
thickness of the ERC and IFCTX (Table 3-1). Neuronal packing
densities were then determined utilizing an MCID image
analysis system for the ERC, the IFCTX and the LSTR. There
was no statistically significant difference in neuronal
density measurements in any brain region examined. Therefore
the decrease seen in [^h]MK-801 binding represents a loss of
NMDA receptors within these brain regions rather than a loss
of neurons.
In this study, [3H]MK-801 binding density was expressed
in pmol/mg protein. Therefore, changes in protein
concentration in specific brain regions as a function of age
may effect these values. Burnett and Zahniser (1989)
performed a quantitative autoradiographic study that examined
age-related changes in a-1 adrenergic receptors in F-344 rat
brain. Protein levels were measured in the same tissue
sections by using a staining procedure described by Miller et
al. (1988). They found no significant differences in protein
concentration between age groups (i.e., 3- to 4-month-old,
16- to 18-month-old and 24- to 28-month-old) in the thalamus,
cerebral cortex, hippocampus, striatum, cerebellum,


55
brainstem, and olfactory tubercle. Since the ages and strain
of rats used in the present study were identical to Burnett
and Zahniser (1989) it is expected that total protein would
also remain unchanged as a function of age. Therefore, it is
probable that age-related differences in [3H]MK-801 binding
density in specific brain regions is due to alterations in
receptor number and not in protein concentration.
It should be noted that finding an age-related effect on
[3H]MK-801 binding in only three of eight brain regions
analyzed may reflect the inability of MK-801 to label all of
the NMDA receptors, since ligand binding can only assay
surface-presented receptors. Studies investigating mRNA
coding for the NMDA receptor is presented in Chapter 5. The
density of NMDA protein is also presented in Chapter 6.
These further studies address the hypotheses that the density
of mRNA coding for the NMDA receptor and the density of NMDA
protein change as a function of age.
These [3h]MK-801 binding experiments have determined
some of the age-dependent changes taking place in the NMDA
receptor/channel complex. The decrease in NMDA receptor
density seen in specific brain regions may account for some
of the deleterious effects of aging on learning and memory
(Barnes, 1979; Barnes and McNaughton, 1985). Gonzales et al.
(1991) studied NMDA receptor-mediated responses in the
hippocampus, cortex, and striatum of F-344 rats of various
ages (3- to 5-, 12- to 14-, and 24- to 28-months-of-age) to
determine whether aging alters some of the functional


56
properties of this receptor complex. They examined NMDA-
stimulated release of norepinephrine and dopamine as indices
of NMDA receptor function and found that NMDA-mediated
responses were attenuated with increasing age. In Chapter 4,
the effects of increasing age on L-glutamate's ability to
enhance [3h]MK-801 binding is studied. This approach was
taken to see if there were differences in the Emax (maximal
response or density of [3H]MK-801 bound) or EC50 (the
concentration of L-glutamate producing a half-maximal
response) as a function of age.
Many of the processes underlying learning and memory are
known to utilize, to a great extent, NMDA receptors (Danysz
et al., 1988; Morris et al., 1986). Therefore, elucidation
of age-dependent changes in NMDA receptors may help target
potential pharmaceutical interventions aimed at alleviating
age-related cognitive decline. It has been suggested that
pharmacological manipulation of glutamatergic
neurotransmission may prove beneficial for cognitive
enhancement (Ingram et al., 1994). However, the utilization
of NMDA receptor agonists may be an unsafe strategy due to
the potential neurotoxic effects associated with
overstimulation of the NMDA receptor (Cotman and Monaghan,
1988). Therefore, indirect activation through other
modulatory sites identified on the NMDA receptor complex
could be investigated, such as the co-agonist glycine site
and/or the polyamine modulatory sites.


57
Milacemide and D-cycloserine are two strong candidates
for agonists at the glycine site and have both been shown to
enhance memory performance in rats (Flood et al., 1992;
Quartermain et al., 1991). A relatively new class of
chemical substances, nootropics, were developed specifically
to alleviate age-related cognitive deficits and have been
shown to enhance NMDA receptor density (10 to 25%) in aged
brain (Cohen and Muller, 1992; Davis et al., 1993; Fiore and
Rampello, 1989). Phosphatidylserine is one example of a
nootropic drug. Cohen and Muller (1992) showed that chronic
treatment with phosphatidylserine ameliorated age-associated
deficits of the NMDA receptor in the forebrain of aged mice
by restoring the density of NMDA receptors to levels similar
to young animals and also by normalizing the sensitivity of
aged animals for the stimulating effects of L-glutamate and
glycine. Cohen and Muller (1993) also showed significant
enhancement (20%) of NMDA receptor density in aged mice
treated with another nootropic drug, piracetam. The kinetic
constants for L-glutamate stimulation of [3H]MK-801 binding
in the aged animals were not significantly different from
untreated young mice (Cohen and Muller, 1993). In addition,
the nootropic agent L-acetylcarnitine was shown to attenuate
the age-dependent decrease of NMDA-sensitive glutamate
receptors in the rat hippocampus (Davis et al., 1993; Fiore
and Rampello, 1989). Taken together, these studies imply
that specific pharmacological targeting of the NMDA receptor


58
may indeed prove to be a viable treatment strategy for
restoration of cognitive deficits in the aged population.


CHAPTER 4
EFFECT OF AGE ON L-GLUTAMATE STIMULATION OF [3H]MK-801
BINDING IN F-344 RAT BRAIN
Introduction
In Chapter 3, a decrease in the number of NMDA receptors
in specific brain regions in the F-344 rat was shown. Other
studies have described a similar reduction in NMDA receptors
in various species (Anderson et al., 1989; Ingram et al.,
1992; Magnusson and Cotman, 1993; Peterson and Cotman, 1989;
Serra et al., 1994; Tamaru et al., 1991; Wenk et al., 1991)
including humans (Piggott et al., 1992). In Chapter 3,
[3H]MK-801 was utilized to study the NMDA receptor/channel
complex. This antagonist binds with high affinity to a site
within the channel of the NMDA receptor complex, thus MK-801
binding will only take place if the NMDA receptor is in the
transmitter-activated (open) state (Huettner and Bean, 1988).
Channel activation by an NMDA agonist such as L-glutamate, in
the presence of glycine allows MK-801 binding to take place.
Glycine alone cannot activate the NMDA receptor. In addition
to the glycine site on the NMDA receptor/complex, there is an
allosteric polyamine recognition site. The polyamines
spermine and spermidine have been shown to increase both
maximum NMDA response amplitude (Lerma, 1992; McGurk et al.,
59


60
1990; Ransom and Deschenes, 1990; Sprosen and Woodruff, 1990)
as well as binding of MK-801 to the NMDA receptor channel
(Ransom and Stec, 1988).
L-glutamate is the endogenous transmitter for the NMDA
receptor channel complex. In this study, an evaluation of
the efficacy of L-glutamate to stimulate MK-801 binding
within the NMDA receptor/channel complex was performed by
using various concentrations of L-glutamate. These studies
examined the efficacy and potency with which L-glutamate
produces receptor activation and channel opening as a
function of increasing age by comparing dose-response curves
from 6-, 12-, and 24-month-old animals.
The hypothesis tested in these experiments was that
there would be an age-related decrease in the maximal
response (Emax) elicited by L-glutamate. It was also
hypothesized that there would not be a concomitant change in
the concentration of L-glutamate producing a half-maximal
response (EC50) as a function of age. in these experiments,
the response of the NMDA receptor was defined as the density
of [3H]MK-801 binding determined as a function of L-glutamate
concentration.
Many studies have shown that there are decreased numbers
of NMDA receptors in aged animals (Anderson et al., 1989;
Ingram et al., 1992; Magnusson and Cotman, 1993; Peterson and
Cotman, 1989; Piggott et al., 1992; Serra et al., 1994;
Tamaru et al.,1991; Wenk et al., 1991). Consequently, there
are fewer NMDA receptors available for occupation by ligand


61
in aged rats. Because there are fewer NMDA receptors
available for occupation by ligand in aged rats it would be
expected that there would be a decreased maximal response
([3H]MK-801 bound) in aged rats due to fewer NMDA receptor
channels available to be activated (opened) by agonist. In
addition, previous studies have shown an age-related decrease
in the functional capacity of the NMDA receptor/channel
complex as defined by a reduction in various NMDA-mediated
responses (Gonzales et al., 1991; Pittaluga et al., 1993;
Serra et al., 1994). The ability of L-glutamate to stimulate
[3H]MK-801 binding was therefore compared across varying age
groups in the F-344 rat.
Methods
Tissue Preparation
The male F-344 rats, 6-, 12- and 24-months-of-age, that
were used in these experiments were the same as those
described in Chapter 3. For a more detailed description of
the tissue preparation see Chapter 2.
r-^HIMK-801 Binding Assay
For a detailed description of this protocol, see Chapter
2. Varying concentrations of L-glutamate (i.e., 0.0 pH, 0.05
p, 0.1 pM, 0.25 pM, 0.5 pM, 1.0 pM, 2.5 pH, 5.0 pM, 10 p,
and 20 phi) were added to the incubation step in order to
examine glutamate's ability to stimulate [3H]MK-801 binding.


62
A total of four slides, from each block of animals, were
analyzed per assay (n=6) for each L-glutamate concentration.
Image analysis
See Chapter 2 for a detailed description of image
analysis.
Data Analysis
The data consist of the EC50 and Emax values obtained
from binding isotherm plots. These plots were generated from
average density measurements of [3H]MK-801 binding (pmol/mg
protein) in the presence of increasing concentrations of L-
glutamate. Binding densities were obtained from bilateral
brain regions for each of the 6-,12-, and 24-month-old rats
(n=6 for each age). One "group" consisted of one 6-month-,
one 12-month-, and one 24-month-old animal mounted per series
of slides. Each [3H]MK-801 binding assay was run with the
three ages of animals mounted per slide. Four slides, from
each of these groups, were analyzed per assay per L-glutamate
concentration, with each [3H]MK-801 binding assay being
performed on one group of animals (n=6). Two slides per
assay were analyzed for non-specific binding and these values
were subtracted from each specific binding value in order to
obtain total binding. Brain regions that were analyzed
included the outer and inner frontal cortices (OFCTX, IFCTX);
the entorhinal cortex (ERC); the molecular layer of the
dentate gyrus (DG); hippocampal area CA1 stratum radiatum


63
(CAI); the lateral septum (LSEP); the lateral striatum
(LSTR); and the lateral thalamus (LTHAL).
Binding Isotherm Plots
INPLOT (GraphPad Software, San Diego, CA) was used to
generate linear regression plots from the density values
obtained in these experiments. Plots gave estimated EC50
(effective concentration at 50%) and Emax (maximal [3h]MK-801
binding elicited by L-glutamate) values for L-glutamate
stimulation of [3H]MK-801 binding. Estimates were then used
to generate rectangular hyperbola plots or binding isotherms.
Binding isotherm plots then yielded the true EC50 and Emax
values for each age group and brain region analyzed. As the
concentration of L-glutamate increased, there was a
concominant increase in the density of [3H]MK-801 binding
(Figure 4-1). EC50 and Emax values obtained from [3H]MK-801
binding isotherm plots for the 6-month-old animals were used
to normalize the 12- and 24-month-old animal values.
Statistical Analysis
Six blocks of animals, each block consisting of an
animal from each age group, were assessed. A different block
of 3 animals, 1 per age group, was assessed on each of 6
experimental days. The experimental design consisted of a
within-animal factor with 8 levels (brain region), a
guantitative between-animal fixed factor with 3 levels (age),
and a between-animal random blocking factor with 6 levels


64
(experimental day). Repeated measures analysis of variance
(ANOVA) with between-animal randomized blocks and age
considered as a linear covariate were used to model the EC50
and Emax data as a linear function of age within each brain
region and to determine if this pattern differed
significantly among brain regions. The change in EC50 and
Emax per month-of-age (i.e. the linear slope) was estimated
and assessed for statistical significance within each brain
region while blocking on experimental day. A general
comparison of these slopes among brain regions was then
performed by testing for the presence of a significant
interaction between age and brain region EC50 and Emax
effects. Mauchly's sphericity test was used to determine if
a multivariate F test (Wilks' Lambda) or a univariate F test
should be used for the age x brain region interaction test.
In assessing the validity of ANOVA assumptions, it was noted
that experimental day age profiles were relatively parallel
within each brain region and that between-subject variability
as estimated by the standard deviation was similar among age
groups within each brain region. Plots of residuals versus
predicted values, residual histograms, and residual normal
probability plots were examined to assess goodness of fit in
the ANOVA models.
Results
[3H]MK-801 binding increased as a function of L-
glutamate concentration in all the age groups and a


65
representative autoradiogram is presented in Figure 4-1.
Significant differences between the Emax values in nearly all
brain regions were found when middle-aged and aged animals
were compared to the young rats (Table 4-1). The only region
where Emax did not differ in middle-aged animals, when
compared to young-adults, was the OFCTX. The areas showing
the greatest percent decline in aged animals when compared to
young were the LSTR (26.4%), ERC (24.7%), and IFCTX (21.5%).
Areas showing the greatest percent decline in middle-aged
animals when compared to young were the LSTR (12.9%), LSEP
(10.3%), and IFCTX (9.3%).
No significant differences were found in the EC50 values
when comparing middle-aged and aged rats to young (Table 4-
2).


66
TABLE 4-1. Emax values (pmol/mg protein) from [^h]MK-801
binding isotherms.
BRAIN REGION
6 month
12 month
24 month
OFCTX
1.588 + 0.29
1.468 + 0.29
1.441 + 0.24*
IFCTX
0.959 + 0.16
0.870 + 0.17*
0.753 + 0.14*
ERC
0.830 + 0.12
0.758 + 0.14*
0.625 + 0.12*
DG
1.874 + 0.37
1.737 + 0.40*
1.669 + 0.39*
CAl
1.712 + 0.36
1.618 + 0.41*
1.537 + 0.40*
LSEP
0.951 + 0.20
0.853 + 0.19*
0.850 + 0.21*
LSTR
0.780 + 0.15
0.679 + 0.14*
0.574 + 0.12*
LTHAL
0.793 + 0.15
0.747 + 0.15*
0.665 + 0.14*
MEAN + SEM
MEAN + SEM
MEAN + SEM
n=6
n=6
n=6
Asterisks (*) indicates significance when compared to 6-
month (p < 0.05 ANOVA; F-test). Abbreviations: OFCTX=outer
frontal cortex; IFCTX=inner frontal cortex; ERC=entorhinal
cortex; DG=molecular layer of the dentate gyrus;
CAl=hippocampal area CAl; LSEP=lateral septum; LSTR=lateral
striatum; LTHAL=lateral thalamus


67
TABLE 4-2. EC50 values (pM) from [3H]MK-801 binding
isotherms.
BRAIN REGION
6 month
12 month
24 month
OFCTX
0.764 + 0.14
0.758 + 0.18
0.798 + 0.22
IFCTX
1.311 + 0.23
1.085 + 0.26
1.148 + 0.39
ERC
1.108 + 0.17
1.197 + 0.32
0.921 + 0.25
DG
0.951 + 0.21
0.936 + 0.25
1.040 + 0.20
CA1
0.782 + 0.17
0.660 + 0.17
0.725 + 0.20
LSEP
0.432 + 0.11
0.444 + 0.14
0.410 + 0.14
LSTR
0.578 + 0.13
0.586 + 0.15
0.599 + 0.23
LTHAL
0.993 + 0.20
0.861 + 0.18
0.892 + 0.23
MEAN + SEM
MEAN + SEM
MEAN + SEM
n=6
n=6
n=6
No significant differences were found when
.2-, and 24-
month-old rats were compared to 6-month-old rats (p > 0.05
ANOVA; F-test). Abbreviations: OFCTX=outer frontal cortex;
IFCTX=inner frontal cortex; ERC=entorhinal cortex;
DG=molecular layer of the dentate gyrus; CAl=hippocampal area
CA1; LSEP=lateral septum; LSTR=lateral striatum;
LTHAL=lateral thalamus


Figure 4-1. Total [3H]MK-801 binding in a range of
concentrations of L-glutamate and a constant
concentration of [3H]MK-801 (10 nM). Concentration of L-
glutamate was as follows: (A), 0.0 ^/M; (B), 0.25 pM;
(C), 2.5 pM; (D), 10 pM. (A) Note that only background
levels of binding were present in sections incubated
without L-glutamate. (B-D) As the concentration of L-
glutamate increased, [3H]MK-801 binding increased in a
dose-dependent manner. Autoradiographs are shown in
inverse exposure so that white indicates high density of
binding and black indicates low levels of binding.


c


70
Discussion
The lateral striatum (LSTR), entorhinal cortex (ERC) and
inner frontal cortex (IFCTX) were the three brain regions
that were shown in Chapter 3 to undergo an age-related
decrease in [3h]MK-801 binding when maximal levels of
glutamate, glycine and spermine were present. These regions
also showed the greatest percentage decrease in Emax values
without concomitant changes in EC50 values as a function of
age. Because both middle-aged and aged animals displayed a
significant decrease in Emax values without differences in
their EC50 values, it appears that there is an age-related
effect on the maximum density of [3H]MK-801 bound as a
function of L-glutamate concentration.
The maximal effect that L-glutamate has on stimulating
[3H]MK-801 binding is decreased in aged rats. This age-
dependent decrease in Emax may be the result of a down-
regulation of NMDA receptors in specific brain regions.
These brain regions may undergo down-regulation to protect
against excitotoxic damage. In a developmental study, Oster
and Schramm (1993) demonstrated that the process of down-
regulation of NMDA receptor activity occurs in rat cerebellar
granule cells. In these experiments, NMDA was added to cell
cultures derived from postnatal day 8 rats. This resulted in
suppression of NMDA receptor-mediated 4^ca uptake without
affecting the viability or total cell protein of the cultured
neurons. The down-regulation also rendered the neurons


71
resistant to NMDA toxicity. They proposed that a similar
form of down-regulation may play a role in adjusting the
activity of postsynaptic NMDA receptors following
synaptogenesis.
Jakoi et al. (1992) showed that activation of EAA
receptors in cultured hippocampal neurons caused a down-
regulation of the protein ligatin at both physiologic and
excitotoxic levels of glutamate stimulation. This down-
regulation was shown to be mediated by the NMDA receptor and
it was hypothesized that EAA receptor activation may alter
expression of NMDA receptors. This effect on NMDA receptor
expression may be a central mechanism that underlies some of
the long-lasting functional and pathophysiological effects of
EAA receptor activation on cell function (Jakoi, et al.,
1992). Therefore, it is possible that down-regulation of
NMDA receptors in the aging CNS may affect the efficacy of L-
glutamate to maximally enhance the receptor's binding
capacity in the aged brain.
Neurons bearing NMDA receptors are vulnerable to
excitotoxic injury associated with an excessive concentration
of extracellular glutamate or related agonists (Choi, 1987).
An age-related increase in basal glutamate release in mouse
striatal and hippocampal slices has been reported (Freeman
and Gibson, 1987). Low affinity glutamate uptake into rat
cerebral cortical slices (Matsumoto et al., 1982) and brain
mitochondria (Victorica et al., 1985) has been shown to be
reduced as a function of age. Furthermore, an age-related


72
loss in the number of high affinity glutamate transport
(uptake) sites of rat striatal (Price et al., 1981) and
cortical synaptosomes (Wheeler and Ondo, 1986) has been
reported. Taken together, these reports suggest that
extracellular glutamate levels are elevated with aging.
Therefore, the age-related down-regulation in NMDA receptors
may essentially be a functional trade-off in order to protect
cells bearing NMDA receptors from excitotoxic insult.
A recent study reported data that contradict some of the
findings here (Serra et al., 1994). Using hippocampal,
striatal and cerebral cortical brain homogenates from 3-, 18-
and 24-month-old male Wistar Kyoto rats a decrease (20 to
25%) was seen in the total number of NMDA receptors in 18-
and 24-month-old rats. No significant differences were found
in Kp between young and aged rats. In addition, no
difference was seen in the sensitivity of [3H]MK-801 binding
as a function of glutamate concentration in the cerebral
cortex, striatum or hippocampus of the aged rats. However,
in the hippocampus of 18-month-old rats, glycine and
glutamate stimulated [3H]MK-801 with a higher efficacy than
in 3- or 24-month-old rats. This group speculated that the
loss of NMDA receptors in the hippocampus of 18-month-old
rats is counteracted by a physiological compensatory
mechanism. This mechanism somehow increases NMDA receptor
activity and thereby prevents a decline in cognitive function
in the "early phase of aging" (e.g., 18-months-of-age). The
compensation ceases to function adequately during, what this


73
group termed, the "late phase of aging" (i.e. 24 months of
age) (Serra et al., 1994). One possible explanation for the
differences in these findings is that 18-month-old animals
were not examined in the present study. Although Serra et
al. (1994) used thorough washing techniques on the rat
membrane homogenates they speculated that the sensitivity of
[3H]MK-801 binding to endogenous concentrations of agonists
and allosteric modulators of the NMDA receptor may have
affected their results. Therefore, another possible
explanation for this discrepancy is the aging-induced changes
in the brain concentrations of glycine and glutamate and/or
other modulators which could affect [3H]MK-801 binding
(Freeman and Gibson, 1987).
Gonzales et al. (1991) found an age-related decrease in
NMDA receptor function in aged animals by analyzing NMDA-
stimulated neurotransmitter release in rat cortex,
hippocampus and striatum. They showed that NMDA-stimulated
[3H]NE (norepinephrine) and [3H]DA (dopamine) release were
decreased as a function of age. In a similar study,
Pittaluga et al. (1993) showed an age-related decrease in
NMDA receptor-mediated noradrenaline release in rat
hippocampus.
It should be noted that there are advantages to using
quantitative autoradiographic analysis over brain tissue
homogenates in that it allows for a more detailed analysis of
any anatomical distributional changes occurring in the rat
CNS. However, although there are other methods that can also


74
test the NMDA receptor's functional capacity (e.g.
electrophysiological patch clamping techniques, molecular
biological oocyte expression systems etc.), the experiments
performed in this study tested the ability of glutamate to
enhance [3H]MK-801 binding. It should be noted that a
confounding variable possibly influencing these results is
the phenomenon of receptor desensitization, which is defined
as the lack or decline of a response as a result of previous
activation. Since brain slices in these experiments were
exposed to glutamate for long periods of time, the receptors
may have desensitized. Therefore, responses of NMDA
receptors may have been examined in the desensitized state.
The sections used in this study were rinsed for a total
of 60 minutes at 30C prior to incubation with [3H]MK-801.
Previous studies performing [3H]MK-801 binding assays have
shown that prewashing sections for as little as thirty
minutes removed substantial amounts of endogenous amino acids
since [3H]MK-801 binding following incubation was stimulated
significantly with the addition of exogenous glutamate
(Sakurai et al., 1990). Therefore, any potential effects of
desensitization were probably not seen prior to the
incubation with radiolabeled MK-801. It should also be noted
that the time-course for the amount of [3H]MK-801 bound to
reach equilibrium in the binding assay occured prior to the
one hour incubation period utilized in these experiments (the
incubation step is when the brain sections are exposed to
[3H]MK-801 in the presence of glutamate, glycine and


75
spermine). Since, at equilibrium, the receptors have reached
their greatest maximal response, comparisons can be made
between the various age groups with any observed differences
in the density of [3H]MK-801 binding being attributed to the
age factor. Monaghan (1991) examined the differential
stimulation of [3H]MK-801 binding to subpopulations of NMDA
receptors and stated that since excess concentrations of
glycine and spermine were present in all incubations and both
sites analyzed could be 'activated' neither site appeared to
be in a desensitized form. However, if desensitization
occurs within minutes or seconds of glutamate application,
then age-related differences in [3H]MK-801 binding may not be
able to be interpreted as clearly due to potential age-
related differences in densensitization mechanisms. In order
to alleviate some of these potential problems in these
experiments, desensitization will be defined as a decreased
density of [3H]MK-801 (response) as a function of increased
glutamate concentration. It is important to note that the
density of [3H]MK-801 bound did not decrease after reaching
maximal levels in any of the age groups, suggesting that
desensitization may not be a significant factor in these
studies.
Consistent with L-glutamate's ability to open the ion
channel associated with the NMDA receptor, it was shown in
this study that [3H]MK-801 binding increased as a function of
L-glutamate concentration in all age groups. However, these
findings suggest that there is an effect of age on the


76
maximal increase in [3H]MK-801 binding induced by L-
glutamate. These age-dependent changes in the maximal
response elicited by L-glutamate may be due to some
functional alteration in the NMDA receptor/channel complex.
Hollenberg (1985) proposed that receptors can be modified by
reactions leading to either covalent or noncovalent bond
formation. In covalent modifications, reactions involving
receptor phosphorylation, disulfide-sulfhydryl exchange and
receptor proteolysis are all possible mechanisms involved in
receptor function (Hollenberg, 1985). Non-covalent
interactions would include changes in membrane potential,
receptor distribution (patching, capping), allosteric
interactions involving either protein-protein (e.g. mobile
receptor model) or small ligand (cations, anions,
nucleotides, etc.) receptor interactions, and alterations in
the membrane lipid environment (e.g. lipid methylation or
hydrolysis) (Hollenberg, 1985). All of these processes could
result in changes in the functional modification of the NMDA
receptor. In conclusion, there is probably a combination of
both an age-dependent decrease in the total number of NMDA
receptors available for binding as well as some age-related
change in the functional capacity of the remaining NMDA
receptors. This may account for some of the differences
found in the maximal increase in [3H]MK-801 induced by L-
glutamate as a function of age.


