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Loss of NMDA, but not GABA-A, binding in the brains of aged rats and monkeys
Neurobiologyof Aging. Vol. 12. pp. 93-98. ©PergamonPress plc, 1991. Printedin the U.S.A.
0197-4580/91 $3.00 + .00
Loss of NMDA, But Not GABA-A, Binding in the Brains of Aged Rats and Monkeys G A R Y L. W E N K , *l L A R Y C. W A L K E R , ? D O N A L D L. PRICE~: A N D L I N D A C. C O R K §
*Neuromnemonics Laboratory, Department of Psychology The Johns Hopkins University, Baltimore, MD 21218 "~Neuropathology Laboratory, Department of Pathology The Johns Hopkins University School of Medicine, Baltimore, MD 21205 ~:Neuropathology Laboratory, Departments of Pathology, Neurology and Neuroscience The Johns Hopkins University School of Medicine, Baltimore, MD 21205 #Division of Comparative Medicine and Department of Pathology The Johns Hopkins UniversiO, School of Medicine, Baltimore, MD 21205 Received 20 October 1989; Accepted 10 October 1990
WENK, G. L., L. C. WALKER, D. L. PRICE AND L. C. CORK. Loss of NMDA, but not GABA-A,bindingin the brainsof aged rats' and monkeys. NEUROBIOL AGING 12(2) 93-98, 1991.--In this quantitative neurochemical study we investigated age-related changes in the GABAergic, glutamatergic, and cholinergic neurotransmitter systems in rats and rhesus monkeys. Sixteen young (5 months) and 20 aged (24 months) rats and seven "young" (4-9 years), six "adult" (20-25 years), and five "aged" (29-34 years) monkeys were studied. NMDA-displaceable l-[3H]glutamate binding was significantly decreased in many neocortical and subcortical regions examined in aged rats and monkeys. The level of choline acetyltransferase (CHAT) activity and [3H]muscimol binding were unchanged in aged animals. Acetylcholine
Glutamate
NMDA
GABA
Receptors
GLUTAMATE and gamma-aminobutyric acid (GABA) are major excitatory and inhibitory neurotransmitters, respectively, in the central nervous system, l-Glutamate is the principal neurotransmitter of the corticocortical associational pathways, and several hippocampal pathways, including the entorhinal, intrahippocampal, and hippocampal efferent pathways (16,35). N-methyl-D-aspartate (NMDA) selectively stimulates one class of glutamate receptors that mediates excitatory synaptic transmission within these pathways (61). NMDA receptors may play a critical role in many neurophysiological processes that underlie neuronal plasticity, long-term potentiation, and learning and memory (15, 29, 39, 42). GABA is a neurotransmitter in neurons of many regions of brain, including a population of intrinsic neocortical interneurons (31). Most neocortical GABA is located within intrinsic interneurons where it is frequently colocalized with specific neuropeptides (36), although a small amount of GABA may arise from subcortical neurons (23, 24, 58, 60). GABAergic neurons regulate the activity of central noradrenergic, serotonergic, dopaminergic and acetylcholinergic neurons (37, 38, 56, 62, 68) and therefore may influence many different cognitive processes and behaviors. The GABA-A receptor subtype is a major site for the inhibitory activities of GABA; this receptor is present on the ma-
Aging
Rats
Monkeys
jority of cerebral neurons in mammals (3) and is the site of action for the benzodiazepines and barbiturates (3,12). In aged rats, as compared to young controls, the concentration of glutamate in the brain (30,57), high affinity glutamate uptake in the striatum (52) and cortex (66), and the conversion of radioactively labeled glucose into glutamate (57) were decreased. In contrast, the total number of [3H]glutamate binding sites was increased in the brains of aged rats (7) and monkeys (65). The present study was designed to further investigate the nature of these changes by examination of the specific NMDA-sensitive glutamate binding site. Evidence for age-related changes in GABAergic systems is contradictory. In aging rats, the concentration of GABA was either slightly increased (30) or unchanged, the number of GABAA receptors was unchanged (12, 26, 40, 45), and the activity of glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA, was either unchanged (32) or decreased (33). In aging humans, the number of GABA receptors increased, the density of GABAergic terminals decreased, the clinical response to benzodiazepines was enhanced (2,21), and GAD activity was either decreased in selected regions (43,44) or unchanged (10). The level of choline acetyltransferase (CHAT) activity, a specific biochemical marker for cholinergic neurons, was unchanged
tRequests for reprints should be addressed to Gary L. Wenk at his present address: Division of Neural Systems, Memory and Aging, 384 Life Sciences North, University of Arizona, Tucson, AZ 85724.