CHAPTER 5
AGE-RELATED CHANGES IN THE LEVELS OF mRNA CODING FOR SPECIFIC
NMDA RECEPTOR SUBUNITS IN THE CNS OF F-344 RATS.
Introduction
Two NMDA receptor subunit families have been cloned and
are named NMDAR1 (NRl) and NMDAR2 (NR2)(Monyer et al., 1992;
Moriyoshi et al., 1991). NMDARl has at least eight
alternatively spliced forms and these are differentiated from
each other by an insertion at the extracellular amino-
terminal region, deletion at two carboxy-terminal regions, or
by combinations of both (Moriyoshi et al., 1991). NMDARl and
its isoforms have been shown in Xenopus oocyte expression
systems to exhibit electrophysiological and pharmacological
responses characteristic of the NMDA receptor. These include
agonist and antagonist selectivity, glycine modulation,
permeability to calcium, voltage-dependent channel block by
magnesium as well as inhibition by zinc (Moriyoshi et al.,
1991).
NMDAR2 subunit family members have been shown to
potentiate the electrophysiological responses of NMDARl but
are not functional in homomeric configurations with each
other. NMDA-induced currents in oocytes expressing NRl and
NR2A, NR2B or NR2C are approximately 100 times larger than
they are in oocytes expressing homomeric NRl channels. It
77


78
should be noted that these currents more closely resemble
native NMDA receptors and thus native NMDA receptors probably
represent heteromeric configurations formed from NRl subunits
and members of the NR2 subunit family (Monyer et al., 1992).
The NMDA receptor has been implicated in age-related
learning and memory deficits in humans as well as other
animals (Barnes, 1979; Barnes and McNaughton, 1985).
Detailed in situ hybridization analyses can now be performed
due to the recent isolation of functional cDNA clones for the
rat NMDA receptor NRl (NMDAR1), splice variants of NRl as
well as for the members of the NR2 subunit family (i.e.,
NR2A, NR2B, NR2C and NR2D) (Buller et al., 1993; Kutsuwada et
al., 1992; Meguro et al., 1992; Monaghan et al., 1993; Monyer
et al., 1992; Moriyoshi et al., 1991; Nakanishi, 1992). In
this study, this technique was utilized to examine mRNA
coding for the NMDA receptor subunits in the aged brain.
Methods
In situ Hybridization
Oligonucleotide probes for this study (45 mer) were
constructed from published sequences and specific to NMDARl,
four alternatively spliced versions of NMDARl (NRl-not insert
1 or NRloxx/ NRl-insert 1 or NRlixx; NRl-insert 2 or NRlxix;
and NRl-insert 3 or NRlxxi), and the members of the NMDAR2
subunit family (NR2A, NR2B, NR2C, and NR2D). The probes for
this study were graciously supplied by Dr. Daniel T.


79
Monaghan, University of Nebraska Medical Center, Omaha NE.
See Chapter 2 for details on the probe labeling procedure,
tissue sectioning, fixation and hybridization.
Data Analysis
Computer assisted semi-quantitative densitometric
measurements were performed as described in detail in Chapter
2. Six-month (young), twelve-month (middle-aged) and twenty-
four month (aged) F-344 rat brains were used (n=6 for each
age group). The outer frontal cortex (OFCTX), inner frontal
cortex (IFCTX), lateral septum (LSEP), lateral striatum
(LSTR), lateral thalamus (LTHAL), entorhinal cortex (ERC),
dentate gyrus (DG), and hippocampal areas CA1 and CA3 were
the brain regions analyzed in this study. Relative mRNA
density levels were obtained for each of the oligonucleotide
NMDA probes. Density values from young adults were compared
to the middle-aged and aged animals.
Statistical Analysis
Six blocks of animals, each block consisting of an
animal from each age group, were assessed. The experimental
design consisted of a within-subject factor with nine levels
(brain region), a quantitative between-subject fixed factor
with three levels (age), and each of six levels (experimental
day). Repeated measures analysis of variance (ANOVA) with
between-subject randomized blocks and age considered as a
linear covariate were used to model the density of mRNA per


80
oligonucleotide probe as a function of age within each brain
region and to determine if this pattern differed
significantly among brain regions.
Results
Significant age-related changes were found in NMDAR1, as
well as three of the splice variants of NMDAR1: NRloxx/
NRllxx, and NRlxlx (Figures 5-1,5-2,5-4,5-5 and 5-7). More
specifically, NMDAR1 mRNA measured in 12-month-old rats
showed a significant decrease in the outer frontal cortex
(17.8%), inner frontal cortex (15.0%), hippocampal area CA3
(9.0%) and the lateral striatum (14.1%). In 24-month-old rat
brains, there was a significant decrease seen in all brain
regions analyzed, with the exception of the molecular layer
of the dentate gyrus. Areas showing the greatest percent
decline included the entorhinal cortex (28.1%) > lateral
thalamus (24.2%) > lateral striatum (20.7%) > outer frontal
cortex (18.6%) > inner frontal cortex (17.7%) > hippocampal
areas CA3 (14.5%) and CA1 (14.4%) > lateral septum (10.4%).
Relative mRNA density for NMDARl splice variant NRloxx
(not containing the 21 amino acid N-terminal insert) showed a
statistically significant decrease in the entorhinal cortex
(5.6%) as well as the CA3 region of the hippocampus (9.2%) in
24-month-old animals. This splice variant also showed a
significant increase in mRNA density in hippocampal area CAl
(5.5%) from 12-month-old animals. There was a significant
age-related change in relative mRNA density for two other


81
splice variants of NMDARl. The NRlixx (those NMDARl isoforms
containing the N-terminal insert) showed an increase in the
lateral septum (9.6%) from 12-month-old animals when compared
to 6-month-old animals (Figure 5-5). NRlxlx isoforms (those
splice variants containing the first of the two C-terminal
inserts) also showed an increase in the lateral septum (5.3%)
along with the lateral striatum (3.4%) and lateral thalamus
(4.7%) from 12-month-old animals (Figure 5-7). In contrast,
no significant differences were observed in either NRlixx or
NR1X1X mRNA density in any of the brain regions analyzed from
24-month-old animals (Figures 5-5, 5-6, 5-7, and 5-8). The
density of NMDARl splice variant NRlxxi (containing the
second C-terminal insert) did not show any differences in any
of the brain regions analyzed at any age (Figures 5-9 and 5-
10).
The NMDAR2 subunit family members, NMDAR2A and NMDAR2B,
did not show any age-related differences in relative density
of mRNA (Figures 5-11, 5-12, 5-13 and 5-14). NMDAR2C subunit
mRNA was not present in any of the brain regions examined.
Since this subunit is found only in the cerebellum it was
excluded from these analyses. There was no detectable
NMDAR2D mRNA present in any of the age groups. This may be
due to the fact that this subunit appears to be
developmentally regulated such that there is an apparent
shift in relative expression of NMDAR2D to NMDAR2B expression
at about six months-of-age in the rat CNS. Representative
autoradiograms are seen in Figures 5-15, 5-16 and 5-17.


82
NMDAR1
100 -i
OFCtx IFCtx LSep LStr LThal
i middle-aged
O aged
*- significant
at 95%
Brain Region
Figure 5-1. In situ hybridization analyses of NMDAR1 mRNA in
cortical and subcortical brain regions. All data are the mean
+ S.E.M. Asterisk indicates densitometric values
significantly different than young animals.(OFCtx=outer
frontal cortex; IFCtx=inner frontal cortex; LSep= lateral
septum; LStr= lateral striatum; LThal= lateral thalamus).
NMDAR1
as
C
3
O
>-
T5
& middle-aged
aged
*- significant
at 95%
Brain Region
Figure 5-2. In situ hybridization analyses of NMDARl mRNA in
hippocampus and entorhinal cortex. All data are the mean +
S.E.M. Asterisk indicates densitometric values significantly
different than young animals. (ERC= entorhinal cortex; DG=
molecular layer of the dentate gyrus; CAl & CA3= hippocampal
areas).


83
NRl Oxx
OFCtx IFCtx LSep LStr LThal
Brain Region
Figure 5-3. In situ hybridization analyses of NMDAR1 splice
variant NRlOxx mRNA. All data are the mean + S.E.M. Asterisk
indicates densitometric values significantly different than
young animals. (OFCtx=outer frontal cortex;IFCtx=inner
frontal cortex;LSep=lateral septum;LStr=lateral striatum;
LThal=lateral thalamus).
NRl Oxx
middle-aged
aged
*- significant
at 95%
Brain Region
Figure 5-4. In situ hybridization analyses of the NMDAR1
splice variant NRlOxx mRNA. All data are the mean + S.E.M.
Asterisk indicates densitometric values significantly
different than young animals. (ERC=entorhinal cortex;DG=
molecular layer of the dentate gyrus;CA1 & CA3=hippocampal
areas).


84
NR1 lxx
120 -i
OFCtx IFCtx LSep LStr LThal
Brain Region
Figure 5-5. In situ hybridization analyses of NMDARl splice
variant NRlixx mRNA. All data are the mean + S.E.M. Asterisk
indicates densitometric values significantly different than
young animals. (OFCtx=outer frontal cortex;IFCtx=inner
frontal cortex;LSep= lateral septum;LStr= lateral striatum;
LThal=lateral thalamus).
NR1 lxx
Brain Region
Figure 5-6. In situ hybridization analyses of the NMDARl
splice variant NRlixx mRNA. All data are the mean + S.E.M.
Asterisk indicates densitometric values significantly
different than young animals. (ERC=entorhinal cortex; DG=
molecular layer of the dentate gyrus;CA1 & CA3= hippocampal
areas).


85
NR1 xlx
120 -i
OFCtx IFCtx LSep LStr LThal
middle-aged
I I aged
*- significant
at 95%
Brain Region
Figure 5-7. In situ hybridization analyses of NMDARl splice
variant NRlxix mRNA. All data are the mean + S.E.M. Asterisk
indicates densitometric values significantly different than
young animals. (OFCtx=outer frontal cortex;IFCtx=inner
frontal cortex;LSep=lateral septum;LStr=lateral striatum;
LThal=lateral thalamus).
NRl xlx
120 n
ERC DG CAI CA3
$ middle-aged
n aged
*- significant
at 95%
Brain Region
Figure 5-8. In situ hybridization analyses of the NMDARl
splice variant NRlxix mRNA. All data are the mean + S.E.M.
Asterisk indicates densitometric values significantly
different than young animals. (ERC=entorhinal cortex;
DG=molecular layer of the dentate gyrus;CA1 & CA3=hippocampal
areas).


86
NRl xxl
120 -i
OFCtx IFCtx LSep LStr LThal
i middle-aged
aged
*- significant
at 95%
Brain Region
Figure 5-9. In situ hybridization analyses of NMDAR1 splice
variant NRlXxl mRNA. All data are the mean + S.E.M. Asterisk
indicates densitometric values significantly different than
young animals. (OFCtx=outer frontal cortex;IFCtx=inner
frontal cortex;LSep=lateral septum;LStr=lateral striatum;
LThal=lateral thalamus).
NRl xxl
Brain Region
Figure 5-10. In situ hybridization analyses of the NMDARl
splice variant NRlxxi mRNA. All data are the mean + S.E.M.
Asterisk indicates densitometric values significantly
different than young animals. (ERC=entorhinal cortex;DG=
molecular layer of the dentate gyrus;CA1 & CA3=hippocampal
areas).


87
,'i middle-aged
aged
*- significant
at 95%
Brain Region
Figure 5-11. In situ hybridization analyses of NMDAR2A mRNA.
All data are the mean + S.E.M. Asterisk indicates
densitometric values significantly different than young
animals. (OFCtx=outer frontal cortex;IFCtx=inner frontal
cortex;LSep= lateral septum;LStr= lateral striatum;LThal=
lateral thalamus).
NMDAR2A
Brain Region
Figure 5-12. In situ hybridization analyses of NMDAR2A mRNA.
(ERC=entorhinal cortex; DG=molecular layer of the dentate
gyrus;CA1 & CA3=hippocampal areas). All data are the mean +
S.E.M. Asterisk indicates densitometric values significantly
different than young animals.


88
a
C
3
O
>-
ts
K
a middle-aged
n aged
*- significant
at 95%
Brain Region
Figure 5-13. In situ hybridization analyses of NMDAR2B mRNA.
All data are the mean + S.E.M. Asterisk indicates
densitometric values significantly different than young
animals. (OFCtx=outer frontal cortex;IFCtx=inner frontal
cortex;LSep= lateral septum;LStr=lateral
striatum;LThal=lateral thalamus).
Brain Region
Figure 5-14. In situ hybridization analyses of NMDAR2B mRNA.
All data are the mean + S.E.M. Asterisk indicates
densitometric values significantly different than young
animals. (ERC=entorhinal cortex;DG= molecular layer of the
dentate gyrus;CA1 & CA3=hippocampal areas).


Figure 5-15. The distribution of mRNA specific to NMDARl
in A) 24-month-old; B) 12-month-old; and C) 6-month-old
rat brain.


Full Text
UNIVERSITY OF FLORIDA
3 1262 08554 3402


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FILES


AGING AND N-METHYL-D-ASPARTATE RECEPTORS
By
JOSEPHINE JEAN MITCHELL
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
1995


ACKNOWLEDGMENTS
I would like to thank my advisor, Dr. Kevin J. Anderson,
for sharing his extensive knowledge and for his support
throughout my doctoral studies. My research was supported by
a National Institute of Aging grant (NIA #AG08843). I would
also like to recognize my other committee members: Dr. A.
John MacLennan, Dr. Joanna Peris, Dr. Tom Vickroy and Dr. Don
Walker for their constructive criticisms and suggestions. I
am also very grateful to Dr. Janet Zengel's for her support
and friendship through the years.
Michael S. Sapper deserves a special acknowledgement for
his technical assistance as well as his friendship from the
very beginning. Special thanks go out to Tanya McGraw for
her friendship and support as well as to many of my other
fellow graduate students.
I wish to especially mention my father, Ignaceous J.
Maddalena, who unfortunately passed away just before I began
my doctoral studies. He was and always will be the person
who fueled my love for academics and taught me the importance
of hard work and perseverance in the pursuance of knowledge.
My mother, Helen, has been a loving and supportive figure
throughout my life and I wish to thank her with all of my
heart as well as the rest of my family and friends.
in

Finally, and most importantly, I wish to acknowledge my
husband, Thomas Mitchell (Mitch), who has given me invaluable
support throughout my academic pursuits as the 'one and only
love of my life' and father to our two beautiful sons Kyle
Ryan and Ty Joseph. I could never have achieved all I have
worked for without the constant love and support of my family
and friends.
IV

TABLE OF CONTENTS
ACKNOWLEDGMENTS iii-iv
ABSTRACT vii-ix
CHAPTERS
1 INTRODUCTION 1
Background 1
Previous Studies Examining Age-Related Changes in
NMDA Receptors 4
Molecular Cloning and Characterization of the NMDA
Receptor/Channel Complex 8
The Use of NMDARl and NMDAR2A/B Antisera in the
Analysis of NMDA Receptor/Channel Protein
Density and Distribution in the Rat CNS 17
Cooperative Modulation of [3H]MK-801 Binding to the
NMDA Receptor-ion/Channel Complex by L-
Glutamate, Glycine and Polyamines 19
Specific Aims 22
2 GENERAL METHODS 23
Animal Model 23
[3H]MK-801 Autoradiography 24
Tissue Preparation 24
[3H JMK-801 Binding Assay 25
Image Analysis 26
Statistical Analysis 27
Stereological Determination of Neuronal Density 27
Tissue Preparation 27
Data Analysis 28
Determination of Neuronal Density in the Lateral
Striatum, Entorhinal Cortex and Inner Frontal
Cortex 30
L-Glutamate Stimulation of [3H]MK-801 Binding to
NMDA Receptors 31
Tissue Preparation 31
[3H]MK-801 Binding Assay 31
Image Analysis 32
Data Analysis 33
In situ Hybridization 33
Probe Labeling 33
Tissue Preparation 34
v

Fixation 35
Hybridization 35
Autoradiography 36
Data Analysis 37
Immunocytochemistry 37
Tissue Preparation 37
Antibody Specificity 37
Immunocytochemical Procedure 38
3 AGE-RELATED CHANGES IN [3H]MK-801 BINDING IN F-344
RATS 40
Introduction 40
Methods 42
Statistical Analysis for [3H]MK-801 Binding 42
Results 44
[3H]MK-801 Binding 44
Discussion 51
4 EFFECT OF AGE ON L-GLUTAMATE STIMULATION OF [3H]MK-
801 BINDING IN F-344 RAT BRAIN 59
Introduction 59
Methods 61
Tissue Preparation 61
[3H]MK-801 Binding Assay 61
Image Analysis 62
Data Analysis 62
Binding Isotherm Plots 63
Statistical Analysis 63
Results 64
Discussion 70
5 AGE-RELATED CHANGES IN THE LEVELS OF mRNA CODING
FOR SPECIFIC NMDA RECEPTOR SUBUNITS IN THE CNS OF F-
344 RATS 77
Introduction 77
Methods 78
In Situ Hybridization 78
Data Analysis 79
Statistical Analysis 79
Results 80
Discussion 97
6 AGE-RELATED EFFECTS ON NMDARl AND NMDAR2A/B PROTEIN
LEVELS IN F-344 RAT BRAIN UTILIZING
IMMUNOHISTOCHEMISTRY 102
Introduction 102
Methods 105
Tissue Preparation 105
vi

Antibody Specificity 105
Immunocytochemical Procedure 106
Data Analysis 106
Statistical Analysis 107
Results 108
Discussion 113
7 SUMMARY AND DISCUSSION 117
Research Summary 117
Discussion 121
LIST OF REFERENCES 124
BIOGRAPHICAL SKETCH 136
vil

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
AGING AND THE N-METHYL-D-ASPARTATE RECEPTOR
By
Josephine Jean Mitchell
December, 1995
Chairman: Kevin J. Anderson
Major Department: Neuroscience
Aging is associated with a reduction in cognition and
memory in humans and other animals. Excitatory amino acids
(EAA) play pivotal roles in learning and memory. L-Glutamate
is thought to be the major EAA transmitter in mammalian
brain. L-Glutamate transmitter systems may therefore be
involved in age-related deficits. The N-methyl-D-aspartate
(NMDA) ionotropic glutamate receptor subtype appears
important in learning and memory. It is critical to long¬
term potentiation and NMDA antagonists impair performance of
rats in spatial and reference memory tasks. Antagonism of
NMDA receptor-mediated neurotransmission produces behavioral
deficits strikingly similar to those detected in aged
animals.
Vlll

IX
Thus deficits in NMDA receptor neurotransmission may
underlie age-related changes in neuronal plasticity. One
possible way NMDA neurotransmission could be reduced in aged
animals is by alterations in NMDA receptor/channel complexes.
The central hypothesis tested in this study was that
there are measurable, anatomically specific age-related
changes in the NMDA receptor and its individual subunits. An
initial examination of age-related differences in NMDA
receptor density in brains of 6-, 12- and 24-month-old F344
rats was performed with [3h]MK-801 in vitro autoradiography.
An age-related decrease was found in the entorhinal and inner
frontal cortices and the lateral striatum.
An analysis of L-glutamate's ability to enhance [3h]MK-
801 binding to NMDA receptor channels was performed by
varying L-glutamate concentration. Emax and EC50 values were
obtained for several brain regions showing an age-dependent
decrease in Emax values without changes in EC50.
The possibility that receptor binding changes were due
to decreases in specific NMDA receptor subunits was analyzed
using in situ hybridization. A significant difference was
seen in NMDARl subunit mRNA in all brain regions. Splice
variants NRloxxr NRlixx and NRlxlx changed in fewer regions
while NMDAR2 subunits did not change with age.
No age-related differences in NMDARl and NMDAR2A/B
protein density levels were found using immunocytochemistry.
These results indicate the NMDA receptor undergoes
regionally specific changes during aging and these changes

X
may account for some of the cognitive deficits in the aging
population.

CHAPTER 1
INTRODUCTION
Background
Ramon y Cajal, as early as the turn of the century,
proposed the connective foundation of neural memory (Cajal,
1911). Many years later, Hebb (1949) formulated his
principles of memory formation which was based upon
facilitation of contacts between neurons. Both Cajal and
Hebb focused on the synapse as the location of plastic change
in the formation of memory. They also theorized that the
processes of learning and memory result from changes in the
strength of synaptic transmission (Cajal, 1911; Hebb, 1949).
Bliss and Lomo (1973) found that in the hippocampus, a
brain region known to be important for learning (Nicoll et
al., 1988), brief repetitive activation of excitatory
pathways resulted in a substantial increase in synaptic
strength that lasted for many hours and, in vivo, even for
weeks. Since its initial discovery, this synaptic
enhancement, referred to as long-term potentiation (LTP), has
provided a model in vertebrate brain for a cellular mechanism
of learning and memory (Bliss and Lomo, 1973). LTP is
defined as an electrophysiological phenomenon of persistent
change in synaptic strength or efficacy as a result of
1

2
impulse transmission across synapses. These activity-induced
changes in the efficacy of existing synapses are thought to
be mediated by excitatory synaptic transmission throughout
the central nervous system (Bliss and Lomo, 1973).
Excitatory glutamatergic synapses are abundant
throughout the central nervous system (CNS), especially in
the hippocampus and cerebral cortex (Westbrook and Jahr,
1989). There are at least three types of ionotropic
glutamate receptors classified by the potent, selective
agonists N-methyl-D-aspartate (NMDA), kainate (KA) and a-
amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)
(Foster and Fagg, 1984). During low-frequency synaptic
transmission, glutamate is released from the presynaptic
terminal and acts on both the NMDA receptor subtypes as well
as the non-NMDA receptor subtypes (KA and AMPA). AMPA and KA
receptors are permeable to K+ and Na+ and provide a voltage-
independent means of depolarizing the postsynaptic neuron.
However, the NMDA receptor has unique biophysical
characteristics making it highly relevant to neural
plasticity and memory formation (Collingridge, 1987; Cotman
and Iversen, 1987; Harris et al., 1984). During low
frequency stimulation, ion flux through the NMDA channel is
blocked by Mg+2 in a voltage-dependent manner. Mg+2 ions are
expelled from membrane channels after the postsynaptic
membrane has reached a certain level of depolarization.
Following relief of the Mg+2 block, the receptor channel is
permeable to Na+ and Ca+2. Furthermore, NMDA receptor-

3
mediated current increases as a function of the degree of
depolarization are longer in duration than those from non-
NMDA receptors. The necessity for both presynaptic release
of transmitter to bind to the NMDA receptor as well as
sufficient postsynaptic depolarization to relieve the
voltage-dependent channel block by Mg+2 give this receptor
Hebb-like properties.
It is now thought that the activation of NMDA receptors
is responsible for the induction of LTP in the hippocampus
(see Nicoll et al., 1988 for review). NMDA antagonists have
been shown to block the development of LTP in the CA1 region
of the hippocampus during high-frequency tetanic stimulation
of the Schaffer commissural pathway (Collingridge et al.,
1983) as well as LTP in the dentate gyrus after perforant
path stimulation (Errington et al., 1987). In addition, a
variety of NMDA antagonists, both competitive (e.g., 2-amino-
5-phosphonovalerate [AP5], CGS 19755) and noncompetitive
(e.g., MK-801, phencyclidine [PCP]), have been shown to
impair performance of rats in the acquisition of spatial
working and reference memory tasks (Shapiro and Caramanos,
1990). NMDA receptor channel antagonism by MK-801 also
impairs performance of rats in aversively motivated complex
maze tasks (Spangler et al., 1991). These results provide
evidence for the NMDA receptor playing a role in memory and
spatial learning and LTP (see also Danysz et al., 1988;
Morris et al., 1986).

4
Aging is associated with a reduction in cognition and
memory in humans as well as other animals (Barnes, 1979;
Barnes and McNaughton, 1985). Senescent rats are slower than
young rats in learning various spatial tasks (Barnes, 1979;
DeToledo-Morrell et al., 1984) which may be due to age-
related changes in NMDA receptor-mediated neurotransmission.
In support of this, it has been shown that antagonism of NMDA
receptor-mediated neurotransmission produces behavioral
deficits strikingly similar to those detected in aged animals
(Bonhaus et al., 1990).
Taken together, the above evidence suggests that a
reduction in NMDA receptor-mediated neurotransmission may
underlie age-related changes in neuronal plasticity. One
means by which NMDA neurotransmission could be reduced in
aged animals is by an alteration in the NMDA receptor/channel
complex itself.
Previous Studies Examining Age-Related Changes in NMDA
Receptors
Previous studies have examined the effects of aging on
the density of central excitatory amino acid (EAA) receptors
in brain membrane preparations. For example, Tamaru et al.
(1991) reported a reduction in the number of NMDA receptors
in the cerebral cortex and hippocampus of aged rats.
Specifically, they found a significant decrease in
[3H]glutamate binding displaceable by NMDA, in strychnine-
insensitive [3H]glycine binding and in [3H]MK-801 binding in

5
both brain regions. Scatchard analysis revealed that the
reduction was due to a decrease in the number of binding
sites, not to an alteration in affinity. The reduction in
the number of receptors was apparently not due to a relative
increase of insoluble protein in membrane fractions since the
protein concentrations and ratio of protein concentration to
tissue wet weight were not significantly different between
young and aged animals. Similar results were obtained by
Ingram et al. (1992), who observed a marked (>50%) age-
related reduction in NMDA receptor binding in rat hippocampal
brain homogenates, and by Peterson and Cotman (1989) who
found reductions in NMDA-displaceable [^HjL-glutamate binding
density in two strains of aged mice.
The effect of increasing age on the binding of [3H]L-
glutamate to NMDA receptors in the brain of the BALB/c and
C57B1 mouse strains was also determined using in vitro
guantitative autoradiographic analysis (Magnusson and Cotman,
1993). In this study, a significant decrease in binding to
NMDA receptors occurred with increasing age (ranging from 3
through 30 months). NMDA receptors, as opposed to non-NMDA
receptors, were selectively affected by age-related changes
in the majority (17 of 20) of the cortical, subcortical and
hippocampal regions assayed in both strains of mice.
[3H]Kainate and [3H]AMPA (non-NMDA) binding, on the other
hand, was decreased in only 7 of 21 and 4 of 21 regions,
respectively. They concluded that the NMDA receptor is

6
selectively vulnerable to the aging process throughout most
cerebral cortical, subcortical and hippocampal regions.
A similar autoradiographic study has also demonstrated
that specific brain regions in aged animals exhibit a decline
in NMDA receptor density (Anderson et al., 1989). The
density of NMDA receptors labeled with [3H]L-glutamate in
young-adult (4-month-old) Fischer 344 rats was compared to
aged (24- to 26-month-old) rats. The areas that showed a
greater than 30% decrease in aged rats included the lateral
striatum, inner layers of the entorhinal cortex and the
lateral septal nucleus. The receptor density in inner
parietal cortex and anterior cingulate cortex was decreased
by 10% in aged rats when compared to young-adults. Within a
given brain region there was no significant difference in the
affinity (Kd) of the NMDA receptor for [^H]L-glutamate.
However, a significant decrease in the Bmax was seen within
the brain regions most affected by increasing age which
indicated that there was a decrease in the total number of
receptors (Anderson et al., 1989). This group also utilized
[]glycine binding to probe the associated allosteric
activating site on the NMDA receptor. Areas that showed the
greatest degree of loss of [^H]glycine binding included the
lateral striatum, parietal cortex and the entorhinal cortex.
There was no significant decrease in the lateral septal
nucleus and the loss of glycine binding sites in aged rats
was more profound in the parietal cortex and outer portions

7
of the entorhinal cortex when compared to [3H]L-glutamate
binding.
Age-related changes in the glutamatergic neuro¬
transmitter systems in rats and rhesus monkeys has been
examined utilizing NMDA-displaceable [3H]L-glutamate binding
to brain homogenates (Wenk et al., 1991). [3H]L-Glutamate
binding density was decreased in many brain regions in aged
rats (24 month-old) when compared to young (5 month-old)
rats. Specifically, the sensory-motor cortex, the parietal-
occipital cortex, the hippocampus and the caudate nucleus all
showed a significant decrease in binding density. In monkey
brains, NMDA-displaceable [3H]L-glutamate binding was
decreased in most brain regions analyzed and particularly
noticeable in the frontal and temporal lobes of the aged
monkeys (29-34 years) when compared to young (4-9 years)
monkeys.
Senescence-accelerated mice (SAM-P/8) have been used as
a murine model of aging and memory dysfunction (Kitamura et
al., 1992). This strain of mice shows an age-related
deterioration of learning and memory at an earlier age when
compared with control mice. In brain homogenates of
hippocampus and cerebral cortex from SAM-P/8 mice there was a
significant increase in the content of glutamate and
glutamine when compared to controls. Potassium-evoked
endogenous glutamate release from the brain slices of SAM-P/8
mice was increased in comparison with the control strains at
9 and 11 months. Additionally, the Bmax of [3H]dizocilpine

8
(MK-801) binding in the cerebral cortex was decreased in SAM
P/8 but not in controls. This suggests that synaptic
dysfunctions in the glutamatergic system occur in the CNS of
the SAM-P/8 mouse strain (Kitamura et al., 1992).
Molecular Cloning and Characterization of the NMDA
Receptor/Channel Complex
A functional cDNA clone for the rat NMDA receptor
(NMDARl) was first isolated in 1991 (Moriyoshi et al., 1991)
The single protein encoded by the cDNA was shown to form a
functional receptor/ion-channel complex with
electrophysiological and pharmacological properties
characteristic of the NMDA receptor; i.e. agonist and
antagonist selectivity, modulation by glycine, Ca2+
permeability, a voltage-dependent channel block by Mg2+, and
Zn2+ inhibition (Moriyoshi et al., 1991).
Three cDNAs encoding different NMDA receptor subunits
were isolated by polymerase chain reaction (PCR) (Monyer et
al., 1992). Two degenerate oligonucleotide primers were
designed after largely conserved peptide sequences in
ionotropic EAA receptor subunits, with which NMDARl shares
several small sequence islands around putative transmembrane
(TM) segments. These primers were used to PCR amplify
homologous sequences from rat brain cDNA. Three full-length
cDNAg, having sequences identified from the PCR products,
were named NMDAR2A (NR2A), NR2B and NR2C. The predicted
proteins were between 55% (NR2A and NR2C) and 70% (NR2A and

9
NR2B) identical to each other, but were only about 20%
identical to homologous AMPA-selective glutamate receptor
subunits (GluRs) and NMDARl (Monyer et al., 1992).
The functional properties of the expressed NR2 subunit
were examined using a Xenopus oocyte expression system. No
detectable calcium currents were recorded after bath
application of glutamate or NMDA to oocytes expressing one or
two NR2 subunits, which indicated that NR2 subunits may not
form functional homomeric or heteromeric channels. However,
large currents were measured in oocytes coexpressing NR1 and
any one oí the NR2 subunits. On average, the NMDA-induced
currents in oocytes expressing NRl and NR2A, NR2B or NR2C
were 100 times larger than they were in oocytes expressing
homomeric NRl channels. These currents also more closely
resemble native NMDA receptors. This indicated that
heteromeric configurations are likely to form from NRl
subunits and members of the NR2 subunit family (Monyer et
al., 1992).
NMDARl and NMDAR2 subunits carry an asparagine residue
in the putative channel forming region TMII, whereas a
glutamine or arginine residue resides in the homologous
position of the AMPA-selective glutamate receptor subunits
(Nakanishi et al., 1992). The importance of the asparagine
residue in the regulation of Ca+2 permeability and channel
blockade was shown by electrophysiological characterization
of receptors in which the asparagine was replaced with either
glutamine or arginine (Nakanishi et al., 1992). These

10
substitutions reduced or abolished Ca+2 permeability and
inhibition by Mg+2, Zn+2, and MK-801. Thus, this particular
asparagine residue may constitute a distinctive functional
determinant in subunits belonging to the NMDA receptor
(Monyer et al., 1992).
Previous in situ hybridization studies have revealed
that NMDAR1 messenger RNA (mRNA) in adult rat brain is
expressed in almost all neuronal cells throughout the brain
(Moriyoshi et al., 1991). This group observed prominent
expression of NMDAR1 mRNA in the cerebellum, hippocampus,
cerebral cortex and olfactory bulb. High expression was also
seen in the granular layer of the cerebellum, in granule
cells of the hippocampal dentate gyrus and in pyramidal cells
throughout hippocampal areas CA1-CA4.
The anatomical distribution of mRNA for NMDAR2 subunits
has been examined with in situ hybridization (Buller et al.,
1993; Kutsuwada et al., 1992; Meguro et al., 1992; Monaghan
et al., 1993; Monyer et al., 1992; Moriyoshi et al., 1991;
Nakanishi, 1992). NMDAR2A mRNA is widely expressed in many
brain regions, and this expression is prominent in the
cerebral cortex, hippocampus, olfactory bulb, some thalamic
nuclei, pontine nuclei, inferior olivary nuclei and
cerebellar cortex. The NMDAR2B mRNA expression is prominent
in most of the telencephalic and thalamic regions but
relatively low in the hypothalamus, cerebellum and lower
brain stem. The distribution of the NMDAR2C mRNA is more
discrete; its expression is extremely high only in the

11
granular layer of the cerebellum. NMDAR2D mRNA is mainly
expressed in the diencephalic and lower brain stem regions.
Recently, the distribution of NMDA receptor subtypes, as
identified with autoradiography, have been shown to
correspond to specific receptor subunits (Monaghan et al.,
1993). Early studies had indicated that there were two
populations of NMDA receptors; agonist-preferring (striatal-
type) and antagonist-preferring (thalamic-type) (Monaghan et
al., 1988). These populations were classified by whether
they displayed a higher affinity for agonist or for
antagonist. Monaghan (1991) showed that low concentrations
of L-glutamate selectively promoted the binding of [3h]MK-801
to striatal-type NMDA sites. He concluded that low
concentrations of L-glutamate appeared to preferentially
activate striatal NMDA receptors without activating thalamic
and cortical NMDA receptors. Conversely, with higher
concentrations of L-glutamate, [3H]MK-801 will also label
NMDA receptors of the lateral thalamic nuclei and cerebral
cortex (Monaghan and Anderson, 1991).
More recently, two other pharmacologically-distinct
populations of NMDA receptors have been defined by
autoradiography. These receptors were identified in the
midline thalamic nuclei and in the cerebellum (Monaghan et
al., 1993). It has been shown that the anatomical
distribution of each of these four native receptor
populations corresponds to a specific NMDAR2 subunit
(Monaghan et al., 1993). The NMDAR2A transcript has a

12
distribution very similar to the "antagonist-preferring" NMDA
receptor subtype. The NMDAR2B subunit mRNA is the only
NMDAR2 species found in regions enriched in "agonist-
preferring" sites. NMDAR2C subunits are largely restricted
to the cerebellum which contains a pharmacologically-distinct
receptor subtype and the NMDAR2D subunits are restricted to
the midline-thalamic nuclei NMDA receptor subtype (Monaghan
et al., 1993). These data suggest that NMDAR2 subunits may
contribute to the pharmacological diversity of native NMDA
receptors.
Alternative splicing has been shown to generate several
functionally distinct NMDARl subunits (Anatharam et al.,
1992; Durand et al., 1992; Nakanishi et al., 1992; Sugihara
et al., 1992). Structures and properties of seven isoforms
of the NMDARl receptor are differentiated from each other by
an insertion at the extracellular amino-terminal regions or
deletions at two different carboxy-terminal regions, or by
combinations of the insertion and deletions. All of these
isoforms have been shown in the Xenopus oocyte expression
system to induce electrophysiological responses to NMDA and
respond to various antagonists selective to the NMDA receptor
(Sugihara et al., 1992). The nomenclature of Durand et al.
(1993) denotes each NMDARl splice variant by the presence or
absence of the three alternatively spliced exons in the 5' to
3' direction. A subscripted o denotes exclusion of an exon
while a subscripted i denotes its inclusion. For example,

13
NRlin has all three exons, NRIqoo has none, and NRlioo has
only the N-terminal insert (Figure 1-1).
insert! insert2 insert3
â–¡ coding
non-coding
j coding when insert3 is absent
Figure 1-1. Proposed gene structure of the NMDARl receptor
(Durand et al., 1993). Three putative inserts can be spliced
in or out to form the mature mRNA. The NMDARl splice
variants are denoted by subscripts indicating the presence or
absence (1 or 0) of the three inserts in the 5’ to 3'
direction.
A recent study by Buller et al. (1994) described the
anatomical distributions of NMDARlixx (the NMDARl splice
variant containing insert 1) and NMDARloxx (the NMDARl splice
variant lacking insert 1). They found NMDARlixx mRNA density
varied across cortical regions with the parietal, temporal,
and superficial entorhinal cortices displaying threefold
higher levels of NMDARlixx mRNA than the anterior cingulate,
perirhinal, and insular cortices. In contrast, higher levels
of NMDARl oxx niRNA were found in the anterior cingulate,
perirhinal, and insular cortices. They concluded that the
localization of NMDARlixx and NMDARloxx mRNA between cortical
regions paralleled the distribution of antagonist-preferring
and agonist-preferring NMDA receptors, respectively.

14
NMDARlixx mRNA displayed a lateral-to-medial gradient pattern
within the striatum whereas NMDARlqXx was shown to be
moderately higher (15%) in the medial striatum than the
lateral striatum. NMDARlixx was present in low levels
throughout the septum while high levels of NMDARloXx mRNA were
found in this region. NMDARlixx mRNA was present at high
levels throughout the thalamus with low levels of NMDARIqxx*
Overall, this study showed that agonist-preferring NMDA
receptors are found predominately in the subset of brain
regions that contain both NMDAR2B and NMDARl oXx mRNA whereas
the antagonist-preferring NMDA receptors are found
predominately in brain regions containing both NMDAR2A and
NMDARlixx mRNA. Monaghan and Buller (1994) also found that
NMDARl mRNAs that are alternatively spliced at the second and
third inserts have distribution patterns that are dissimilar
to that of previously described NMDA receptor subtypes. No
differences in NMDA receptor pharmacological properties have
been found after studying homomeric receptors of NMDARl mRNA
that contain alternatively spliced exons at the second and
third insert sites (Durand et al., 1993; Nakanishi et al.,
1992). Because of these findings, alternative splicing at
these C-terminal sites is thought to have less of an effect
upon NMDA receptor pharmacology than N-terminal alternative
splicing events.
Expression cloning in Xenopus oocytes isolated two
different cDNAs encoding functional NMDA receptor subunits.
These receptor subunits were termed NMDA-R1A (NRIqh) and -RIB

15
(NRlm) (Durand et al., 1992). The two subunits displayed
different pharmacologic properties as a consequence of
alternative exon addition within the putative ligand-binding
domain. The splicing choice is regulated such that NRlm is
the predominate form of the receptor in the cerebellum,
whereas NRIqh predominates in the cerebral cortex,
hippocampus and olfactory bulb. Durand et al. (1992) showed
that the functional differences between NRlm and NRIqh are
marked and include differences in agonist affinity and
potentiation by spermine. Clearly, alternative splicing
contributes to NMDA receptor diversity. The expression of
distinct NMDA receptors with different electrophysiological
properties and anatomical distribution may be responsible for
the different forms of activity-dependent synaptic plasticity
in the mammalian brain such as the generation of different
forms of LTP (e.g., hippocampal associative LTP and
motorcortical LTP) (Nakanishi et al., 1992).
Recent studies examining the transmembrane topology of a
glutamate receptor GluRl (an AMPA subunit) showed that the N-
terminus is extracellular, whereas the C-terminus is
intracellular (Hollmann et al., 1994). In addition, three
transmembrane domains (TMD), (designated TMD A, TMD B, and
TMD C) corresponded to the previously proposed TMDs I, III,
and IV, respectively. It was found by N-glycosylation
tagging that, contrary to earlier models (Barnard et al.,
1987; Hollmann et al., 1989), the putative channel-lining
hydrophobic domain TMD II does not span the membrane.

16
Instead, this domain was suggested to either lie in close
proximity to the intracellular face of the plasma membrane or
loop into the membrane without traversing it. Furthermore,
the region between TMDs III and IV, in previous models
thought to be intracellular, is an entirely extracellular
domain (Hollmann et al., 1994).
Previously, Durand et al. (1993) showed that the 21-
amino acid insert in the N-terminal domain reduced the
apparent affinity of homomeric NMDARl receptors for NMDA and
nearly abolished potentiation by spermine and glycine. For
both of these properties, the N-terminal insert was the
determining structural feature; the C-terminal domain
produced only a very minor effect. These observations
correlate with a three transmembrane spanning topology where
the N-terminal domain is extracellular and therefore should
affect the ligand binding affinity characteristics of the
NMDA receptor. Therefore, it is not surprising that the C-
terminus would have minimal, if any, effect on ligand binding
characteristics due to its intracellular location. This was
demonstrated by Durand et al. (1993) when they examined the
electrophysiological characteristics of six splice variants
of NMDARl receptors and concluded that variants differing
only in their C-terminal domain showed little change in
agonist affinity or spermine potentiation.