93
94
WENK, WALKER, PRICE AND CORK
NMDA Receptors 700.
TABLE 1
,J "
I~
I
II
* p
/
II
5 months
I
-L
F oo
0 Ant.Ct
Post.Ct
Hippo
Cerebel
Coud
[3H]Muscimol Binding
80-
['-7 5 months B B 24. months
70-
"i 6030-
NMDA-DISPLACEABLEL-[3H]GLUTAMATEBINDING SITES§ IN MONKEYBRAIN Brain Region Gyrus Rectus Dorsal Fr. Pole Ant. Sup. Cing. Gyr. Superior Fr. Gyr. Middle Fr. Gyr. Inf. Fr. Gyr. Medial Orbital Gyr. Ant. Inf. Cing. Gyr. Precentral Gyr. Postcentral Gyr. Inf. Parietal Lobule Sup. Temporal Gyr. Middle Temporal Gyr. Parahippocampal Gyr. Hippocampus Dorsal Occipital Pole
4-9 Years
20-25 Years
29-34 Years
211.5 ~ 26.9 180.1 ± 37.6 206.5 ± 56.8 162.4 _+ 19.9 168.1 _+ 12.0 139.1 + 22.9 121.1 _+ 21.8 189.1 _+ 58.0 155.4 -+- 22.3 124.8 _+ 26.7 126.1 _+ 38.6 206.0 -+ 48.2 221.5 -+ 31.2 212.7 ___ 74.7 146.7 + 32.2 175.4 + 30.1
269.2 ___ 33.9 306.2 __+ 57.4 260.2 + 63.2 257.9 __. 33.8 205.9 _ 51.1 324.3 -+ 39.1 368.4 + 63.1 150.6 _+ 59.0 187.9 ± 37.6 230.9 ___ 15.4 183.4 _+_ 31.0 325.6 -+ 67.0 276.5 _+ 31.8 284.6 -- 44.0 436.9 _+ 39.7 338.5 _.+ 47.7
131.4 _+ 8.5* 153.8 ± 35.8 122.3 - 22.7 138.9 _+ 33.7* 61.1 _+ 15.55 80.6 -+ 9.1" 51.6 _+ 6.6t 48.3 --- 21.3 128.9 - 41.6 90.3 _+ 18.8t 93.2 --- 26.8 112.1 -+ 17.7:~ 146.1 ~ 32.5* 68.7 _+ 8.6* 70.3 _+ 20.8# 167.7 _ 30.2t
50-
*p<0.05, +p<0.01 by ANOVA. $p<0.05 vs. "Adult" levels, by t-test. Each value represents the mean - S.E.M. of seven young (4-9 years), six adult (20-25 years), and five aged (29-34 years) rhesus monkeys. §fmol/mg protein.
40-
E
20100 Ant.Ct
Post.Ct
Hippo
Cerebel (xl0)
Coud
ChATActivity 5O
I-'-I 5 months B B 24 months
40
~
3o
o.
: Ant.Ct
:All Post.Ct
Hippo
Cerebel
fll
(,lo)
Coud
FIG. 1. The number of NMDA-displaceable 1-[3H]glutamate and [3H]muscimol binding sites, and the level of ChAT activity in five brain regions of rats. Each value represents the mean--+S.E.M. of 16 young (5 months) or 20 aged (24 months) Fisher 344 rats. *Indicates a significant difference, p<0.05, as compared to levels in young rats. ( x 10) Indicates that the unit of measurement for this region should be multiplied by 10. Ant.C, sensory-motor cortex; Post.Ct, parietal-occipital cortex; Hippo, hippocampus; Cerebel, cerebellum; Caud, caudate nucleus.