17
The Use of NMDAR1 and NMDAR2A/B Antisera in the Analysis of
NMDA Receptor/Channel Protein Density and Distribution in the
Rat CNS
One of the first studies examining the NMDARl protein in
the rat CNS was provided by Hennegriff et al. (1992).
Polyclonal antibodies were raised against synthetic peptides
corresponding to the carboxyterminal region of the putative
NMDA receptor cloned by Nakanishi (1991). The affinity
purified antibodies to the NMDA receptor subunit labeled
antigens of 75 and 81 kDa. In order to determine the
regional distribution of NMDARl, the 75/81 kDa doublet
protein was quantitated by laser densitometry from Western
blots in homogenate samples prepared from seven brain
regions. They found the doublet to be most abundant in
neocortex, followed by hippocampus > striatum » thalamus >
olfactory bulb > cerebellum » brain stem. The same regional
distribution was found in synaptosomes using antibodies to
NMDARl and resembled earlier autoradiographic studies
measuring NMDA-sensitive [ 3H]glutamate binding sites
(Monaghan and Cotman, 1985). A more thorough light and
electron microscopic analysis of the distribution of the NMDA
receptor subunit NMDARl in the rat CNS was performed by
Petralia et al. (1994a). This group made a polyclonal
antiserum that recognized four of the seven splice variants
of NMDARl and subsequently utilized this antiserum to perform
a comprehensive immunohistochemical survey of the
distribution of this antigen. They showed that the NMDARl

18
subunit is widespread throughout the rat CNS. The most
densely stained cells included the pyramidal and hilar
neurons of the CA3 region of the hippocampus, Purkinje cells
of the cerebellum and paraventricular neurons of the
hypothalamus. Ultrastructural localization of NMDAR1 antigen
showed labeling present in postsynaptic densities in a
pattern consistent with the synthesis, processing and
transport of this protein. Staining was seen in the
cytoplasm of dendrites and concentrated in patches associated
with groups of microtubules and/or the surface of one pole of
a mitochondrion. In addition, patches of staining in the
cell bodies were found to form similar associations with
microtubules and mitochondria, as well as an association with
rough endoplasmic reticulum, Golgi apparatus, and the nuclear
envelope. No staining was found in the synaptic cleft. The
antiserum did not cross-react with extracts from transfected
cells expressing other glutamate subunits, nor did it label
non-neuronal tissues. The pattern of staining correlated
closely with previous in situ hybridization studies but
differed somewhat from binding studies (Petralia et al.,
1994a).
Another study by this laboratory (Petralia et al.,
1994b) examined the histological and ultrastructural
localization patterns of the NMDA receptor subunits NMDAR2A
and NMDAR2B. They made a polyclonal antiserum to a C-
terminus peptide of NMDAR2A. In analysis of membranes from
transfected cells, this antiserum recognized NMDAR2A and

19
NMDAR2B, and to a slight extent, NMDAR2C and NMDAR2D.
Immunostained sections of rat brain showed significant
labeling throughout the CNS that was similar to that seen
previously with their antiserum to NMDARl. Dense staining
was present in postsynaptic densities in the cerebral cortex
and hippocampus. Since there is physiological evidence that
both NMDARl and NMDAR2 subunits coexist in the native NMDA
receptor, their findings are consistent with this idea
(Petralia et al., 1994b).
Cooperative Modulation of r^HlMK-801 Binding to the NMDA
Receptor-ion/Channel Complex by L-Glutamate, Glycine and
Polyamines
L-Glutamate binding to the NMDA receptor, in the
presence of glycine, produces channel opening. MK-801 is an
NMDA receptor antagonist that binds with high affinity (in
the low nanomolar range) to a site located within the NMDA
channel. L-Glutamate markedly stimulates [3H]MK-801 binding
by increasing the affinity for the ligand. Other NMDA
receptor agonists (e.g. NMDA, D-aspartate) are capable of
enhancing this binding as well, but the non-NMDA receptor
agonists (AMPA and kainate) have no effect on [3h]MK-801
binding (Foster and Wong, 1987). Because MK-801 binding has
been shown to take place only if the NMDA receptor channel is
in the transmitter-activated state (Huettner and Bean, 1988),
the efficacy with which L-glutamate produces receptor
activation and channel opening can be measured by determining

20
the amount of [3H]MK-801 binding as a function of glutamate
concentration.
Glycine appears to be a constitutive co-agonist for NMDA
receptor activation. Glycine alone is ineffective in opening
NMDA-linked cation channels. However, glycine greatly
potentiates the frequency of channel opening in response to
NMDA receptor agonists (Johnson and Ascher, 1987; Reynolds et
al., 1987). Glycine also potentiates NMDA-stimulated Ca2+
influx in cultured striatal neurons (Reynolds et al., 1987).
An allosteric role for glycine in NMDA receptor function is
supported by autoradiographic studies. These studies showed
that the distribution of strychnine-insensitive [2H]glycine
binding sites in supraspinal brain regions mirrored the
regional distribution of NMDA-sensitive L-glutamate sites
(Monaghan and Cotman, 1985). Since glycine has been shown to
promote channel activation in response to agonist, it can be
predicted to enhance [3H]MK-801 binding since MK-801 prefers
to bind to the activated state of the channel.
Along with the allosteric co-agonist glycine site on the
NMDA receptor/complex, there is also a polyamine recognition
site. The polyamines spermine and spermidine have been shown
to increase both the maximum NMDA response amplitude (Lerma,
1992; McGurk et al., 1990; Ransom and Deschenes, 1990;
Sprosen and Woodruff, 1990) as well as the binding of [3H]MK-
801 to the NMDA receptor channel (Ransom and Stec, 1988).
Because spermine does not activate NMDA receptors in the
absence of glutamate and glycine, it has been suggested to

21
act at an allosteric site independent from the glycine site
(Ransom and Stec, 1988).
The mechanism by which L-glutamate and these other
agonists affect [3H]MK-801 binding affinity has not been
critically addressed. In response to varying agonist
concentrations, [3H]MK-801 binding sites may undergo a
continuum of conformational changes expressing a smooth range
of different affinities for [3H]MK-801. However, it seems
more probable that [3H]MK-801 binding is an all (open
channel) or nothing (closed channel) phenomenon since most
ion channels have not been shown to exhibit broad conductance
ranges (i.e. they are either open or closed [Ransom and Stec,
1988]). This would be more consistent with the
electrophysiological characterization of MK-801 as a use-
dependent, open channel blocker (Foster and Wong, 1987). A
more likely explanation for the observed changes in Kp for
[3H]MK-801 is that they result from changes in the length of
time a channel spends in the activated state (Ransom and
Stec, 1988). At high agonist concentrations, channels are
opened for a proportionately greater amount of time and the
affinity for [3H]MK-801 is high. At low agonist
concentrations, the freguency with which the channel opens is
comparatively low. The result is an apparent reduction in
[3H]MK-801 binding affinity since there is a much lower
probability, due to decreased access, that an infreguently
opened channel will bind radioligand (Ransom and Stec, 1988).

Specific Aims
The central hypothesis tested in this dissertation
project was that there are measurable, anatomically specific
aging-related changes in the NMDA receptor/channel complex
and its individual subunits. Four specific hypotheses were
addressed within the framework of the central hypothesis.
The first hypothesis was that there are age-related
differences in the density of the NMDA receptor in F-344 rat
brain as a function of age. This hypothesis was addressed
using in vitro quantitative [3H]MK-801 binding analyses
(Chapter 3). The second hypothesis was that there are age-
related differences in the ability of L-glutamate to enhance
[3h]MK-801 binding to NMDA receptors. [3H]MK-801 binding as
a function of L-glutamate concentration was performed to test
this hypothesis (Chapter 4). The third hypothesis was that
there are selective changes in mRNA coding for subunits of
the NMDA receptor as a function of age. In situ
hybridization analyses were performed with cDNA probes
specific to NMDA receptor subunits (Chapter 5). The fourth
hypothesis was that there are specific age-related changes in
NMDARl and NMDAR2 protein density in the rat CNS.
Immunocytochemistry was performed with antisera specific to
the NMDA receptor subunit protein (Chapter 6).

CHAPTER 2
GENERAL METHODS
A description of the general methods that were used
throughout this dissertation will follow and will be referred
to when describing individual experiments.
Animal Model
Fischer 344 (F-344) male rats 6-, 12-, and 24-months-of-
age were used for these experiments. These animals were
obtained from the National Institute on Aging (NIA) breeding
colony (Harlan). In 1981, the Committee on Animal Models for
Research on Aging described the life span characteristics of
two rat strains, the F-344 and the Brown Norway (BN) rat, and
recommended their use as models for aging research. Among
the reasons for choosing F-344 and BN rat strains over other
rat strains was that these rats displayed delayed onset of
kidney problems (nephropathy) as well as certain tumors.
Currently, the NIA recommends and maintains a total of three
rat strains for aging studies, the F-344, the BN, and the Fi
F-344/BN hybrid. The F-344 was chosen for these studies
because it has long been a standard model for aging research
and thus has been extremely well characterized. Survival
curves for the F-344 rat show that 24-month-old males have a
23

24
40% probability of survival and the probability of survival
curve drops rapidly to only 10% by 27-months-of-age.
Therefore, 24-month-old animals represent advanced stages of
aging in this rat strain and 12-month-old animals can be
classified as middle-aged. 6-month-old animals represent
young-adults and are used throughout these studies for
comparisons to middle-aged and aged rats.
r^HlMK-801 Autoradiography
Tissue Preparation
Male F-344 rats 6-, 12- and 24-months-of-age (n=6 per
age group) were decapitated, their brains rapidly removed and
frozen with powdered dry ice. Brains were stored at -80 °C
until used. Sections (6pm thick) were cut on a cryostat and
representative sections from each age group were thaw-mounted
onto chromic acid washed and gelatin-subbed slides. A rat
brain atlas (Paxinos and Watson, 1982) was referenced in
order to delineate the brain regions for analyses. The
brains were sectioned in the horizontal plane. The bregma
coordinate corresponded to the dorsoventral distance of the
sections from the horizontal plane passing through bregma and
lambda on the surface of the skull. The start and stop
bregma coordinates were -4.1 mm and -6.1 mm, respectively.
This anatomical range was used to thoroughly analyze brain
regions of interest that were present on each sectional
profile. Every fifth serial section was taken and a total of

25
fifty sections were cut from each rat brain. One section
from each of the three age groups was mounted onto each slide
which represented one block of animals. Ten slides were used
per block of animals for each [3H]MK-801 binding assay. Two
slides per assay were used to determine non-specific binding
and these values were subtracted from each total binding
value in order to obtain specific binding. These tissue
sections were used immediately or stored for no longer than
24 hours at -20 °C prior to the [3H]MK-801 binding assay.
r3H1MK-801 Binding Assay
Slides were thawed and preincubated at room temperature
for 10 minutes in 50 mM Tris acetate with 1.0 mM EDTA and
0.1% saponin (pH 7.7). Sections were then rinsed at 30 °C
for 60 minutes in 50 mM Tris-acetate buffer (pH 7.7). This
treatment removes endogenous glutamate, glycine and various
ions. Sections were then incubated for 60 minutes at room
temperature in 10nM [3H]MK-801 (30 Ci/mmol;New England
Nuclear, Boston,MA,U.S.A.) in 50 mM Tris-acetate buffer
containing 20 pM D-2-amino-5-phosphonopentanoic acid (D-
AP5), 250 ¡jM spermine, 25 ¡uñ glycine and 20 ^/M L-glutamate.
D-AP5, a competetive NMDA antagonist, was added to all
[3H]MK-801 incubations to give lower and more consistent
basal binding levels (Monaghan 1991). The addition of
spermine, glycine and L-glutamate ensures a maximal degree of
stimulation of the NMDA receptor/ channel complex and
therefore optimal [3H]MK-801 binding. Sections were then

26
washed for 60 minutes in ice-cold Tris-acetate buffer
containing 20 jjM D-AP5. Nonspecific binding was defined in
sections treated identically in the presence of 50 jjIA MK-801.
Following the rinse, the sections were dried under an air
stream. The sections were then placed in x-ray cassettes and
apposed to tritium-sensitive film (Hyperfilm, Amersham).
Tritium standards calibrated against brain paste were
included in the cassettes (Microscales, Amersham). The film
was exposed for six weeks and then developed using Kodak D-19
developer.
Receptor densities (expressed in pmol/mg protein) from
the 12- and 24-month rat brains were normalized against
values derived from 6-month-old animals. Data from the 12-
and 24-month-old rats were presented as a percentage of the
receptor density values from the 6-month-old animals.
Image Analysis
Each slide contained a representative horizontal section
from each age group. Ten slides were analyzed per block of
animals, with each block consisting of an animal from each
age group. Autoradiograms were analyzed, with the
investigator blind to age, by computer assisted densitometry
with a Microcomputer Imaging Device (MCID, Imaging Research,
Inc., St. Catherines, Ont.). Densitometric measurements were
converted on line to pmol/mg protein binding.

27
Statistical Analysis
Six blocks of animals, each block consisting of an
animal from each age group, were assessed. The experimental
design consisted of a within-subject factor with eight levels
(brain region), a between-subject fixed factor with three
levels (age), and each of six levels (experimental day).
Repeated measures analysis of variance (ANOVA) with between-
subject randomized blocks and age considered as a linear
covariate were used to model the amount of receptor density
as a function of age within each brain region and to
determine if this pattern differed significantly among brain
regions.
The brain regions that were analyzed included the outer
and inner frontal cortices (OFCTX and IFCTX); the entorhinal
cortex (ERC); the molecular layer of the dentate gyrus (DG);
hippocampal area CAl stratum radiatum (CA1); the lateral
septum (LSEP); the lateral striatum (LSTR); and the lateral
thalamus (LTHAL).
Stereoloqical Determination of Neuronal Density
Tissue Preparation
Thirty micron sections of brains from 6-, 12-, and 24-
month-old F-344 rats (n=4 per age) were cut on a cryostat.
One section from each age group was thaw-mounted on each
slide with a total of fifty slides for each block of animals.

28
The brain regions of interest in these studies were those
that showed an age-related decrease in [3h]MK-801 binding as
determined by the first experiment in Chapter 3. These
regions were the lateral striatum (LSTR), the inner frontal
cortex (IFCTX) and the entorhinal cortex (ERC). Sections
were stained with Cresyl violet in order to view neuronal
cell bodies.
A rat brain atlas (Paxinos and Watson, 1982) was
referenced in order to delineate the stereotaxic coordinates
of brain regions for analyses. Brains were sectioned in the
horizontal plane. Bregma coordinates correspond to the
dorsoventral distance of the sections from the horizontal
plane passing through bregma and lambda on the surface of the
skull. The start and stop bregma coordinates were -3.1 mm
and -7.9 mm, respectively. This broader anatomical range was
chosen to thoroughly analyze the dorsal to ventral extent of
the lateral striatum as well as to view the entorhinal and
inner frontal cortices. Two serial sections were taken every
120pm throughout the stereotaxic range.
Data Analysis
Five blocks of animals were used for these analyses with
each block containing one 6-month-, one 12-month- and one 24-
month-old rat. An initial analysis was performed to
ascertain whether the lateral striatum (LSTR) underwent
volumetric changes by sampling this brain region through its
dorsoventral extent. The volume of this structure could be

29
readily determined because its entire anatomical distribution
could be viewed throughout the series of sections. The LSTR
was sampled at five separate anatomical levels (2 serial
slides per level) with computer assisted image analysis
(Microcomputer Imaging Device, Imaging Research, Inc.)* A
determination was then made as to whether there was a
significant age-related change in the overall volume (mm2) of
this structure. In addition, a quantitative analysis of the
laminar thickness of the inner frontal cortex (IFCTX) and the
entorhinal cortex (ERC) in 6-, 12- and 24-month-old animals
was performed. This study was performed to determine whether
there were changes in laminar thickness in these cortical
structures in the aged brain when compared to the young
animals. Calculations of laminar thickness were used because
both the entorhinal cortex and inner frontal cortex have
highly organized laminar structures. There were no
significant age-related differences in the volume of the LSTR
or the laminar thickness of the ERC or the IFCTX.
Neuronal packing density units are in neurons/mm2. No
significant age-related differences in the volume of the LSTR
or in the laminar thickness of the ERC or IFCTX were found,
therefore packing density should not vary as a function of
age. Therefore, any change seen in neuronal packing density
would be attributable to differences in the number of neurons
per mm2. In support of this, Coleman et al. (1987) examined
the volumes of the components of the hippocampus in the aging
F-344 rat and found that there was no change in volume

30
between 12 and 37 months. They suggested that neuronal
packing density (expressed as the number of neurons per mm2)
could be compared in aged animals independent of a volumetric
effect.
Determination of Neuronal Density in the Lateral Striatum,
Entorhinal Cortex and Inner Frontal Cortex
An MCID impage analysis system was used to determine
neuronal packing density in three brain regions. A fully
automated sampling scheme was utilized where the general
target characteristics were defined. The two target
definitions that were used were target density and target
size. For the first criteria of target density, MCID was
given upper and lower density thresholds (segmentation
ranges) and all pixels darker or lighter than the threshold
values were ignored. Pixels that were lying within the
segmentation range were valid targets. The second criteria
for target acceptance was defined as a range of target
lengths where the target neurons smaller or larger than the
defined size range ( >5pm and <20pm) would be ignored with
those targets fulfilling the length criteria being counted.
After the system found the defined targets, post-scan editing
of the digitized image was performed using editing tools.
This enabled the investigator to separate any neuronal
targets that may have been overlapping.
The area searched for targets (scan area) was also
guantified, thus neuronal packing density (number of

31
neurons/mm3) was determined for each brain region. Ten
serial sections were analyzed from each age group with the
investigator blind to age. Neuronal density from 6-month-old
animals was used to normalize counts from 12-, and 24-month-
old animals. Results were then statistically compared
between age groups (one-way repeated measures ANOVA, paired
t-tests).
L-Glutamate Stimulation of r3HlMK-801 Binding to NMDA
Receptors
Tissue Preparation
Male F-344 rats 6-, 12- and 24-months-of-age (n=6 per
age) were decapitated and their brains were rapidly removed
and frozen with powdered dry ice. The brains were stored at
-80 °C until used. Fifty 6¿jm sections were cut on a cryostat
and representative sections from each age group were thaw-
mounted onto acid washed and gelatin-subbed slides. These
tissue sections were used immediately or stored for no longer
than 24 hours at -20 °C prior to the [3H]MK-801 binding
assay.
r 3H1MK-801 Binding Assay
The [3H]MK-801 binding assay was the same as described
previously with the exception that varying concentrations of
L-glutamate were added to the incubation step in order to
examine glutamate's ability to stimulate [3H]MK-801 binding.

32
The following concentrations (in micromolar) of L-glutamate
were used: 0.0, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5, 10 and 20.
Non-specific binding was defined by the addition of 50 pM
unlabeled MK-801 with 20pM L-glutamate. Each slide contained
one section from each of the three age groups. A total of
four slides, from each block of animals, were analyzed per
assay (n=6) for each L-glutamate concentration.
Receptor density (expressed in pmol/mg protein) from 12-
and 24-month rat brains was normalized against 6-month-old
rats. Data from 12- and 24-month-old animals were
statistically compared to receptor density values from 6-
month-old animals.
Image Analysis
Each slide consisted of a horizontal section from each
age group taken from between bregma coordinates -4.1 mm and
-6.1 mm (the dorsoventral distance of the sections from the
horizontal plane passing through bregma and lambda on the rat
skull). This stereotaxic range was used for a thorough
analysis of the brain regions of interest that were present
on each sectional profile. A total of fifty sections were
cut for each block of animals, with every fifth section being
taken throughout the stereotaxic range. Autoradiograms were
analyzed, blind to age, by computer assisted densitometry
with an MCID system. Four sections from each block of
animals, for each concentration of L-glutamate, were
analyzed. Two slides per assay were analyzed for non-

33
specific binding and these values were subtracted from each
total binding value in order to obtain specific binding.
Densitometric measurements obtained were converted on line to
pmol/mg protein binding.
Data Analysis
The brain regions that were analyzed included the OFCTX
and IFCTX; the ERC; the molecular layer of the DG;
hippocampal area CAl (stratum radiatum); the LSEP; the LSTR;
and the LTHAL.
INPLOT (GraphPad Software, San Diego, CA) was used to
generate rectangular hyperbola plots or binding isotherms.
These binding isotherm plots yielded EC50 (effective
concentration at 50%) and Emax (maximal effect) values. EC50
values and Emax values were obtained for each age group and
each brain region. Statistical analyses were then performed
to ascertain significant age-related differences in these
parameters (repeated measures ANOVA; F tests).
In situ Hybridization
Probe Labeling
Oligonucleotide probes for this study were graciously
supplied by Dr. Daniel T. Monaghan, University of Nebraska
Medical Center, Omaha NE. These probes are 45 nucleotides in
length (45 mer) and are specific to NMDARl, four
alternatively spliced versions of NMDARl (NRloxx» NRlixx/

34
NRlxix t NRlxxi) and the members of NMDAR2 subunit family
(NMDAR2A, NMDAR2B, NMDAR2C, NMDAR2D). The specificity of
these probes was previously confirmed by Buller and Monaghan
et al. (1994) by incubation of the radiolabeled
oligonucleotides in the presence of excess (100 nM )
unlabeled probe. In this study, only oligonucleotides with
the same sequence inhibited probe hybridization.
The oligonucleotide probes were labeled using a New
England Nuclear (NEN) labeling kit and following the methods
described by Wisden et al. (1991). The probes were labeled
at the 3'- end using [35S]dATP (1000-1500 Ci/mmol, NEN) and 3'
terminal deoxynucleotidyl transferase (NEN). After labeling,
the probes were used immediately or stored for a limited time
at -70 °C.
Tissue Preparation
Frozen brains from 6-, 12- and 24-month-old rats (n=6
per age) were cut at 12 pm, in the horizontal plane, on a
cryostat and thaw-mounted on Fisher Superfrost Plus glass
slides. The stereotaxic bregma coordinates were from -4.1 mm
to -6.1 mm. Sections were refrozen in the cryostat. One
section from each age group was mounted per slide for a total
of three sections per slide. A total of fifty slides was
prepared per block of animals. Four slides per probe, each
containing a section from each age group, were used. Gloves
were worn at all times during sectioning of the brains and

35
handling of the slides in order to avoid contamination with
ribonucleases.
Fixation
The sections were fixed immediately after cutting by
placing the slides in 4% paraformaldehyde at 4°C for 5-15
minutes. Slides were washed in 0.1M phosphate-buffered
saline (PBS) for 1 minute and 70% EtOH (RNase-free) for
several minutes. Slides were then stored in 95% EtOH in a
cold room (4°C) until needed. All solutions were made with
diethylprocarbonate (DEPC)-treated water.
Hybridization
Slides were removed from storage and air dried at room
temperature. Each labeled probe was dissolved in appropriate
amounts of hybridization buffer (NEN, Boston, MA), containing
0.2 M dithiothreitol to achieve a final concentration of 2000
cpm/pl. 100 pi of this solution was applied to the slide and
covered with a glass coverslip. Slides were incubated at 42
°C overnight with a parafilm wrap. The following day,
parafilm and coverslips were removed and the slides were
placed in lx SSC (saline sodium citrate buffer) at 60 °C for
20 minutes. Finally, the sections were rinsed again in lx
SSC at 60 °C for 5 seconds and then rapidly dried under an
air stream.
In situ hybridization is an experimental method that can
be used to determine the relative density of individual mRNA

36
species. Each 45 mer probe was constructed to be highly
specific and hybridizes to its particular NMDA subunit mRNA.
One potential drawback is that only the relative density of
mRNA can be obtained from this method. Therefore, only semi-
quantitative analyses were performed. Another drawback is
that each probe corresponding to specific NMDA receptor
subunits could only be compared across each age group. No
quantitative comparisons could be made between various
probes. In other words, the relative density of the NMDAR1
mRNA could only be compared between 6-, 12- and 24-month-old
animals from the same assay. This information could not be
compared to the data obtained for any of the other mRNAs
(i.e. NMDAR2A-D and the four alternatively spliced versions
of NMDAR1). Another drawback is that the analyses gave an
assessment of the relative density of mRNA while not all mRNA
is necessarily translated into protein.
Autoradiography
Dried slides were placed in an x-ray cassette and
apposed to B-max film (Hyperfilm, Amersham) for two weeks,
developed in D-19 (Kodak) developer and fixed with Rapid Fix
(Kodak). Autoradiograms were analyzed by computer assisted
densitometry with a Microcomputer Imaging Device, Imaging
Research, Inc.(MCID) image processing system.