basalis of Meynert was not changed (14). The level of ChAT activity is usually unchanged in the brains of healthy elderly humans (5). Experimental destruction of basal forebrain cholinergic cells in rats and monkeys produces memory deficits similar to those associated with normal aging and Alzheimer's disease (AD) (1, 34, 53, 54, 64). In AD, the amount of glutamate and GABA, the number of glutamate terminals (17,28), and various biochemical markers of cholinergic function (18) are decreased in many cortical and subcortical brain regions. The decline in cholinergic function in AD correlates somewhat with the degeneration of acetylcholine-containing cells in the basal forebrain, the density of neuropathological changes in the cortex and hippocampus, and the degree of dementia (9,64). An investigation of age-associated changes in glutamatergic and GABAergic systems in nonhuman primates would provide valuable information that cannot be reliably obtained from aged humans. In humans, extensive postmortem delays and antemortem treatments with various pharmacotherapies can significantly alter many neurochemical markers. The present study investigated ChAT activity, as a measure of the integrity of cholinergic presynaptic terminals, and NMDA-type glutamate and bicuculline-sensitive GABA-A binding in the brains of young and aged rats and monkeys. If neurons giving rise to long corticocortical pathways are most vulnerable to aging, then there should be a decline in glutamatergic, but not GABAergic, markers in the neocortex of aged animals. METHOD
throughout the neocortex and hippocampus in aged rats (19,22) and decreased only in the frontal pole and motor cortex of aged monkeys (65). The cholinergic cells in the basal forebrain that project to these cortical regions may decrease in size and number with aging in rats (22); measures of neuronal size have not been reported in aged humans, but the number of cells in the nucleus
Subjects The subjects were 16 young (5 months) and 20 aged (24 months) male F344 rats. Each rat was housed in a metal hanging cage and maintained on a 16:8 h light/dark cycle with lights on at 07:00 h. Eighteen rhesus monkeys (Macaca mulatta) were studied, in-
NMDA BINDING AND AGING
95
TABLE 2 ChAT ACTIVITY* IN MONKEY BRAIN
Brain Region Gyrus Rectus Dorsal Fr. Pole Ant. Sup. Cing. Gyr. Superior Fr. Gyr. Middle Ft. Gyr. Inf. Fr. Gyr. Medial OrbitalGyr. Ant. Inf. Cing. Gyr. Caudate Nuc., Head Rostral Putamen Precentral Gyr. Postcentral Gyr. Inf. Parietal Lobule Sup. Temporal Gyr. Middle Temporal Gyr. Parahippocampal Gyr. Hippocampus Dorsal Occipital Pole
4-9 Years 8.71 8.89 12.2 7.64 10.7 9.60 11.6 14.4 112.8 141.5 11.7 8.27 9.26 10.3 7.98 13.9 20.9 5.26
± 0.84 ± 1.10 ± 2.2 ± 1.51 ± 1.9 _+ 1.50 --- 1.8 ± 2.6 ± 11.5 ± 24.5 ± 1.4 _+ 0.86 ± 1.42 --- 0.6 ± 0.71 ± 1.6 ± 1.9 ± 0.79
20-25 Years 10.7 8.08 17.2 10.9 11.8 12.9 13.6 14.9 140.1 232.9 13.5 10.8 11.5 9.04 6.70 12.7 20.1 6.01
± 0.7 ± 0.52 ± 0.6 ± 0.4 ± 0.7 z 1.7 ± 1.1 +-- 2.8 +_. 43.1 + 85.3 --- 1.4 --- 0.6 ± 0.9 ± 0.59 ± 0.26 ± 2.0 ± 1.7 -+ 0.50
29-34 Years 7.61 8.48 11.0 9.13 7.73 7.26 14.1 13.9 76.2 150.1 8.45 7.87 8.69 9.01 6.89 12.8 20.7 3.53
+-- 0.42 ± 1.18 -4- 0.7 ± 2.19 ± 1.32 ± 0.84 ± 2.3 ± 1.4 ± 12.2 --- 26.1 --- 0.49 - 0.91 ± 0.67 ± 1.35 -+ 0.52 ± 3.4 ± 4.2 ± 0.77
Each value represents the mean ± S.E.M. of seven young (4-9 years), six adult (20-25 years), and five aged (29-34 years) rhesus monkeys. *nmol acetylcholine/mg protein/h.
cluding seven " y o u n g " (4-9 years), six " a d u l t " (20-25 years), and five " a g e d " monkeys (29-34 years). All of the young and adult monkeys, and two of the aged monkeys, had known birthdates. The ages of the other two aged monkeys were minimum estimates based on their years in captivity and their sexual maturity when captured. Because rhesus monkeys reach sexual maturity between 3--4 years of age (3-4 kg body weight), animals weighing more than 3 - 4 kg, or which were sexually mature at capture, were assumed to be at least 3 years of age.
Tissue Preparation Each rat was sacrificed by decapitation (between 09:00 and 11:00 h) and the brain was quickly removed and dissected on ice. Five areas were sampled from each rat brain, including the anterior (sensory-motor) and posterior (parietal-occipital) cerebral cortex, the entire hippocampus, caudate nucleus and cerebellum. Each monkey was restrained with ketamine, euthanized with a lethal dose of sodium pentobarbital, and then perfused transcardially for 3-5 minutes with cold (4°C) isotonic saline. The brain was quickly blocked stereotaxically in a cold room (4°C) and removed; the blocks were frozen on dry ice and stored at - 8 0 ° C .
TABLE 3 [3H]MUSCIMOL BINDING SITES* IN MONKEY BRAIN
Brain Region Frontal Pole Middle Temporal Gyrus Occipital Pole Cerebellum
4-6 Years 171.1 103.1 147.6 278.5
_± 17.4 ± 27.7 --. 23.5 ± 39.9
31-34 Years 145.1 125.1 147.5 250.2
± 17.7 +-- 23.7 ± 20.7 ± 57.8
Each value represents the mean __. S.E.M. of 4 young (4-6 years) or 4 aged (31-34 years) rhesus monkeys. *fmol/mg protein.
Samples of specific regions were later taken from the frozen blocks at - 2 0 ° C and then returned to - 8 0 ° C until assayed. Each monkey had a complete autopsy after tissues were taken for analysis. No significant pathological c h a n g e s , e . g . , brain tumors, cerebrovascular or metabolic diseases, were found that might affect the interpretation of the neurochemical data. Tissues from all rats and monkeys were processed identically. Each sample was homogenized in 0.32 M sucrose and stored at - 8 0 ° C until assayed. Repeated freeze-thawing of the tissues does not inactivate, and may improve, the NMDA-sensitive 1[3H]glutamate (50) and [3H]muscimol (49) binding. For these binding assays, the membranes were extensively washed in order to remove any endogenous ligands present in the tissue. In order to perform all three biochemical analyses on each small tissue sample, only single-point determinations were made for the two receptor sites. Therefore, changes in binding could reflect either a change in the number and/or affinity of the receptors. Proteins were measured by standard methods (41).
GABA Receptor Binding Studies [3H]Muscimol binding to the bicuculline-sensitive G A B A - A binding site was performed according to the method of Williams and Risley (67). The incubation was conducted in a sodium-free medium in order to inactivate high affinity G A B A uptake sites. Membrane preparations were incubated in 50 mM Tris-citrate buffer (pH 7.1) with [3H]muscimol (2 nM) in the presence or absence of GABA (10 txM) to define nonspecific binding.
NMDA Receptor Binding Studies NMDA-displaceable 1-[3H]glutamate binding sites were defined according to a modified method of Monahan and Michel (46). These sites are probably associated with postsynaptic, Na+-independent, NMDA-type glutamate binding sites (8,46). The tissue preparation has been described previously (63). Mem-
96
WENK, WALKER, PRICE AND CORK
brane preparations were incubated in 50 mM Tris-acetate buffer (pH 7.4) with l-[3H]-glutamate (10 nM) in the presence or absence of NMDA (0.5 raM) to define nonspecific binding.
ChAT Activity' A 40-1xl sucrose aliquot was combined with 10 ~1 of a solution containing 2% Triton X-100 and 50 mM EDTA (pH 7.4). ChAT activity was determined (25) using [~4C]acetyl Coenzyme-A. RESULTS NMDA-displaceable 1-[3H]glutamate binding was decreased in many brain regions in aged rats, and particularly in the frontal and temporal lobes of the aged monkeys (see Fig. 1 and Table 1). NMDA binding tended to increase, but not significantly, between ages 4-25 years; in the middle frontal gyrus and superior temporal gyrus the decline in NMDA binding sites was only significant as compared to these elevated levels. [3H]Muscimol binding and the level of ChAT activity in rats and monkeys did not change with age (see Fig. 1 and Tables 2 and 3). DISCUSSION The primary finding of this investigation was a widespread decline in NMDA-displaceable 1-[3H]glutamate binding in the brains of aged rats and monkeys. The decline in NMDA binding in the aged rat and monkey brain is similar to that reported in aged humans with AD (17,28) and may be due to the loss of neurons that give rise to corticocortical associational pathways (11,35), the entorhinal pathways into the hippocampus (16), cortical afferents (20), and/or the cortical projections to the caudate nucleus (55). Two recent reports determined that the total number of [3H]glutamate binding sites, including both NMDA and nonNMDA sites, was increased in the brains of aged