37
Data Analysis
The brain regions analyzed included the OFCTX, IFCTX,
LSEP, LSTR, LTHAL, ERC, DG and hippocampal areas CAl and CA3.
Brain regions were compared between the three age groups and
the relative density of each subunit mRNA was normalized
against the values obtained for young animals. Probes were
labeled simultaneously and labeling experiments were grouped
such that variability was kept to a minimum. A semi-
quantitative measurement of mRNA levels and the distribution
of the different subunits was compared between ages.
Immunocytochemistry
Tissue Preparation
Brains from 6-, 12- and 24-month rats (n=5) were
serially sectioned at 30 pm on a cryostat and thaw-mounted
onto subbed microscope slides and then refrozen in the
cryostat. Every fifth section was taken for a total of fifty
slides. One brain section from each block of animals was
mounted per slide n=5.
Antibody Specificity
AB1516 (Chemicon Inti. Inc., Temecula, CA), an antiserum
raised against a synthetic peptide corresponding to the C-
terminus of rat NMDA receptor subunit (NMDAR1), is selective
for splice variants NRIqh, NRlm, NRlQOl/ NRlioi- These

38
appear to be the major splice variants expressed in rat brain
(Hollmann et al., 1993). It has been shown previously that
there is no cross reactivity of this antiserum with other
glutamate receptor subunits (Petralia et al.f 1994a). AB1548
(Chemicon Inti. Inc., Temecula, CA), an antiserum raised
against a synthetic peptide corresponding to the C-terminus
of rat NMDAR2A receptor subunit, recognizes both NMDAR2A and
NMDAR2B subunits equally. This antiserum shows no cross
reactivity with NMDARl or other glutamate receptor subunits
(Petralia et al., 1994b).
Immunocytochemical Procedure
An optimal antibody concentration was determined
empirically. Previous studies had used an AB1516 antibody
concentration between 2 and 4 pg/ml (Petralia et al., 1994a)
and AB1548 antibody concentration of 0.5-1.5 pg/ml (Petralia
et al., 1994b). In the present experiments, a concentration
of 2.5 pg/ml was found to produce optimal results with both
NMDARl and NMDAR2A/B antibodies. Sections were incubated in
10% normal goat serum in PBS (pH 7.4) for 1 hour and then
kept overnight in primary antisera (AB1516 or AB1548) in PBS.
Sections were then washed and incubated in biotinylated goat
anti-rabbit antisera (1:250 dilution) for 1 hour, washed,
incubated in avidin-horse radish-peroxidase (1:200 dilution)
for 1 hour, washed, treated for 15 minutes with 3',3-
diaminobenzidine tetrahydrochloride (0.5 mg/ml PBS + 5 pl/ml
of 0.6% hydrogen peroxide), and washed. All washes were

39
performed with PBS (2 x 15 min.). Sections with PBS
substituted for the primary antibody (PBS controls) were also
used in order to correct for nonspecific staining.
Staining density was semi-quantitated by an MCID image
analysis system. Data were presented as raw optical
densities. Raw optical density of NMDARl and NMDAR2A/B
immunoreactivity from 6-month-old animals was used to
normalize data from 12- and 24-month-old animals. Data from
middle-aged and aged animals were presented as a percentage
of the six-month-old animals.

CHAPTER 3
AGE-RELATED CHANGES IN [3H]MK-801 BINDING IN F-344 RATS
Introduction
Aging is associated with a reduction in many physiologic
forms of neuronal plasticity. Recent findings indicate a
direct correlation in age-dependent deficits in spatial
memory and in hippocampal kindling (a measure of neuronal
plasticity) as measured in 26-month-old Fischer 344 rats
(DeToledo-Morrell et al.f 1984). N-Methyl-D-aspartate (NMDA)
receptor-mediated glutamatergic neurotransmission has been
shown to be important for learning and memory in animals and
man (Collingridge, 1987; Cotman and Iversen, 1987; Harris,
1984). Profound learning and memory impairments are seen in
a variety of animal species after NMDA-receptor activation is
blocked by either competitive or non-competitive antagonists
(Morris et al., 1986). There are also some striking
similarities between NMDA antagonist induced memory
impairments and age-related cognitive deficits, suggesting
that age-related alterations in NMDA receptors may be
involved in cognitive decline with increasing age (Bonhaus et
al., 1990; Ingram et al., 1992).
40

41
To test the hypothesis that NMDA receptors decline
during aging, an examination of the density of NMDA receptors
was performed in aged Fischer 344 rats. In this study,
[ ](+)-5-methyl-l0,1l-dihydro-5H-dibenzo(a,d)-cycloheptan-
5,10-iminehydrogen maléate (MK-801 or dizocilpine), a highly
specific non-competitive NMDA receptor antagonist was used to
determine the density of NMDA receptors. This antagonist
binds with high affinity (low nanomolar range KD) to a site
located within the NMDA receptor ion channel. Thus, MK-801
binding occurs only if the NMDA receptor is in the
transmitter-activated (open) state (Huettner and Bean, 1988).
Channel activation by an NMDA receptor agonist like L-
glutamate, in the presence of glycine, promotes high-affinity
MK-801 binding. Glycine alone cannot open NMDA receptor
channels, but is a constitutive co-agonist. Glycine, in the
presence of L-glutamate or other NMDA receptor agonists
(i.e., NMDA or D-aspartate) has been shown to greatly
potentiate the frequency of channel opening (Johnson and
Ascher, 1987; Reynolds et al., 1987). In addition, the
polyamines spermine and spermidine increase both the maximum
NMDA response amplitude as well as binding of MK-801 to the
NMDA receptor. Alone, polyamines do not activate the NMDA
receptor, suggesting the presence of an allosteric binding
site independent from the other co-agonist sites (Lerma,
1992; McGurk et al., 1990; Ransom and Deschenes, 1990;
Sprosen and Woodruff, 1990).

42
Because of the high degree of specificity and affinity
of MK-801 for the NMDA receptor channel complex, [3H]MK-801
is an ideal ligand to examine age-related changes in NMDA
receptors in the CNS. Also, because MK-801 only binds to the
activated state of the NMDA receptor, the total number of
available NMDA receptors can be determined by using optimal
agonist and co-agonist concentrations.
Methods
For a detailed description of the experimental tissue
used in these studies, the [3H]MK-801 binding assay
procedure, and image analyses see Chapter 2.
Statistical Analysis forr3H1MK-801 Binding
The data acquired in this study consisted of
quantitative analyses of the average density of [3H]MK-801
binding (pmol/mg protein) obtained from bilateral brain
regions for each of the 6-,12-,and 24-month-old rats (n=6 for
each age). One "group" consisted of one 6-month-, one 12-
month-, and one 24-month-old animal mounted per series of
slides. Ten slides, from each of these groups, were analyzed
per assay. The regions that were analyzed included the outer
frontal cortex (OFCTX), inner frontal cortex (IFCTX),
entorhinal cortex (ERC), dentate gyrus of the hippocampus
(DG), hippocampal area CA1, lateral septum (LSEP), lateral
striatum (LSTR) and the lateral thalamus (LTHAL).

43
Repeated measures analysis of variance (ANOVA) with
between-animal randomized blocks and age considered as a
linear continuous covariate was used to model the pattern of
MK-801 binding as a linear function of age within each brain
region. F-tests were also performed for the presence of a
significant interaction between age and brain region receptor
density effects. In assessing the validity of ANOVA
assumptions, it was noted that experimental day age profiles
of receptor binding were parallel within each brain region
and that between-animal variability as estimated by the
standard deviation was similar among age groups within each
brain region. This indicated that the patterns of [3H]MK-801
binding obtained for each group of 6-, 12-, and 24-month-old
rats were the same, independent of which experimental day
each group was assayed. Within age groups however, brain
region between-animal standard deviations tended to increase
as the mean level of receptor density in those regions
increased. ANOVA models assume that standard deviations do
not vary in this manner. Therefore, receptor binding density
values were transformed logarithmically, prior to analysis,
in order to appropriately model this inherent pattern of
variability within the ANOVA framework. Plots of residuals
versus predicted values, residual histograms, and residual
normal probability plots were examined to assess goodness of
fit in the ANOVA model. The statistical analyses determined
which mean age profiles of MK-801 binding density appeared to

44
be linearly decreasing in specific brain regions and which
profiles had slopes which were significantly less than zero.
Results
r 3H1MK-801 Binding
The IFCTX, ERC and LSTR were the only regions analyzed
that underwent a significant change (decrease) in [3H]MK-801
binding when 24-month-old animals were compared to 6-month-
old animals. The mean age profiles for the IFCTX, ERC and
the IFCTX all linearly decreased and had slopes significantly
less than 0 with p values of .0026, .0151 and .0018
respectively (Figure 3-1). The other brain regions analyzed,
the OFCTX, the LSEP, the LTHAL, the molecular layer of the DG
and hippocampal area CAl, did not change as a function of
increasing age (Figures 3-2 and 3-3). A representative
autoradiograph shows the distribution of [3h]MK-801 binding
in the rat brain (Figure 3-4).

45
1.0 i
0.8-
0.6-
0.4-
0.2-
0.0
â–  6 mo.
E3 12 mo.
® 24 mo.
* p< .003
** p< .01
***p< .002
IFCTX ERC LSTR
Brain Region
Figure 3-1. Brain regions that underwent a significant age-
related decrease in [3H]MK-801 binding. (IFCTX=inner frontal
cortex; ERC=entorhinal cortex; LSTR=lateral striatum).
Repeated measures ANOVA with between-animal randomized blocks
and age as a linear covariate presented as a function of
increasing age. The p values represent mean age profiles with
slopes significantly less than 0.

46
2 n
OFCTX LSEP LTHAL
Brain Region
Figure 3-2. Brain regions that did not undergo a significant
age-related change in [3H]MK-801 binding. (OFCTX= outer
frontal cortex; LSEP= lateral septum; LTHAL= lateral
thalamus). Repeated measures ANOVA with between subject
randomized blocks and age as a linear covariate presented as
a function of increasing age.

47
O
O
CO O)
¿ £
£ I
DG CAI
6 mo.
12 mo.
24 mo.
Brain Region
Figure 3-3. Brain regions that did not undergo a significant
age-related change in [3H]MK-801 binding. (DG= molecular
layer of the hippocampal dentate gyrus; CA1= hippocampal area
CAl stratum radiatum). Repeated measures ANOVA with between
subject randomized blocks and age as a linear covariate
presented as a function of increasing age.

Figure 3-4. NMDA receptor distribution as determined by
[3H]MK-801 binding in : (A) 6-month-old, (B) 12-month-
old, and (C) 24-month-old rat brain.

49

50
TABLE 3-1. Laminar thickness (mm), volume (mm3), and
neuronal density (neurons/mm2) values from Fischer 344 rat
brain.
6-month-old
12-month-old
24-month-old
Laminar
Thickness
(mm)
Frontal Cortex
Mean= 0.1952
SEM = 0.0013
0.19561
0.00211
0.1945
0.0017
Entorhinal
Cortex
Mean= 0.2988
SEM = 0.0031
0.2940
0.0037
0.2980
0.0026
Volume (mm3)
Lateral
Striatum
Mean=41.8491
SEM = 1.2064
41.8147
1.0065
41.3812
0.5626
Neuronal
Density
(per mm2)
Entorhinal
Cortex
Mean=1343.86
SEM = 36.60
1300.38
127.03
1319.50
41.33
Inner Frontal
Cortex
Mean=1285.50
SEM = 35.17
1279.89
10.86
1285.31
33.81
Lateral
Striatum
Mean=1284.23
SEM = 24.01
1284.71
51.28
1297.88
21.01
n=5 animals
per age qroup
No statistically significant differences found with ANOVA
(multi-comparison significance level at 95%, repeated
measures).

51
Discussion
These studies showed that the number of NMDA receptors,
as determined by [3H]MK-801 binding, is reduced in the ERC,
the LSTR and the IFCTX in aged F-344 rats. However, receptor
density in the OFCTX, the LSEP, the LTHAL, the molecular
layer of the DG as well as hippocampal area CA1 did not
change as a function of increasing age.
It is interesting that the decrease in the density of
[3h]MK-801 binding was limited to only three of the eight CNS
structures analyzed. The ERC was one structure that did
undergo an age-related decrease in MK-801 binding. The ERC
projects to the hippocampal formation, a component of the
limbic system associated with brain mechanisms for memory.
More specifically, the ERC is the major source of extrinsic
afferents to the dentate gyrus granule cells and pyramidal
cells of the hippocampus proper (Amaral and Witter, 1989).
There are decreased numbers of NMDA receptors in the ERC of
aged rats and this may affect the connectivity from this
structure to the hippocampus. This may result in an
alteration in NMDA receptor-mediated neurotransmission and
subsequently alter some aspects of memory (e.g., spatial
information processing) (Barnes and McNaughton, 1985; Morris
et al., 1978). It has been shown that excitotoxic lesions of
the rat ERC impair the retention of reference memory tasks
(Levisohn and Isacson, 1991). Age-dependent deficits in
spatial memory have been shown to be related to impairments

52
in hippocampal "kindling" (used as a measure of neuronal
plasticity) in F-344 rats (DeToledo-Morrell et al., 1984).
The kindling phenomenon is dependent upon sequential synaptic
modification in a cascading neural system (DeToledo-Morrell
et al., 1984), and would be expected to be especially
vulnerable to age-related alterations in postsynaptic
mechanisms such as a decrease in NMDA receptors in the
entorhinal cortex. The ERC, in addition to having
connections with the hippocampus, has reciprocal connections
with a wide range of cortical association areas (Amaral and
Witter, 1989). The decrease in MK-801 binding in the ERC
could therefore also affect cortical circuitry by decreasing
the overall amount of NMDA receptor-mediated
neurotransmission.
The decrease in MK-801 binding observed in the IFCTX of
the aged animals may contribute to some alteration in the
circuitry necessary for learning and memory as well.
Cortical-striatal projections have been shown to utilize NMDA
receptors (Cherubini et al., 1988), and a decreased density
of NMDA receptors in both IFCTX and the LSTR may affect the
NMDA receptor-mediated neurotransmission between these
projections. Piggott et al. (1992) found that [3H]MK-801
binding declined with age in human frontal cortical
membranes. More recently, Serra et al. (1994) found that the
total number of binding sites for [3H]MK-801 was decreased in
the hippocampus, cerebral cortex and striatum of 18- and 24-
month-old rats, relative to 3-month-old animals. Other

53
groups have also shown a significant age-related decrease in
[3H]MK-801 binding in cerebral cortical and hippocampal brain
homogenates (Kitamura et al., 1992; Tamaru et al., 1991). In
another study, the ERC, IFCTX and LSTR were among various
brain regions that were shown to undergo the greatest percent
decline in NMDA-displaceable [3H]L-glutamate binding when 30-
month-old mice were compared to 3-month-olds (Magnusson and
Cotman, 1993). These regions may be some of the most
vulnerable to the effects of aging. Since these regions are
implicated in normal memory function and plasticity of the
CNS (DeToledo-Morrell et al., 1984; Gonzales et al., 1991;
Petit, 1988), an age-related decrease in NMDA receptors in
these regions suggests that some of the cellular mechanisms
encoding memory are subsequently impaired.
It was determined that the age-related decrease seen in
[3h]MK-801 binding was not a result of a decrease in neuronal
density in the ERC, IFCTX or the LSTR. Age comparisons were
made in neuronal packing densities for each of these brain
regions. As West et al. (1993) stated, the volume and/or
potential shrinkage (laminar thickness) of specific brain
regions needs to be determined in order to obtain accurate
measurements of changes that may be occurring in specific
neuronal populations. He stated that only when these
parameters are known can neuron packing density be used,
instead of total neuronal number, in a meaningful discussion
of the functional state of a neural structure (West et al,
1993). In the present study, neuronal packing density was

54
measured as the number of neurons per square millimeter.
Therefore, this measurement would only be valid after
determining that these brain regions did not undergo an age-
related change in overall volume (LSTR) or laminar thickness
(ERC and IFCTX) (i.e., the mm^ denominator value was not
affected by age). No significant age-dependent differences
were found in the volume of the LSTR or in the laminar
thickness of the ERC and IFCTX (Table 3-1). Neuronal packing
densities were then determined utilizing an MCID image
analysis system for the ERC, the IFCTX and the LSTR. There
was no statistically significant difference in neuronal
density measurements in any brain region examined. Therefore
the decrease seen in [^h]MK-801 binding represents a loss of
NMDA receptors within these brain regions rather than a loss
of neurons.
In this study, [3H]MK-801 binding density was expressed
in pmol/mg protein. Therefore, changes in protein
concentration in specific brain regions as a function of age
may effect these values. Burnett and Zahniser (1989)
performed a quantitative autoradiographic study that examined
age-related changes in a-1 adrenergic receptors in F-344 rat
brain. Protein levels were measured in the same tissue
sections by using a staining procedure described by Miller et
al. (1988). They found no significant differences in protein
concentration between age groups (i.e., 3- to 4-month-old,
16- to 18-month-old and 24- to 28-month-old) in the thalamus,
cerebral cortex, hippocampus, striatum, cerebellum,

55
brainstem, and olfactory tubercle. Since the ages and strain
of rats used in the present study were identical to Burnett
and Zahniser (1989) it is expected that total protein would
also remain unchanged as a function of age. Therefore, it is
probable that age-related differences in [3H]MK-801 binding
density in specific brain regions is due to alterations in
receptor number and not in protein concentration.
It should be noted that finding an age-related effect on
[3H]MK-801 binding in only three of eight brain regions
analyzed may reflect the inability of MK-801 to label all of
the NMDA receptors, since ligand binding can only assay
surface-presented receptors. Studies investigating mRNA
coding for the NMDA receptor is presented in Chapter 5. The
density of NMDA protein is also presented in Chapter 6.
These further studies address the hypotheses that the density
of mRNA coding for the NMDA receptor and the density of NMDA
protein change as a function of age.
These [3h]MK-801 binding experiments have determined
some of the age-dependent changes taking place in the NMDA
receptor/channel complex. The decrease in NMDA receptor
density seen in specific brain regions may account for some
of the deleterious effects of aging on learning and memory
(Barnes, 1979; Barnes and McNaughton, 1985). Gonzales et al.
(1991) studied NMDA receptor-mediated responses in the
hippocampus, cortex, and striatum of F-344 rats of various
ages (3- to 5-, 12- to 14-, and 24- to 28-months-of-age) to
determine whether aging alters some of the functional

56
properties of this receptor complex. They examined NMDA-
stimulated release of norepinephrine and dopamine as indices
of NMDA receptor function and found that NMDA-mediated
responses were attenuated with increasing age. In Chapter 4,
the effects of increasing age on L-glutamate's ability to
enhance [3h]MK-801 binding is studied. This approach was
taken to see if there were differences in the Emax (maximal
response or density of [3H]MK-801 bound) or EC50 (the
concentration of L-glutamate producing a half-maximal
response) as a function of age.
Many of the processes underlying learning and memory are
known to utilize, to a great extent, NMDA receptors (Danysz
et al., 1988; Morris et al., 1986). Therefore, elucidation
of age-dependent changes in NMDA receptors may help target
potential pharmaceutical interventions aimed at alleviating
age-related cognitive decline. It has been suggested that
pharmacological manipulation of glutamatergic
neurotransmission may prove beneficial for cognitive
enhancement (Ingram et al., 1994). However, the utilization
of NMDA receptor agonists may be an unsafe strategy due to
the potential neurotoxic effects associated with
overstimulation of the NMDA receptor (Cotman and Monaghan,
1988). Therefore, indirect activation through other
modulatory sites identified on the NMDA receptor complex
could be investigated, such as the co-agonist glycine site
and/or the polyamine modulatory sites.

57
Milacemide and D-cycloserine are two strong candidates
for agonists at the glycine site and have both been shown to
enhance memory performance in rats (Flood et al., 1992;
Quartermain et al., 1991). A relatively new class of
chemical substances, nootropics, were developed specifically
to alleviate age-related cognitive deficits and have been
shown to enhance NMDA receptor density (10 to 25%) in aged
brain (Cohen and Muller, 1992; Davis et al., 1993; Fiore and
Rampello, 1989). Phosphatidylserine is one example of a
nootropic drug. Cohen and Muller (1992) showed that chronic
treatment with phosphatidylserine ameliorated age-associated
deficits of the NMDA receptor in the forebrain of aged mice
by restoring the density of NMDA receptors to levels similar
to young animals and also by normalizing the sensitivity of
aged animals for the stimulating effects of L-glutamate and
glycine. Cohen and Muller (1993) also showed significant
enhancement (20%) of NMDA receptor density in aged mice
treated with another nootropic drug, piracetam. The kinetic
constants for L-glutamate stimulation of [3H]MK-801 binding
in the aged animals were not significantly different from
untreated young mice (Cohen and Muller, 1993). In addition,
the nootropic agent L-acetylcarnitine was shown to attenuate
the age-dependent decrease of NMDA-sensitive glutamate
receptors in the rat hippocampus (Davis et al., 1993; Fiore
and Rampello, 1989). Taken together, these studies imply
that specific pharmacological targeting of the NMDA receptor

58
may indeed prove to be a viable treatment strategy for
restoration of cognitive deficits in the aged population.

CHAPTER 4
EFFECT OF AGE ON L-GLUTAMATE STIMULATION OF [3H]MK-801
BINDING IN F-344 RAT BRAIN
Introduction
In Chapter 3, a decrease in the number of NMDA receptors
in specific brain regions in the F-344 rat was shown. Other
studies have described a similar reduction in NMDA receptors
in various species (Anderson et al., 1989; Ingram et al.,
1992; Magnusson and Cotman, 1993; Peterson and Cotman, 1989;
Serra et al., 1994; Tamaru et al., 1991; Wenk et al., 1991)
including humans (Piggott et al., 1992). In Chapter 3,
[3H]MK-801 was utilized to study the NMDA receptor/channel
complex. This antagonist binds with high affinity to a site
within the channel of the NMDA receptor complex, thus MK-801
binding will only take place if the NMDA receptor is in the
transmitter-activated (open) state (Huettner and Bean, 1988).
Channel activation by an NMDA agonist such as L-glutamate, in
the presence of glycine allows MK-801 binding to take place.
Glycine alone cannot activate the NMDA receptor. In addition
to the glycine site on the NMDA receptor/complex, there is an
allosteric polyamine recognition site. The polyamines
spermine and spermidine have been shown to increase both
maximum NMDA response amplitude (Lerma, 1992; McGurk et al.,
59

60
1990; Ransom and Deschenes, 1990; Sprosen and Woodruff, 1990)
as well as binding of MK-801 to the NMDA receptor channel
(Ransom and Stec, 1988).
L-glutamate is the endogenous transmitter for the NMDA
receptor channel complex. In this study, an evaluation of
the efficacy of L-glutamate to stimulate MK-801 binding
within the NMDA receptor/channel complex was performed by
using various concentrations of L-glutamate. These studies
examined the efficacy and potency with which L-glutamate
produces receptor activation and channel opening as a
function of increasing age by comparing dose-response curves
from 6-, 12-, and 24-month-old animals.
The hypothesis tested in these experiments was that
there would be an age-related decrease in the maximal
response (Emax) elicited by L-glutamate. It was also
hypothesized that there would not be a concomitant change in
the concentration of L-glutamate producing a half-maximal
response (EC50) as a function of age. in these experiments,
the response of the NMDA receptor was defined as the density
of [3H]MK-801 binding determined as a function of L-glutamate
concentration.
Many studies have shown that there are decreased numbers
of NMDA receptors in aged animals (Anderson et al., 1989;
Ingram et al., 1992; Magnusson and Cotman, 1993; Peterson and
Cotman, 1989; Piggott et al., 1992; Serra et al., 1994;
Tamaru et al.,1991; Wenk et al., 1991). Consequently, there
are fewer NMDA receptors available for occupation by ligand

61
in aged rats. Because there are fewer NMDA receptors
available for occupation by ligand in aged rats it would be
expected that there would be a decreased maximal response
([3H]MK-801 bound) in aged rats due to fewer NMDA receptor
channels available to be activated (opened) by agonist. In
addition, previous studies have shown an age-related decrease
in the functional capacity of the NMDA receptor/channel
complex as defined by a reduction in various NMDA-mediated
responses (Gonzales et al., 1991; Pittaluga et al., 1993;
Serra et al., 1994). The ability of L-glutamate to stimulate
[3H]MK-801 binding was therefore compared across varying age
groups in the F-344 rat.
Methods
Tissue Preparation
The male F-344 rats, 6-, 12- and 24-months-of-age, that
were used in these experiments were the same as those
described in Chapter 3. For a more detailed description of
the tissue preparation see Chapter 2.
r-^HIMK-801 Binding Assay
For a detailed description of this protocol, see Chapter
2. Varying concentrations of L-glutamate (i.e., 0.0 pH, 0.05
pñ, 0.1 pM, 0.25 pM, 0.5 pM, 1.0 pM, 2.5 pM, 5.0 pM, 10 pM,
and 20 phi) were added to the incubation step in order to
examine glutamate's ability to stimulate [3H]MK-801 binding.

62
A total of four slides, from each block of animals, were
analyzed per assay (n=6) for each L-glutamate concentration.
Image analysis
See Chapter 2 for a detailed description of image
analysis.
Data Analysis
The data consist of the EC50 and Emax values obtained
from binding isotherm plots. These plots were generated from
average density measurements of [3H]MK-801 binding (pmol/mg
protein) in the presence of increasing concentrations of L-
glutamate. Binding densities were obtained from bilateral
brain regions for each of the 6-,12-, and 24-month-old rats
(n=6 for each age). One "group" consisted of one 6-month-,
one 12-month-, and one 24-month-old animal mounted per series
of slides. Each [3H]MK-801 binding assay was run with the
three ages of animals mounted per slide. Four slides, from
each of these groups, were analyzed per assay per L-glutamate
concentration, with each [3H]MK-801 binding assay being
performed on one group of animals (n=6). Two slides per
assay were analyzed for non-specific binding and these values
were subtracted from each specific binding value in order to
obtain total binding. Brain regions that were analyzed
included the outer and inner frontal cortices (OFCTX, IFCTX);
the entorhinal cortex (ERC); the molecular layer of the
dentate gyrus (DG); hippocampal area CA1 stratum radiatum

63
(CAI); the lateral septum (LSEP); the lateral striatum
(LSTR); and the lateral thalamus (LTHAL).
Binding Isotherm Plots
INPLOT (GraphPad Software, San Diego, CA) was used to
generate linear regression plots from the density values
obtained in these experiments. Plots gave estimated EC50
(effective concentration at 50%) and Emax (maximal [3h]MK-801
binding elicited by L-glutamate) values for L-glutamate
stimulation of [3H]MK-801 binding. Estimates were then used
to generate rectangular hyperbola plots or binding isotherms.
Binding isotherm plots then yielded the true EC50 and Emax
values for each age group and brain region analyzed. As the
concentration of L-glutamate increased, there was a
concominant increase in the density of [3H]MK-801 binding
(Figure 4-1). EC50 and Emax values obtained from [3H]MK-801
binding isotherm plots for the 6-month-old animals were used
to normalize the 12- and 24-month-old animal values.
Statistical Analysis
Six blocks of animals, each block consisting of an
animal from each age group, were assessed. A different block
of 3 animals, 1 per age group, was assessed on each of 6
experimental days. The experimental design consisted of a
within-animal factor with 8 levels (brain region), a
guantitative between-animal fixed factor with 3 levels (age),
and a between-animal random blocking factor with 6 levels

64
(experimental day). Repeated measures analysis of variance
(ANOVA) with between-animal randomized blocks and age
considered as a linear covariate were used to model the EC50
and Emax data as a linear function of age within each brain
region and to determine if this pattern differed
significantly among brain regions. The change in EC50 and
Emax per month-of-age (i.e. the linear slope) was estimated
and assessed for statistical significance within each brain
region while blocking on experimental day. A general
comparison of these slopes among brain regions was then
performed by testing for the presence of a significant
interaction between age and brain region EC50 and Emax
effects. Mauchly's sphericity test was used to determine if
a multivariate F test (Wilks' Lambda) or a univariate F test
should be used for the age x brain region interaction test.
In assessing the validity of ANOVA assumptions, it was noted
that experimental day age profiles were relatively parallel
within each brain region and that between-subject variability
as estimated by the standard deviation was similar among age
groups within each brain region. Plots of residuals versus
predicted values, residual histograms, and residual normal
probability plots were examined to assess goodness of fit in
the ANOVA models.
Results
[3H]MK-801 binding increased as a function of L-
glutamate concentration in all the age groups and a

65
representative autoradiogram is presented in Figure 4-1.
Significant differences between the Emax values in nearly all
brain regions were found when middle-aged and aged animals
were compared to the young rats (Table 4-1). The only region
where Emax did not differ in middle-aged animals, when
compared to young-adults, was the OFCTX. The areas showing
the greatest percent decline in aged animals when compared to
young were the LSTR (26.4%), ERC (24.7%), and IFCTX (21.5%).
Areas showing the greatest percent decline in middle-aged
animals when compared to young were the LSTR (12.9%), LSEP
(10.3%), and IFCTX (9.3%).
No significant differences were found in the EC50 values
when comparing middle-aged and aged rats to young (Table 4-
2).

66
TABLE 4-1. Emax values (pmol/mg protein) from [^H]MK-801
binding isotherms.
BRAIN REGION
6 month
12 month
24 month
OFCTX
1.588 + 0.29
1.468 + 0.29
1.441 + 0.24*
IFCTX
0.959 + 0.16
0.870 + 0.17*
0.753 + 0.14*
ERC
0.830 + 0.12
0.758 + 0.14*
0.625 + 0.12*
DG
1.874 + 0.37
1.737 + 0.40*
1.669 + 0.39*
CAl
1.712 + 0.36
1.618 + 0.41*
1.537 + 0.40*
LSEP
0.951 + 0.20
0.853 + 0.19*
0.850 + 0.21*
LSTR
0.780 + 0.15
0.679 + 0.14*
0.574 + 0.12*
LTHAL
0.793 + 0.15
0.747 + 0.15*
0.665 + 0.14*
MEAN + SEM
MEAN + SEM
MEAN + SEM
n=6
n=6
n=6
Asterisks (*) indicates significance when compared to 6-
month (p < 0.05 ANOVA; F-test). Abbreviations: OFCTX=outer
frontal cortex; IFCTX=inner frontal cortex; ERC=entorhinal
cortex; DG=molecular layer of the dentate gyrus;
CAl=hippocampal area CAl; LSEP=lateral septum; LSTR=lateral
striatum; LTHAL=lateral thalamus

67
TABLE 4-2. EC50 values (pM) from [3H]MK-801 binding
isotherms.
BRAIN REGION
6 month
12 month
24 month
OFCTX
0.764 + 0.14
0.758 + 0.18
0.798 + 0.22
IFCTX
1.311 + 0.23
1.085 + 0.26
1.148 + 0.39
ERC
1.108 + 0.17
1.197 + 0.32
0.921 + 0.25
DG
0.951 + 0.21
0.936 + 0.25
1.040 + 0.20
CA1
0.782 + 0.17
0.660 + 0.17
0.725 + 0.20
LSEP
0.432 + 0.11
0.444 + 0.14
0.410 + 0.14
LSTR
0.578 + 0.13
0.586 + 0.15
0.599 + 0.23
LTHAL
0.993 + 0.20
0.861 + 0.18
0.892 + 0.23
MEAN + SEM
MEAN + SEM
MEAN + SEM
n=6
n=6
n=6
No significant differences were found when
.2-, and 24-
month-old rats were compared to 6-month-old rats (p > 0.05
ANOVA; F-test). Abbreviations: OFCTX=outer frontal cortex;
IFCTX=inner frontal cortex; ERC=entorhinal cortex;
DG=molecular layer of the dentate gyrus; CAl=hippocampal area
CA1; LSEP=lateral septum; LSTR=lateral striatum;
LTHAL=lateral thalamus

Figure 4-1. Total [3H]MK-801 binding in a range of
concentrations of L-glutamate and a constant
concentration of [3H]MK-801 (10 nM). Concentration of L-
glutamate was as follows: (A), 0.0 ^/M; (B), 0.25 pM;
(C), 2.5 pM; (D), 10 pM. (A) Note that only background
levels of binding were present in sections incubated
without L-glutamate. (B-D) As the concentration of L-
glutamate increased, [3H]MK-801 binding increased in a
dose-dependent manner. Autoradiographs are shown in
inverse exposure so that white indicates high density of
binding and black indicates low levels of binding.


70
Discussion
The lateral striatum (LSTR), entorhinal cortex (ERC) and
inner frontal cortex (IFCTX) were the three brain regions
that were shown in Chapter 3 to undergo an age-related
decrease in [3h]MK-801 binding when maximal levels of
glutamate, glycine and spermine were present. These regions
also showed the greatest percentage decrease in Emax values
without concomitant changes in EC50 values as a function of
age. Because both middle-aged and aged animals displayed a
significant decrease in Emax values without differences in
their EC50 values, it appears that there is an age-related
effect on the maximum density of [3H]MK-801 bound as a
function of L-glutamate concentration.
The maximal effect that L-glutamate has on stimulating
[3H]MK-801 binding is decreased in aged rats. This age-
dependent decrease in Emax may be the result of a down-
regulation of NMDA receptors in specific brain regions.
These brain regions may undergo down-regulation to protect
against excitotoxic damage. In a developmental study, Oster
and Schramm (1993) demonstrated that the process of down-
regulation of NMDA receptor activity occurs in rat cerebellar
granule cells. In these experiments, NMDA was added to cell
cultures derived from postnatal day 8 rats. This resulted in
suppression of NMDA receptor-mediated 4^ca uptake without
affecting the viability or total cell protein of the cultured
neurons. The down-regulation also rendered the neurons

71
resistant to NMDA toxicity. They proposed that a similar
form of down-regulation may play a role in adjusting the
activity of postsynaptic NMDA receptors following
synaptogenesis.
Jakoi et al. (1992) showed that activation of EAA
receptors in cultured hippocampal neurons caused a down-
regulation of the protein ligatin at both physiologic and
excitotoxic levels of glutamate stimulation. This down-
regulation was shown to be mediated by the NMDA receptor and
it was hypothesized that EAA receptor activation may alter
expression of NMDA receptors. This effect on NMDA receptor
expression may be a central mechanism that underlies some of
the long-lasting functional and pathophysiological effects of
EAA receptor activation on cell function (Jakoi, et al.,
1992). Therefore, it is possible that down-regulation of
NMDA receptors in the aging CNS may affect the efficacy of L-
glutamate to maximally enhance the receptor's binding
capacity in the aged brain.
Neurons bearing NMDA receptors are vulnerable to
excitotoxic injury associated with an excessive concentration
of extracellular glutamate or related agonists (Choi, 1987).
An age-related increase in basal glutamate release in mouse
striatal and hippocampal slices has been reported (Freeman
and Gibson, 1987). Low affinity glutamate uptake into rat
cerebral cortical slices (Matsumoto et al., 1982) and brain
mitochondria (Victorica et al., 1985) has been shown to be
reduced as a function of age. Furthermore, an age-related

72
loss in the number of high affinity glutamate transport
(uptake) sites of rat striatal (Price et al., 1981) and
cortical synaptosomes (Wheeler and Ondo, 1986) has been
reported. Taken together, these reports suggest that
extracellular glutamate levels are elevated with aging.
Therefore, the age-related down-regulation in NMDA receptors
may essentially be a functional trade-off in order to protect
cells bearing NMDA receptors from excitotoxic insult.
A recent study reported data that contradict some of the
findings here (Serra et al., 1994). Using hippocampal,
striatal and cerebral cortical brain homogenates from 3-, 18-
and 24-month-old male Wistar Kyoto rats a decrease (20 to
25%) was seen in the total number of NMDA receptors in 18-
and 24-month-old rats. No significant differences were found
in Kp between young and aged rats. In addition, no
difference was seen in the sensitivity of [3H]MK-801 binding
as a function of glutamate concentration in the cerebral
cortex, striatum or hippocampus of the aged rats. However,
in the hippocampus of 18-month-old rats, glycine and
glutamate stimulated [3H]MK-801 with a higher efficacy than
in 3- or 24-month-old rats. This group speculated that the
loss of NMDA receptors in the hippocampus of 18-month-old
rats is counteracted by a physiological compensatory
mechanism. This mechanism somehow increases NMDA receptor
activity and thereby prevents a decline in cognitive function
in the "early phase of aging" (e.g., 18-months-of-age). The
compensation ceases to function adequately during, what this

73
group termed, the "late phase of aging" (i.e. 24 months of
age) (Serra et al., 1994). One possible explanation for the
differences in these findings is that 18-month-old animals
were not examined in the present study. Although Serra et
al. (1994) used thorough washing techniques on the rat
membrane homogenates they speculated that the sensitivity of
[3H]MK-801 binding to endogenous concentrations of agonists
and allosteric modulators of the NMDA receptor may have
affected their results. Therefore, another possible
explanation for this discrepancy is the aging-induced changes
in the brain concentrations of glycine and glutamate and/or
other modulators which could affect [3H]MK-801 binding
(Freeman and Gibson, 1987).
Gonzales et al. (1991) found an age-related decrease in
NMDA receptor function in aged animals by analyzing NMDA-
stimulated neurotransmitter release in rat cortex,
hippocampus and striatum. They showed that NMDA-stimulated
[3H]NE (norepinephrine) and [3H]DA (dopamine) release were
decreased as a function of age. In a similar study,
Pittaluga et al. (1993) showed an age-related decrease in
NMDA receptor-mediated noradrenaline release in rat
hippocampus.
It should be noted that there are advantages to using
quantitative autoradiographic analysis over brain tissue
homogenates in that it allows for a more detailed analysis of
any anatomical distributional changes occurring in the rat
CNS. However, although there are other methods that can also

74
test the NMDA receptor's functional capacity (e.g.
electrophysiological patch clamping techniques, molecular
biological oocyte expression systems etc.), the experiments
performed in this study tested the ability of glutamate to
enhance [3H]MK-801 binding. It should be noted that a
confounding variable possibly influencing these results is
the phenomenon of receptor desensitization, which is defined
as the lack or decline of a response as a result of previous
activation. Since brain slices in these experiments were
exposed to glutamate for long periods of time, the receptors
may have desensitized. Therefore, responses of NMDA
receptors may have been examined in the desensitized state.
The sections used in this study were rinsed for a total
of 60 minutes at 30°C prior to incubation with [3H]MK-801.
Previous studies performing [3H]MK-801 binding assays have
shown that prewashing sections for as little as thirty
minutes removed substantial amounts of endogenous amino acids
since [3H]MK-801 binding following incubation was stimulated
significantly with the addition of exogenous glutamate
(Sakurai et al., 1990). Therefore, any potential effects of
desensitization were probably not seen prior to the
incubation with radiolabeled MK-801. It should also be noted
that the time-course for the amount of [3H]MK-801 bound to
reach equilibrium in the binding assay occured prior to the
one hour incubation period utilized in these experiments (the
incubation step is when the brain sections are exposed to
[3H]MK-801 in the presence of glutamate, glycine and

75
spermine). Since, at equilibrium, the receptors have reached
their greatest maximal response, comparisons can be made
between the various age groups with any observed differences
in the density of [3H]MK-801 binding being attributed to the
age factor. Monaghan (1991) examined the differential
stimulation of [3H]MK-801 binding to subpopulations of NMDA
receptors and stated that since excess concentrations of
glycine and spermine were present in all incubations and both
sites analyzed could be 'activated' neither site appeared to
be in a desensitized form. However, if desensitization
occurs within minutes or seconds of glutamate application,
then age-related differences in [3H]MK-801 binding may not be
able to be interpreted as clearly due to potential age-
related differences in densensitization mechanisms. In order
to alleviate some of these potential problems in these
experiments, desensitization will be defined as a decreased
density of [3H]MK-801 (response) as a function of increased
glutamate concentration. It is important to note that the
density of [3H]MK-801 bound did not decrease after reaching
maximal levels in any of the age groups, suggesting that
desensitization may not be a significant factor in these
studies.
Consistent with L-glutamate's ability to open the ion
channel associated with the NMDA receptor, it was shown in
this study that [3H]MK-801 binding increased as a function of
L-glutamate concentration in all age groups. However, these
findings suggest that there is an effect of age on the

76
maximal increase in [3H]MK-801 binding induced by L-
glutamate. These age-dependent changes in the maximal
response elicited by L-glutamate may be due to some
functional alteration in the NMDA receptor/channel complex.
Hollenberg (1985) proposed that receptors can be modified by
reactions leading to either covalent or noncovalent bond
formation. In covalent modifications, reactions involving
receptor phosphorylation, disulfide-sulfhydryl exchange and
receptor proteolysis are all possible mechanisms involved in
receptor function (Hollenberg, 1985). Non-covalent
interactions would include changes in membrane potential,
receptor distribution (patching, capping), allosteric
interactions involving either protein-protein (e.g. mobile
receptor model) or small ligand (cations, anions,
nucleotides, etc.) receptor interactions, and alterations in
the membrane lipid environment (e.g. lipid methylation or
hydrolysis) (Hollenberg, 1985). All of these processes could
result in changes in the functional modification of the NMDA
receptor. In conclusion, there is probably a combination of
both an age-dependent decrease in the total number of NMDA
receptors available for binding as well as some age-related
change in the functional capacity of the remaining NMDA
receptors. This may account for some of the differences
found in the maximal increase in [3H]MK-801 induced by L-
glutamate as a function of age.