rats (7) and monkeys (65). In the present study, a general widespread increase in NMDA-displaceable 1-[3H]glutamate binding was observed between ages 4 and 25 years, although the increase was not statistically significant. Taken together, these results suggest that future investigations should examine this age group more closely, with particular emphasis on the number and distribution of non-NMDA type glutamate binding sites. NMDA-displaceable 1-[3H]glutamate binding in hippocampus and neocortex of young rats correlated significantly with performance in tasks that require learning and memory (63), and pharmacological antagonism of NMDA receptors, using AP5, impaired the performance of rats in tasks that require learning and memory (47). Similar to the effects of AP5, the loss of NMDA bind-
ing sites, coupled with a decrease of glutamate synthesis (57), could contribute to the cognitive decline observed in aged rats (32,33), aged monkeys (6, 48, 51, 59) and humans with AD. The overall stability of the levels of ChAT activity with age confirms previous reports on aged rats (19), monkeys (65), and humans (10). We had previously shown that there is considerable variability in the individual levels of ChAT activity in monkey brain, and that the selection of cases might influence whether an age-related statistical decline in ChAT activity is observed (65). Fischer et al. (22) report a degeneration of cholinergic basal forebrain neurons without the loss of cortical or hippocampal levels of ChAT activity, suggesting that cholinergic cells that survive the age-associated neurodegenerative processes may augment ChAT activity to compensate for the loss of cholinergic terminals within the cortex. The elevated levels of cholinergic synthetic enzyme may underlie the finding that selected cholinergic agents can facilitate performance in aged, memory-impaired monkeys (4). Increased levels of neuronal ChAT would allow the cholinergic cell to respond to increased levels of precursors, provided by choline or lecithin in the diet, and thereby produce more acetylcholine for release. GABA may provide postsynaptic inhibition of interneurons via GABA-A receptors (37). GABA may also inhibit transmitter release presynaptically via GABA-A or GABA-B receptors on the terminals of glutamatergic, noradrenergic or serotonergic cortical afferent pathways (37,38). In the present study, [3H]muscimol binding to GABA-A sites was unchanged in aged rats and rhesus monkeys. In rats, aging was associated with a decreased number of [3H]GABA receptors in the substantia nigra, but not in the cerebral cortex (26,27). In humans, aging was associated with an increased number of [3H]muscimol receptors in the temporal lobe, decreased GAD activity in the temporal cortex, and decreased [3H]GABA uptake in the frontal neocortex (2). These results are consistent with the loss of a selected population of GABAergic terminals with normal aging in humans. In patients with AD, the number of GABA-A receptors was unchanged, while the density of GABA-B receptors was decreased by 68% (13). The apparent sparing of GABA-A sites, the loss of GABAergic terminals, and the significant loss of NMDA sites are all consistent with the hypothesis that smaller intracortical interneurons are spared with normal aging in rats, monkeys, and aged humans with or without AD, and that corticocortical associational and selected cortical afferent pathways, many of which may be glutamatergic (36), are lost (11, 13, 20). ACKNOWLEDGEMENTS We thank Anu Durr for typing the manuscript and Irene Malak for excellent technical assistance. This work was supported by PHS grants (NIH NS 20471 and AG 07735).
REFERENCES 1. Aigner, T. G.; Mitchell, S. J.; Aggleton, J. P.; DeLong, M. R.; Struble, R. G.; Price, D. L.; Wenk, G. L,; Mishkin, M. Effects of scopolamine and physostigmine on recognition memory in monkeys with ibotenic acid lesions of the nucleus basalis of Meynert. Psychopharmacology (Berlin) 92:292-300; 1987. 2. Allen, S. J.; Benton, J. S.; Goodhardt, M. J.; Haan, E. A.; Sims, N. R.; Smith, C. C. T.; Spillane, J. A.; Bowen, D. M.; Davison, A. N. Biochemical evidence of selective nerve cell changes in the normal ageing human and rat brain. J. Neurochem. 41:256-265; 1983. 3. Barnard, E. A.; Darlison, M. G.; Seeburg, P. Molecular biology of the GABA-A receptor: the receptor/channel superfamily. Trends Neurosci. 10:502-509; 1987. 4. Bartus, R. T.; Dean, R. L.; Beer, B. Memory deficits in aged Cebus monkeys and facilitation with central cholinomimetics. Neurobiol. Aging 1:145-152; 1980.