CHAPTER 5
AGE-RELATED CHANGES IN THE LEVELS OF mRNA CODING FOR SPECIFIC
NMDA RECEPTOR SUBUNITS IN THE CNS OF F-344 RATS.
Introduction
Two NMDA receptor subunit families have been cloned and
are named NMDAR1 (NRl) and NMDAR2 (NR2)(Monyer et al., 1992;
Moriyoshi et al., 1991). NMDARl has at least eight
alternatively spliced forms and these are differentiated from
each other by an insertion at the extracellular amino-
terminal region, deletion at two carboxy-terminal regions, or
by combinations of both (Moriyoshi et al., 1991). NMDARl and
its isoforms have been shown in Xenopus oocyte expression
systems to exhibit electrophysiological and pharmacological
responses characteristic of the NMDA receptor. These include
agonist and antagonist selectivity, glycine modulation,
permeability to calcium, voltage-dependent channel block by
magnesium as well as inhibition by zinc (Moriyoshi et al.,
1991).
NMDAR2 subunit family members have been shown to
potentiate the electrophysiological responses of NMDARl but
are not functional in homomeric configurations with each
other. NMDA-induced currents in oocytes expressing NRl and
NR2A, NR2B or NR2C are approximately 100 times larger than
they are in oocytes expressing homomeric NRl channels. It
77

78
should be noted that these currents more closely resemble
native NMDA receptors and thus native NMDA receptors probably
represent heteromeric configurations formed from NRl subunits
and members of the NR2 subunit family (Monyer et al., 1992).
The NMDA receptor has been implicated in age-related
learning and memory deficits in humans as well as other
animals (Barnes, 1979; Barnes and McNaughton, 1985).
Detailed in situ hybridization analyses can now be performed
due to the recent isolation of functional cDNA clones for the
rat NMDA receptor NRl (NMDAR1), splice variants of NRl as
well as for the members of the NR2 subunit family (i.e.,
NR2A, NR2B, NR2C and NR2D) (Buller et al., 1993; Kutsuwada et
al., 1992; Meguro et al., 1992; Monaghan et al., 1993; Monyer
et al., 1992; Moriyoshi et al., 1991; Nakanishi, 1992). In
this study, this technique was utilized to examine mRNA
coding for the NMDA receptor subunits in the aged brain.
Methods
In situ Hybridization
Oligonucleotide probes for this study (45 mer) were
constructed from published sequences and specific to NMDARl,
four alternatively spliced versions of NMDARl (NRl-not insert
1 or NRloxx/ NRl-insert 1 or NRlixx; NRl-insert 2 or NRlxix;
and NRl-insert 3 or NRlxxi), and the members of the NMDAR2
subunit family (NR2A, NR2B, NR2C, and NR2D). The probes for
this study were graciously supplied by Dr. Daniel T.

79
Monaghan, University of Nebraska Medical Center, Omaha NE.
See Chapter 2 for details on the probe labeling procedure,
tissue sectioning, fixation and hybridization.
Data Analysis
Computer assisted semi-quantitative densitometric
measurements were performed as described in detail in Chapter
2. Six-month (young), twelve-month (middle-aged) and twenty-
four month (aged) F-344 rat brains were used (n=6 for each
age group). The outer frontal cortex (OFCTX), inner frontal
cortex (IFCTX), lateral septum (LSEP), lateral striatum
(LSTR), lateral thalamus (LTHAL), entorhinal cortex (ERC),
dentate gyrus (DG), and hippocampal areas CA1 and CA3 were
the brain regions analyzed in this study. Relative mRNA
density levels were obtained for each of the oligonucleotide
NMDA probes. Density values from young adults were compared
to the middle-aged and aged animals.
Statistical Analysis
Six blocks of animals, each block consisting of an
animal from each age group, were assessed. The experimental
design consisted of a within-subject factor with nine levels
(brain region), a quantitative between-subject fixed factor
with three levels (age), and each of six levels (experimental
day). Repeated measures analysis of variance (ANOVA) with
between-subject randomized blocks and age considered as a
linear covariate were used to model the density of mRNA per

80
oligonucleotide probe as a function of age within each brain
region and to determine if this pattern differed
significantly among brain regions.
Results
Significant age-related changes were found in NMDAR1, as
well as three of the splice variants of NMDAR1: NRloxx/
NRllxx, and NRlxlx (Figures 5-1,5-2,5-4,5-5 and 5-7). More
specifically, NMDAR1 mRNA measured in 12-month-old rats
showed a significant decrease in the outer frontal cortex
(17.8%), inner frontal cortex (15.0%), hippocampal area CA3
(9.0%) and the lateral striatum (14.1%). In 24-month-old rat
brains, there was a significant decrease seen in all brain
regions analyzed, with the exception of the molecular layer
of the dentate gyrus. Areas showing the greatest percent
decline included the entorhinal cortex (28.1%) > lateral
thalamus (24.2%) > lateral striatum (20.7%) > outer frontal
cortex (18.6%) > inner frontal cortex (17.7%) > hippocampal
areas CA3 (14.5%) and CA1 (14.4%) > lateral septum (10.4%).
Relative mRNA density for NMDARl splice variant NRloxx
(not containing the 21 amino acid N-terminal insert) showed a
statistically significant decrease in the entorhinal cortex
(5.6%) as well as the CA3 region of the hippocampus (9.2%) in
24-month-old animals. This splice variant also showed a
significant increase in mRNA density in hippocampal area CAl
(5.5%) from 12-month-old animals. There was a significant
age-related change in relative mRNA density for two other

81
splice variants of NMDARl. The NRlixx (those NMDARl isoforms
containing the N-terminal insert) showed an increase in the
lateral septum (9.6%) from 12-month-old animals when compared
to 6-month-old animals (Figure 5-5). NRlxlx isoforms (those
splice variants containing the first of the two C-terminal
inserts) also showed an increase in the lateral septum (5.3%)
along with the lateral striatum (3.4%) and lateral thalamus
(4.7%) from 12-month-old animals (Figure 5-7). In contrast,
no significant differences were observed in either NRlixx or
NR1X1X mRNA density in any of the brain regions analyzed from
24-month-old animals (Figures 5-5, 5-6, 5-7, and 5-8). The
density of NMDARl splice variant NRlxxi (containing the
second C-terminal insert) did not show any differences in any
of the brain regions analyzed at any age (Figures 5-9 and 5-
10).
The NMDAR2 subunit family members, NMDAR2A and NMDAR2B,
did not show any age-related differences in relative density
of mRNA (Figures 5-11, 5-12, 5-13 and 5-14). NMDAR2C subunit
mRNA was not present in any of the brain regions examined.
Since this subunit is found only in the cerebellum it was
excluded from these analyses. There was no detectable
NMDAR2D mRNA present in any of the age groups. This may be
due to the fact that this subunit appears to be
developmentally regulated such that there is an apparent
shift in relative expression of NMDAR2D to NMDAR2B expression
at about six months-of-age in the rat CNS. Representative
autoradiograms are seen in Figures 5-15, 5-16 and 5-17.

82
NMDAR1
100 -i
OFCtx IFCtx LSep LStr LThal
3 middle-aged
C3 aged
*- significant
at 95%
Brain Region
Figure 5-1. In situ hybridization analyses of NMDARl mRNA in
cortical and subcortical brain regions. All data are the mean
+ S.E.M. Asterisk indicates densitometric values
significantly different than young animals.(OFCtx=outer
frontal cortex; IFCtx=inner frontal cortex; LSep= lateral
.ateral striatum; LThal= lateral thalamus).
NMDARl
middle-aged
septum;
LStr
100 -1
80 -
60 -
c
3
o
>â– 
40 -
T5
â– 
20 -
â–¡ aged
*- significant
at 95%
ERC DG CAI CA3
Brain Region
Figure 5-2. In situ hybridization analyses of NMDARl mRNA in
hippocampus and entorhinal cortex. All data are the mean +
S.E.M. Asterisk indicates densitometric values significantly
different than young animals. (ERC= entorhinal cortex; DG=
molecular layer of the dentate gyrus; CAl & CA3= hippocampal
areas).

83
NR1 Oxx
OFCtx IFCtx LSep LStr LThal
Brain Region
Figure 5-3. In situ hybridization analyses of NMDAR1 splice
variant NRlOxx mRNA. All data are the mean + S.E.M. Asterisk
indicates densitometric values significantly different than
young animals. (OFCtx=outer frontal cortex;IFCtx=inner
frontal cortex;LSep=lateral septum;LStr=lateral striatum;
LThal=lateral thalamus).
NRl Oxx
middle-aged
â–¡ aged
*- significant
at 95%
Brain Region
Figure 5-4. In situ hybridization analyses of the NMDAR1
splice variant NRlOxx mRNA. All data are the mean + S.E.M.
Asterisk indicates densitometric values significantly
different than young animals. (ERC=entorhinal cortex;DG=
molecular layer of the dentate gyrus;CA1 & CA3=hippocampal
areas).

84
NR1 lxx
120 -i
OFCtx IFCtx LSep LStr LThal
Brain Region
Figure 5-5. In situ hybridization analyses of NMDARl splice
variant NRlixx mRNA. All data are the mean + S.E.M. Asterisk
indicates densitometric values significantly different than
young animals. (OFCtx=outer frontal cortex;IFCtx=inner
frontal cortex;LSep= lateral septum;LStr= lateral striatum;
LThal=lateral thalamus).
NR1 lxx
Brain Region
Figure 5-6. In situ hybridization analyses of the NMDARl
splice variant NRlixx mRNA. All data are the mean + S.E.M.
Asterisk indicates densitometric values significantly
different than young animals. (ERC=entorhinal cortex; DG=
molecular layer of the dentate gyrus;CA1 & CA3= hippocampal
areas).

85
NR1 xlx
120 -i
OFCtx IFCtx LSep LStr LThal
middle-aged
I I aged
*- significant
at 95%
Brain Region
Figure 5-7. In situ hybridization analyses of NMDARl splice
variant NRlxix mRNA. All data are the mean + S.E.M. Asterisk
indicates densitometric values significantly different than
young animals. (OFCtx=outer frontal cortex;IFCtx=inner
frontal cortex;LSep=lateral septum;LStr=lateral striatum;
LThal=lateral thalamus).
NRl xlx
120 n
ERC DG CAI CA3
M middle-aged
n aged
*- significant
at 95%
Brain Region
Figure 5-8. In situ hybridization analyses of the NMDARl
splice variant NRlxix mRNA. All data are the mean + S.E.M.
Asterisk indicates densitometric values significantly
different than young animals. (ERC=entorhinal cortex;
DG=molecular layer of the dentate gyrus;CA1 & CA3=hippocampal
areas).

86
NRl xxl
120 -i
OFCtx IFCtx LSep LStr LThal
i middle-aged
â–¡ aged
*- significant
at 95%
Brain Region
Figure 5-9. In situ hybridization analyses of NMDAR1 splice
variant NRlXxl mRNA. All data are the mean + S.E.M. Asterisk
indicates densitometric values significantly different than
young animals. (OFCtx=outer frontal cortex;IFCtx=inner
frontal cortex;LSep=lateral septum;LStr=lateral striatum;
LThal=lateral thalamus).
NRl xxl
Brain Region
Figure 5-10. In situ hybridization analyses of the NMDARl
splice variant NRlxxi mRNA. All data are the mean + S.E.M.
Asterisk indicates densitometric values significantly
different than young animals. (ERC=entorhinal cortex;DG=
molecular layer of the dentate gyrus;CA1 & CA3=hippocampal
areas).

87
-,2¡ middle-aged
â–¡ aged
*- significant
at 95%
Brain Region
Figure 5-11. In situ hybridization analyses of NMDAR2A mRNA.
All data are the mean + S.E.M. Asterisk indicates
densitometric values significantly different than young
animals. (OFCtx=outer frontal cortex;IFCtx=inner frontal
cortex;LSep= lateral septum;LStr= lateral striatum;LThal=
lateral thalamus).
NMDAR2A
Brain Region
Figure 5-12. In situ hybridization analyses of NMDAR2A mRNA.
(ERC=entorhinal cortex; DG=molecular layer of the dentate
gyrus;CA1 & CA3=hippocampal areas). All data are the mean +
S.E.M. Asterisk indicates densitometric values significantly
different than young animals.

88
a»
C
3
O
>-
ts
K
middle-aged
n aged
*- significant
at 95%
Brain Region
Figure 5-13. In situ hybridization analyses of NMDAR2B mRNA.
All data are the mean + S.E.M. Asterisk indicates
densitometric values significantly different than young
animals. (OFCtx=outer frontal cortex;IFCtx=inner frontal
cortex;LSep= lateral septum;LStr=lateral
striatum;LThal=lateral thalamus).
Brain Region
Figure 5-14. In situ hybridization analyses of NMDAR2B mRNA.
All data are the mean + S.E.M. Asterisk indicates
densitometric values significantly different than young
animals. (ERC=entorhinal cortex;DG= molecular layer of the
dentate gyrus;CA1 & CA3=hippocampal areas).

Figure 5-15. The distribution of mRNA specific to NMDARl
in A) 24-month-old; B) 12-month-old; and C) 6-month-old
rat brain.

90
I

Figure 5-16. The distribution of mRNA specific to: (A)
NMDARlOxx (not insertl); (B) NMDARliXx (insertl) in
6-,12- and 24-month-old rat brain.

92

Figure 5-17. The distribution of mRNA specific to (A)
NMDARlxlx (insert2) and (B) NMDARlXxl(insert3) in
6-,12- and 24-month-old rat brain.


Figure 5-18. The distribution of mRNA specific to A)
NMDAR2A and (B) NMDAR2B in 6-,12- and 24-month-old rat
brain.

96

97
Discussion
In summary, results from this study demonstrate that in
virtually all brain regions examined, mRNA for the NMDAR1
subunit was decreased in aged animals. NMDARl mRNA was also
shown to decrease in the outer and inner frontal cortex, CA3
region of the hippocampus and lateral striatum of middle-aged
animals. These data are consistent with many studies that
have shown a widespread decrease in the density of the NMDA
receptor channel complex in aged animals as well as humans
(Anderson et al., 1989; Ingram et al., 1992; Magnusson and
Cotman, 1993; Peterson and Cotman, 1989; Piggott et al.,
1992; Serra et al., 1994; Tamaru et al., 1991; Wenk et al.,
1991). A decrease in NMDA receptor density may be due to a
decrease in the mRNA coding for the main subunit of the NMDA
receptor.
The splice variants that do not contain the N-terminal
insert (NRloxx) were shown to decrease in the entorhinal
cortex (ERC) and the CA3 region of the hippocampus of aged
animals. It is possible that the decrease in [3H]MK-801
binding (Chapter 3) in the entorhinal cortex may be due to a
loss in the NMDARl subunit and in the splice variant NRloxx.
Interestingly, an increase in the density NRloxx mRNA in
the CAl region of the hippocampus in aged animals was found,
with no increase in the NMDARl mRNA. Rather, there was a
significant decrease in NMDARl mRNA in this same brain

98
region. One explanation for this observation may be that
this particular brain region is upregulating the specific
NMDARl isoform, NRloxx, to compensate for decreased levels of
NMDARl subunit in an attempt to maintain NMDA
receptor/channel neurotransmission in aged CNS. In support
of this, splice variants lacking the N-terminal insert, i.e.
NRlOxx/ exhibit a 3- to 5-fold higher affinity for agonist
when expressed in Xenopus oocytes (Durand et al., 1993). It
has been suggested that since the glutamate binding site is
presumably contained within the extracellular N-terminal
domain of the NMDA receptor (although not at the 21-amino
acid insert) the effect on agonist affinity may be the result
of a delocalized conformational change in the N-terminal
domain caused by the insert (Durand et al., 1993).
Therefore, an upregulation of this specific NMDARl isoform
may allow the aging CA1 region of the hippocampus to better
maintain an optimal degree of neurotransmission.
Alternatively, a decrease in this isoform in brain regions
such as the ERC and CA3, may lead to an enhanced deficit in
NMDA receptor-mediated neurotransmission.
A similar argument can be proposed to explain variants
of NMDARl that underwent an increase in mRNA density. The
NMDARl splice variant with the N-terminal insert (NRlixx) and
the splice variant containing the first of the two C-terminal
inserts (NRlxlx) showed a significant increase in the lateral
septum of 12-month-old rats. There were no significant
changes in the same brain region of 24-month-old rats . The

99
splice variant NRlxlx also showed an increase in the lateral
striatum (LSTR) and the lateral thalamus (LTHAL) in middle-
aged animals. It is possible that middle-aged animals are
better able to upregulate these isoforms in these brain
regions where the aged animals cannot, due to an age-related
change in some aspect of transcriptional control. However,
Worley et al. (1993) showed that identical transcription
factor responses to various stimulus patterns were observed
in 6- to 12-month-old and 23- to 26-month-old rats. They
suggested that the synaptic mechanisms involved in the
induction of various transcription factors was preserved in
aged animals. It should be noted that the stimulus
parameters that are used to induce transcription factors are
more intense than are required to produce long-lasting
synaptic enhancement (LTP) (Barnes, 1979). This suggests a
possible dissociation between the conditions sufficient for
LTP induction and those necessary for induction of
transcription factors (i.e., c-fos, c-jun, zif268). Previous
studies have demonstrated that the induction of LTP is
comparable in young and aged rats, whereas the rate of decay
of the enhanced synaptic response is accelerated in aged
animals (Barnes, 1979; deToledo-Morrell and Morrell, 1985).
Therefore, this acceleration of decay of LTP may account for
some of the age-dependent differences in this form of
synaptic plasticity.
The NMDAR1 receptor splice variants that contain the N-
terminal insert show a lower agonist affinity and little or

100
no spermine potentiation at saturating glycine concentrations
(Durand et al., 1993). A possible explanation for an
increase in NRlixx could be that the 12-month-old animals are
upregulating this isoform in specific brain regions to
enhance the NMDA receptor channel's affinity for agonist. It
has been shown that the NMDA receptor channel complex can be
formed by heteromeric as well as homomeric configurations
between the NMDARl subunit as well as its splice variants
(Monyer et al., 1992).
The stability of the mRNA levels for the NMDAR2 family
in aged animals may also represent a compensatory mechanism
to maintain the functionality of the NMDA receptor complex.
NMDAR2 family members are known to potentiate NMDA receptor
neurotransmission when expressed in heteromeric combinations
NMDARl (Monyer et al., 1992). It should be noted that NMDAR2
subunits do not form functional receptors when expressed in
homomeric combinations (Monyer et al., 1992). This may
indicate that the NMDARl subunit and its various isoforms
confer a more important functional role in NMDA receptor-
mediated neurotransmission when compared to NMDAR2 subunits.
This may explain why only changes in mRNA coding for NMDARl
and/or its splice variants were detected as a function of
age.
It should be noted that the presence of NMDARl or NMDAR2
subunit mRNA in particular CNS neurons may not necessarily
indicate NMDA receptor protein is translated. Sucher et al.
(1994) showed the presence of endogeneous NMDARl transcripts

101
without receptor protein in PC12 cells suggesting that the
expression of the NMDARl protein may be controlled by post-
transcriptional mechanisms. A study of the density of NMDARl
and NMDAR2 protein utilizing antibodies raised against the
NMDA receptor subunits is performed in Chapter 6.
In conclusion, the results of these experiments have
demonstrated changes in mRNA coding for the NMDA receptor
channel complex in the aged brain. These results, in
combination with immunocytochemical and ligand binding
studies, have contributed to further characterization of the
NMDA receptor in the aged brain.

CHAPTER 6
AGE-RELATED EFFECTS ON NMDARl AND NMDAR2A/B PROTEIN LEVELS IN
F-344 RAT BRAIN UTILIZING IMMUNOHISTOCHEMISTRY
Introduction
N-methyl-D-aspartate (NMDA) receptor-mediated
glutamatergic neurotransmission has been shown to be
important in the formation of learning and memory in animals
and humans (Danysz et al., 1988; DeToledo-Morrell et al.,
1984; Morris et al., 1986). NMDA receptor density has been
shown to undergo a significant region-specific decrease in
aged animals (Anderson et al., 1989; Ingram et al., 1992;
Magnusson and Cotman, 1993; Peterson and Cotman, 1989;
Piggott et al., 1992; Serra et al., 1994; Tamaru et al.,1991;
Wenk et al., 1991; see Chapter 3). These previous studies
examined the NMDA receptor utilizing various ligand binding
techniques. Recently, two NMDA receptor subunit families
were cloned and named NMDARl (Moriyoshi et al., 1991) and
NMDAR2 (Monyer et al., 1992). NMDARl has at least eight
alternatively spliced forms. NMDARl as well as all of its
isoforms have been shown in Xenopus oocyte expression systems
to exhibit electrophysiological and pharmacological responses
characteristic of the native NMDA receptor (Moriyoshi et al.,
1991). The four members of the NMDAR2 subunit family are
named NMDAR2A, NMDAR2B, NMDAR2C and NMDAR2D (Monyer et al..
102

103
1992). In heteromeric combinations with NMDARl or splice
variants, NMDAR2 subunits have been shown to potentiate
electrophysiological responses to agonist but are not
functional in homomeric configurations with each other.
NMDA-induced currents in Xenopus oocytes expressing NMDARl
and NMDAR2A, NMDAR2B or NMDAR2C are about 100 times larger
than homomeric NMDARl channels. NMDARl/NMDAR2 heteromers
also exhibit the following properties which are
characteristic of native NMDA receptors: 1) voltage-dependent
blockade by extracellular Mg+2; 2) high Ca+2/Na+ permeability
ratio and 3) gating kinetics that are characterized by slow
onset and offset time courses to pulses of high
concentrations of agonist (Monyer et al., 1992). These data
suggest that native NMDA receptors consist of heteromeric
configurations formed from NMDARl subunits and members of the
NMDAR2 subunit family.
Recently, polyclonal antibodies have been raised against
the carboxyterminal peptides of both NMDARl and NMDAR2. One
of the first studies to examine NMDARl protein in the rat CNS
was performed by Hennegriff et al. (1992). They raised
antibodies against the carboxyterminal region of the NMDARl
receptor cloned by Nakanishi (1991) and examined the regional
distribution of NMDARl in Western blots from seven brain
regions. NMDARl was most abundant in neocortex, followed by
hippocampus > striatum » thalamus > olfactory bulb >
cerebellum » brain stem. Petralia et al. (1994a) made a
polyclonal antiserum that recognized four of the seven splice

104
variants of NMDAR1 and showed a wide distribution in the rat
CNS. Densely stained cells included the pyramidal and hilar
neurons of the CA3 region of the hippocampus, Purkinje cells
of the cerebellum and paraventricular neurons of the
hypothalamus. Ultrastructural analyses of NMDAR1 antigen
showed labeling present in neuronal cell bodies, dendrites
and in the postsynaptic densities of synapses. This pattern
is consistent with the synthesis, processing and transport of
this protein (Petralia et al., 1994a). Staining was not seen
in the synaptic cleft. This observation supports an
intracellular location of the C-terminus for NMDARl. The
pattern of staining at the tissue level matched that of
previous in situ hybridization studies but differed somewhat
from ligand binding studies (Petralia et al., 1994a).
This same group examined the histological and
ultrastructural localization of NMDAR2A and NMDAR2B (Petralia
et al., 1994b). They utilized a polyclonal antibody to a C-
terminus peptide of NMDAR2A that recognized NMDAR2A and
NMDAR2B, and to a lesser extent, NMDAR2C and NMDAR2D.
NMDAR2A/B immunostaining was widespread throughout the rat
CNS, and the overall distribution resembled NMDARl (Petralia
et al., 1994a). Many regions contained substantial staining
with NMDAR2A/B antibody in neuropil and less staining in
neuronal cell bodies. In contrast, NMDARl immunostaining was
shown to be substantial in neuronal soma and only light to
moderate in neuropil (Petralia et al., 1994a). Dense
NMDAR2A/B staining was present in postsynaptic densities in

105
the cerebral cortex and hippocampus, similar to NMDARl.
Since functional NMDA receptors appear to require both NMDARl
and NMDAR2 subunit proteins for full function, this study
provided structural evidence for the coexistence of NMDARl
and NMDAR2 in the nervous system (Petralia et al., 1994b).
The following experiments utilized the same antisera
directed against NMDARl and NMDAR2A/B, to examine potential
changes in protein levels as a function of increasing age.
Methods
Tissue preparation
Rat brains from 6-,12- and 24-month-old F-344 rats were
sectioned on a cryostat in the horizontal plane at 30 pm.
One representative brain section from each age group was
mounted per slide with group n=5. See Chapter 2 for a more
detailed description of the tissue preparation.
Antibody Specificity
See Chapter 2 for a detailed description of antibody
specificity. AB1516 (antisera raised against a synthetic
peptide corresponding to the C-terminus of rat NMDA receptor
subunit) is selective for splice variants NRl-la,lb,2a,2b
(Hollmann et al., 1993). These appear to be the major splice
variants expressed in rat brain (Hollmann et al., 1993). It
was shown that there was no cross-reaction with other
glutamate receptor subunits and the antisera labels a single
band corresponding to NMDARl in Western blots (Petralia et

106
al., 1994a). AB1548 (antisera raised against a synthetic
peptide corresponding to the C-terminus of rat NMDAR2A
receptor subunit) recognizes both NMDAR2A and NMDAR2B
subunits equally (Petralia et al., 1994b). This antisera
labels a single band corresponding to NMDAR2A and NMDAR2B.
No cross reactivity with NMDARl or other glutamate receptor
subunits was noted (Petralia et al., 1994b).
Immunocytochemical Procedure
See Chapter 2 for a detailed description of this
procedure.
Data Analysis
Brain regions analyzed included the outer frontal cortex
(OFCTX), inner frontal cortex (IFCTX), lateral septum (LSEP),
lateral striatum (LSTR), lateral thalamus, entorhinal cortex
(ERC), dentate gyrus (DG) and hippocampal areas CAl and CA3.
Brain regions were compared across varying ages and the
relative density of immunostaining was determined by computer
assisted densitometry with a Microcomputer Imaging Device,
Imaging Research, Inc.(MCID) image processing system. A
semi-quantitative measurement of NMDARl and NMDAR2A/B
immunostaining levels, represented as raw optical densities
(ROD), from twelve- and twenty-four-month-old animals in each
brain region were normalized with the values obtained for
young animals. To reduce variability, all three ages were
mounted on one slide with the investigator blind to age.

107
Statistical Analysis
Five blocks of animals, each block consisting of an
animal from each age group, were assessed. A different block
of 3 animals, 1 per age group, was assessed on each of 5
experimental days. The experimental design consisted of a
within-subject factor with 9 levels (brain region), a
quantitative between-subject fixed factor with 3 levels
(age), and a between subject random blocking factor with 5
levels (experimental day). Repeated measures analysis of
variance (ANOVA) with between-subject randomized blocks and
age considered as a linear covariate were used to model the
NMDARl and NMDAR2A/B immunostaining density data as a linear
function of age within each brain region and to determine if
this pattern differed significantly among brain regions. The
change in NMDARl and NMDAR2A/B immunostaining densities per
month of age (i.e. the linear slope) was estimated and
assessed for statistical significance within each brain
region while blocking on experimental day. A general
comparison of these slopes among brain regions was then
performed by testing for the presence of a significant
interaction between age and brain region NMDARl and NMDAR2A/B
immunostaining effects. Mauchly's sphericity test was used
to determine if a multivariate F test (Wilks' Lambda) or a
univariate F test should be used for the age x brain region
interaction test. In assessing the validity of ANOVA
assumptions, it was noted that experimental day age profiles

108
were relatively parallel within each brain region and that
between-subject variability as estimated by the standard
deviation was similar among age groups within each brain
region. Plots of residuals versus predicted values, residual
histograms, and residual normal probability plots were
examined to assess goodness of fit in the ANOVA models.
Results
The pattern of immunostaining obtained for both antisera
was consistent with previous publications (Petralia et al.,
1994a,b). Immunostaining for NMDAR1 was widespread with
moderate to high levels present in the OFCTX and IFCTX, ERC,
hippocampus, LSTR, LSEP and LTHAL. This widespread
expression of NMDARl protein matches closely with the
distribution of NMDARl mRNA (see Chapter 5). There was also
widespread expression of NMDAR2A/B protein that mirrored the
distribution of NMDARl protein (Figure 6-5). This finding is
consistent with the fact that native NMDA receptors appear to
require both NMDARl and NMDAR2A/B subunit proteins for full
function (Monyer et al., 1992; Nakanishi 1992). There were
no significant age-related differences in NMDARl or NMDAR2A/B
immunostaining levels in any of the brain regions analyzed
(Figures 6-1 through 6-4).

109
4 middle-aged
â–¡ aged
*- significant
at 95%
Brain Region
Figure 6-1. Cortical and subcortical brain regions that did
not undergo a significant age-related decrease in NMDARl
antibody staining density. (OFCtx= outer frontal cortex;
IFCtx= inner frontal cortex; LSep= lateral septum; LStr=
lateral striatum; LThal= lateral thalamus). Repeated
measures ANOVA with between subject randomized blocks and age
as a linear covariate. Data are presented as a percentage of
young (6-month-old) animals.
NMDARl (AB1516)
Brain Region
Figure 6-2. Hippocampal and entorhinal cortical brain regions
that did not undergo a significant age-related decrease in
NMDARl antibody staining density. ( ERC= entorhinal cortex;
DG= molecular layer of the dentate gyrus; CA1= hippocampal
area CA1; CA3= hippocampal area CA3). Repeated measures
ANOVA with between subject randomized blocks and age as a
linear covariate. Data are presented as a percentage of
young (6-month-old) animals.

110
uuniDíit/n /aniRAQ\
OFCtx IFCtx LSep LStr LThal
-i middle-aged
D aged
*- significant
at 95%
Brain Region
Figure 6-3. Cortical and subcortical brain regions that did
not undergo a significant age-related decrease in NMDAR2A/B
antibody staining density. (OFCtx= outer frontal cortex;
IFCtx= inner frontal cortex; LSep= lateral septum; LStr=
lateral striatum; LThal= lateral thalamus). Repeated
measures ANOVA with between subject randomized blocks and age
as a linear covariate. Data are presented as a percentage of
young (6-month-old) animals.
¿S middle-aged
â–¡ aged
*- significant
at 95%
Brain Region
Figure 6-4. Hippocampal and entorhinal cortical brain regions
that did not undergo a significant age-related decrease in
NMDAR2A/B antibody staining density. (ERC= entorhinal
cortex; DG= molecular layer of the dentate gyrus; CA1=
hippocampal area CAl; CA3= hippocampal area CA3). Repeated
measures ANOVA with between subject randomized blocks and age
as a linear covariate. Data are presented as a percentage of
young (6-month-old) animals.

Figure 6-5. Representative brain section showing
A) NMDAR1 (AB1516) immunostaining ; and B) NMDAR2A/B
(AB1548) immunostaining in the hippocampus. Note the
identical distribution of both antisera. This over¬
lapping distribution was seen in all brain regions
analyzed.

112
A

113
Discussion
A significant age-related decrease in mRNA levels coding
for the NMDARl subunit of the NMDA receptor was demonstrated
in Chapter 5. However, no concomitant decrease in NMDAR1
protein immunostaining was found. This discrepancy may be
because relative levels of mRNA for an NMDA receptor subunit
in a neuron may not always coincide with expression of the
protein. For example, a recent study using Northern blots
demonstrated that mRNA for the NMDAR1 subunit is abundantly
expressed in PC12 cells yet, no functional NMDA-operated
channels were found (Sucher et al., 1993). Furthermore,
immunodetection with a monoclonal antibody indicated the
presence of only trace amounts of NMDARl protein.
Post-transcriptional mechanisms may be important in the
expression of NMDARl protein. It was shown that there is a
significant decrease in NMDARl mRNA levels in specific brain
regions of aging rats (Chapter 5) without a loss of NMDARl
protein. This may be due, in part, to alterations in some
aspect of post-transcriptional mechanisms as a function of
age. The thresholds for synaptic activation of transcription
factors in the hippocampus and their correlation with long¬
term potentiation (an in vitro model of memory function) has
been described by Worley et al. (1993). The transcription
factor responses to two stimulus patterns were shown to be
blocked by the noncompetitive NMDA receptor antagonist MK-
801. There were identical transcription factor responses

114
observed in adult (6- to 12-month-old) and aged (23 to 26-
month-old) Fischer 344 rats. This study therefore suggested
that the synaptic mechanisms involved in these responses are
preserved in aged animals. Therefore, the discrepancies seen
between NMDARl mRNA levels and NMDARl protein levels may be
due to differences in post-transcriptional or translational
mechanisms.
The cellular control of NMDARl translation into protein
and the subsequent expression of functional NMDA-gated
channels could occur at several levels. Several studies have
suggested that post-transcriptional control of NMDA receptor
expression occurs in the CNS. Adult rat cerebellar Purkinje
cells were shown to express NMDARl mRNA (Moriyoshi et al.,
1991), however functional NMDA receptor channels were only
demonstrated during early stages of development (Rosenmund et
al., 1992). In the peripheral nervous system, NMDARl mRNA
was shown to be expressed in adult dorsal root ganglia with
erratic expression of functional NMDA receptors in dorsal
root ganglion cells (Huettner 1990).
It can be speculated that there may be an upregulation
of translational activity in aged rats to compensate for
their decreased levels of NMDARl mRNA. This upregulation may
explain why there is no age-dependent difference in NMDARl
protein levels. Alternatively, NMDARl protein may be subject
to modification or decreased degradation due to post-
translational mechanisms.

115
A significant decrease in NMDA receptors in the
entorhinal cortex, lateral striatum and inner frontal cortex,
utilizing [3H]MK-801 binding assays, was shown in Chapter 3.
However, no change in NMDARl immunoreactivity was seen these
same brain regions. One possibility for this discrepancy may
be the presence of many receptor molecules in the cytoplasm
of cells that bear few receptor molecules on their plasma
membrane (Petralia et al., 1994a). Only those NMDA receptor
molecules on the plasma membrane, exposed to extracellular
ligands, would be able to be identified in ligand binding
studies.
No age-related differences in NMDARl or NMDAR2A/B
protein levels in the rat CNS suggests that the function of
the NMDA receptor, rather than the number of binding sites
may be responsible for age-related cognitive and memory
deficits. In support of this idea, there is a significant
age-related decrease in the efficacy with which L-glutamate
can maximally enhance [3H]MK-801 binding (Chapter 4). Others
have shown that NMDA mediated responses decrease with age in
the hippocampus, cortex and striatum (Gonzales et al., 1991).
It is important to note that gene expression can be
regulated at each step in the pathway from DNA to RNA to
protein (Alberts et al., 1989). Thus, a cell can control the
proteins it makes by 1) controlling when and how often a
given gene is transcribed (transcriptional control), 2)
controlling how the primary RNA transcript is spliced or
otherwise processed (RNA processing control), 3) selecting

116
which completed mRNAs in the cell nucleus are exported to the
cytoplasm (RNA transport control), 4) selecting which mRNAs
in the cytoplasm are translated by ribosomes (translational
control), 5) selectively destabilizing certain mRNA molecules
in the cytoplasm (mRNA degradation control), or 6)
selectively activating, inactivating, or compartmentalizing
specific protein molecules after they have been made (protein
activity control) (Alberts et al., 1989). There may be age-
related changes occurring at one or more of these
aforementioned steps leading to differences in the production
of fully functional NMDA receptor/channel complexes.
Therefore, when examining age-related changes that occur
in the NMDA receptor, [3H]MK-801 binding analyses may be a
more accurate method than in situ hybridization (NMDA mRNA)
or immunostaining (NMDA antibodies) studies. [3H]MK-801
binding analyses (Chapter 3 and 4) labels NMDA receptors that
are in the open or activated state ('functional').
Therefore, [3H]MK-801 binding may more accurately determine
the number of NMDA receptors that are responsible for NMDA
receptor-mediated neurotransmission as a function of age.
In conclusion, age-related cognitive and memory changes
attributed to the NMDA receptor channel/complex may be the
result of both a decreased number of available NMDA receptors
as well as a decreased functional capacity of the remaining
NMDA receptors.

CHAPTER 7
SUMMARY AND DISCUSSION
Research Summary
A significant amount of evidence has accumulated
suggesting that a reduction in NMDA receptor-mediated
neurotransmission may underlie age-related changes in several
forms of neuronal plasticity, including spatial learning
(Morris et al., 1986) and long-term potentiation
(Collingridge and Bliss, 1987; Danysz et al., 1988). One
means by which NMDA neurotransmission could be reduced in
aged animals is by an alteration in the NMDA receptor/channel
complex itself.
The central hypothesis tested in this research was that
there are measurable, anatomically specific aging-related
changes in the NMDA receptor and its individual subunits.
Within the framework of this general hypothesis are the
following specific hypotheses which were all critically
addressed in specific chapters:
Hypothesis #1 (Chapter 3): There are age-related
differences in the density of the NMDA receptor in brains of
6-, 12- and 24-month-old F-344 rats. In vitro quantitative
autoradiography was performed on young, middle-aged and aged
F-344 rat brains using [3h]MK-801.
117

118
Results: The entorhinal cortex (ERC), lateral striatum
(LSTR) and the inner frontal cortex (IFCTX) showed a
significant decrease in [3H]MK-801 binding in aged animals
when compared to young.
Summary: The ERC, IFCTX, and LSTR are brain regions that
appear to be among the most vulnerable to the effects of
aging (Chapter 3; Magnusson and Cotman, 1993; Serra et al.,
1994). These regions are implicated in normal memory
function and plasticity of the CNS (DeToledo-Morrell et al.,
1984; Gonzales et al., 1991; Petit, 1988). A decrease in
NMDA receptors in these specific regions, as a function of
age, suggests that some of the cellular mechanisms encoding
memory may be impaired.
Hypothesis #2 (Chapter 4): There are age-related
differences in the ability of L-glutamate to enhance [3H]MK-
801 binding to NMDA receptor channels in brains of 6-, 12-
and 24-month-old F-344 rats. Various concentrations of L-
glutamate were used to stimulate MK-801 binding within the
NMDA receptor/channel complex. The ability of L-glutamate to
maximally enhance [3H]MK-801 binding was determined as a
function of age. Age-related comparisons were made between
dose-response curve generated Emax and EC50 values.
Results: All brain regions analyzed (i.e., OFCTX, IFCTX,
ERC, LSTR, LTHAL, LSEP, DG, and CAl) showed an age-dependent
decrease in their Emax values without any change in EC50
values. The ERC, IFCTX, and LSTR showed the greatest percent
decrease in Emax values.

119
Summary: The decrease in L-glutamate's efficacy in
stimulating [3h]MK-801 binding may account for some of the
age-related decreases in learning and memory processes in the
CNS. Down-regulation of NMDA-receptor mediated
neurotransmission results in a decrease in responsiveness of
the NMDA receptor. This down-regulation may be a
compensatory mechanism that ultimately compromises NMDA
receptor function yet may provide protection from potential
excitotoxic processes.
Hypothesis #3 (Chapter 5): There are selective changes
in mRNA coding for subunits of the NMDA receptor in the
brains of aged F-344 rats. In situ hybridization analyses
were performed to determine the density of individual mRNA
species coding for NMDA subunits. The density and anatomical
distribution of the mRNA for the various subunits (i.e.
NMDARl, NMDAR2A, NMDAR2B, NMDAR2C, NMDAR2D and four
alternatively spliced versions of NMDARl) were determined
using brains from young (6-month), middle-aged (12-month) and
aged (24-month) F-344 male rats. The density of each subunit
mRNA was evaluated as a function of increasing age.
Results: NMDARl mRNA levels decreased in all brain
regions analyzed (i.e. OFCTX, IFCTX, ERC, LSTR, LTHAL, LSEP,
DG, CAl and CA3) as a function of age. The NMDARl splice
variant NRloxx showed an age-dependent decrease in the ERC
and the CA3 region of the hippocampus in 24-month-old rats.
There was an increase in this same isoform in 12-month-old
hippocampal area CAl. NRlixx mRNA levels increased in 12-

120
month-old animals' LSEP. NRlxix mRNA levels increased in 12-
month-old rats' LSEP, LSTR and LTHAL. The remaining NMDAR1
subunit mRNA levels did not change as a function of age.
Summary: NMDA receptor subtypes with distinct properties
may subserve different functional assignments in neurons
(Monyer et al., 1994). One likely molecular mechanism for
functional changes may be the regulation of subunit
expression. A progressive age-dependent increase in one NMDA
receptor subunit's expression with a concomitant decline in
the expression of another subunit may account for
modifications in NMDA receptor activity and vice-versa.
Changes in the levels of NMDA subunit mRNA expression may be
controlled by age-dependent genetic programs that ultimately
effect the make-up of the NMDA receptor/channel. Changes in
mRNA may therefore account for some of the deficits in NMDA
receptor-mediated learning and memory processes as a function
of age.
Hypothesis #4 (Chapter 6): There are specific age-
related changes in NMDARl and NMDAR2A/B protein density
levels in the rat CNS. Immunocytochemistry determined the
localization of the NMDA receptor protein within each neuron
in each region of interest in the rat CNS. Any potential
changes in NMDARl and/or NMDAR2A/B protein density levels
associated with increasing age were semi-quantitated using
the currently available polyclonal antibodies.

121
Results: There were no significant age-related
differences seen in NMDAR1 or NMDAR2A/B immunostaining levels
in any of the brain regions analyzed.
Summary: There may be a significant amount of non¬
membrane bound (cytosolic) NMDA receptor protein detected by
immunostaining that is not involved in the formation of
heteromeric NMDA receptor/channel complexes. Only surface-
presented NMDA receptors would be directly involved in NMDA
receptor-mediated neurotransmission. There may be an
overproduction of NMDA receptor protein in the cytoplasm that
do not form functional complexes on the membrane surface.
Therefore, NMDARl and NMDAR2A/B immunohistochemistry may not
have selectively determined the number of NMDA receptors that
are directly involved in NMDA-mediated neurotranmission
processes.
Discussion
Taken together, these results indicate that there were
measurable, anatomically specific aging-related changes in
the NMDA receptor and its individual subunits. The decrease
in [3h]MK-801 binding confirmed that there are age-dependent
changes in the number of NMDA receptors in the aged rat
brain. These differences may be attributable, in part, to
selective changes in specific mRNA coding for the NMDA
receptor as seen in Chapter 5. The decrease in Emax value
seen throughout the CNS of 12- and 24-month-old rats suggests
that there are some differences in the efficacy of L-

122
glutamate to enhance [3H]MK-801 binding to the NMDA
receptor/channel complex in aged rats.
A decrease in cognitive processes will occur in a
significant number of aged individuals (65 years of age or
older). Aged men and women are estimated to comprise 12.7%
of the total United States population and 18.4% of Florida's
population (U.S. Bureau of the Census, Wash., D.C. 1995).
Because of their increasing numbers, a considerable amount of
emphasis should be placed on improving the overall quality of
life for elderly individuals. Possible areas of improvement
must focus on the documented cognitive deficits associated
with advancing age (Barnes, 1979; DeToledo-Morrell et al.,
1984). Specific targeting of the NMDA receptor using drugs
that interact directly with this receptor's binding and
modulatory sites has begun, and cognitive improvements have
been seen in aged individuals (Cohen and Muller, 1992; Davis
et al., 1993; Fiore and Rampello, 1989) (see Chapter 3
discussion). The data obtained from NMDA mRNA analyses may,
in the future, allow researchers to genetically manipulate
the NMDA receptor. The use of antisense oligodeoxy-
nucleotides (ODNs) specific to NMDA receptor subunits may be
useful tools for the dissection of the participation of
specific molecules in the expression of functional NMDA
receptors. The antisense ODNs would suppress translation of
specific neuronal molecules and their effect on NMDA receptor
function could be examined as a function of age. The
characterization of the NMDA receptor using ligand binding

123
assays (Chapter 3 and 4), in situ hybridization (Chapter 5)
and immunohistochemistry (Chapter 6) may help to pinpoint the
changes occurring within this receptor/channel complex as a
function of age. The elucidation of NMDA receptor-specific
changes found in these experiments may help to explain the
NMDA receptor's involvement in learning and memory deficits
associated with advancing age.

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BIOGRAPHICAL SKETCH
Josephine "Jean" Mitchell was born on January 17,1964 in
Shalimar, Florida at Eglin Air Force Base where her family
was stationed while her father was an active duty
officer/pilot in the USAF. Jean's family moved to several
places during her childhood, including Turkey, Ohio and
Pennsylvania and then settled in Shalimar, Florida where her
father retired from active duty. Jean attended Meigs Junior
High School and Choctawhatchee Senior High School where she
was a member of the jazz and marching bands and the golf
team. She graduated in 1982 with honors. It was in Shalimar
that Jean met Thomas "Mitch" Mitchell and later married in
June of 1985. Jean attended Ocean County College in Tom's
River, New Jersey, and graduated with an associate of arts
degree in 1985. Jean and Mitch then moved to Gainesville,
Florida where she attended the University of Florida and
graduated in 1987 with a bachelor's degree in zoology. In
1989, Jean began work in Kevin J. Anderson's laboratory as a
technician and became guite interested in the field of
neuroscience and therefore entered the Neuroscience
Department at the University of Florida College of Medicine
in 1990 to pursue her doctoral studies.
136

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
/
/!.,//
idetscrrfc
'M+ _ .
Kevin J / Anders^rrv Chair
Associate Professor of
Neuroscience and
Physiological Sciences
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
A. John MacLennan
Assistant Professor of
Neuroscience
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
^Wtvvvy^- rJLUV-
Joanna Peris
Associate Professor of
Pharmacodynamics
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Thomas Vickroy
Associate Professor1^
of Veterinary Medicine

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
CO ' 0 ¿¿*4^—-
Don Walker
Professor of Neuroscience
This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate School and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
December 1995
Dean,'College of Medicine
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08554 3402




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