5. Bartus, R. T.; Dean, R. L.; Beer, B.; Lippa, A. S. The cholinergic hypothesis of geriatric memory dysfunction. Science 217:408-417; 1982. 6. Bartus, R. T.; Fleming, D.; Johnson, H. R. Aging in the rhesus monkey: debilitating effects on short-term memory. J. Gerontol. 33: 858-871; 1978. 7. Baudry, M.; Arst, D. S.; Lynch, G. Increased [3H]glutamate receptor binding in aged rats. Brain Res. 223:195-198; 1981. 8. Baudry, M.; Lynch, G. Characterization of two [3H]-glutamate binding sites in rat hippocampal membranes. J. Neurochem. 36:811-820; 1981. 9. Blessed, G.; Tomlimson, B. E.; Roth, M. The association between quantitative measures of dementia and of senile change in the cerebral grey matter of elderly subjects. Br. J. Psychiatry 14:797-811; 1968.
NMDA BINDING AND AGING
10. Bowen, D. M.; Smith, C. B., White, P.; Davison, A. N. Neurotransmitter-related indices of hypoxia in senile dementia and other abiotrophies. Brain 99:459-496; 1976. 11. Brizzee, K. R.; Ordy, J. M.; Bartus, R. T. Localization of cellular changes within multimodal sensory regions in aged monkey brain: Possible implications for age-related cognitive loss. Neurobiol. Aging 1:45-52; 1980. 12. Calderini, G.; Bonetti, A. C.; Aldinio, A.; Savoini, G.; DiPerri, B.; Biggio, G.; Toffano, G. Functional interaction between benzodiazepine and GABA recognition sites in aged rats. Neurobiol. Aging 2: 309-323; 1981. 13. Chu, D. C. M.; Penney, J. B.; Young, A. B. Cortical GABA-B and GABA-A receptors in Alzheimer's disease: A quantitative autoradiographic study. Neurology 37:1454-1459; 1987. 14. Chui, H. C.; Bondareff, W.; Zarow, C.; Slager, U. Stability of neuronal number in the human nucleus basalis of Meynert with age. Neurobiol. Aging 5:83-88; 1984. 15. Collingridge, G. L. The role of NMDA receptors in learning and memory. Nature 330:604-605; 1987. 16. Collingridge, G. L.; Kehl, S. J.; McLennan, H. Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus. J. Physiol. 334:33-46; 1983. 17. Cowburn, R.; Hardy, J.; Roberts, P.; Briggs, R. Presynaptic and postsynaptic glutamatergic function in Alzheimer's disease. Neurosci. Lett. 86:109-113; 1988. 18. Coyle, J. T.; Price, D. L.; DeLong, M. R. Alzheimer's disease: A disorder of cortical cholinergic innervation. Science 219:1184-1190; 1983. 19. Decker, M. W. The effects of aging on hippocampal and cortical projections of the forebrain cholinergic system. Brain Res. Rev. 12: 423-438; 1987. 20. DeKosky, S. T.; Bass, N. H. Aging, senile dementia, and the intralaminar microchemistry of cerebral cortex. Neurology 32:12271233; 1982. 21. Epstein, J. L. Anxiolytics, antidepressants, and neuroleptics in the treatment of geriatric patients. In: Lipton, M. A.; DiMascio, A.; Killam, K. F., eds. Psychopharmacology: A generation of progress. New York: Raven Press; 1978:1517-1523. 22. Fischer, W.; Gage, F. H.; Bjorklund, A. Degenerative changes in forebrain nuclei correlate with cognitive impairments in aged rats. Eur. J. Neurosci. 1:34-45; 1989. 23. Fisher, R. S.; Levine, M. S. Transmitter cosynthesis by corticopetal basal forebrain neurons. Brain Res. 491:163-168; 1989. 24. Fisher, R. S.; Buchwald, N. A.; Hull, C. D.; Levine, M. S. GABAergic basal forebrain neurons project to the neocortex: The localization of glutamic acid decarboxylase and choline acetyltransferase in feline corticopetal neurons. J. Comp. Neurol. 272:489-502; 1988. 25. Fonnum, F. Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities. Biochem. J. 115:465---472; 1969. 26. Govoni, S.; Memo, M.; Saiani, L.; Spano, P. F.; Trabucchi, M. Impairment of brain neurotransmitter receptors in aged rats. Mech. Ageing Dev. 12:39-46; 1980. 27. Govoni, S.; Trabucchi, M.; Bosio, A.; Spano, P. F. Functional changes of neurotransmitter receptors in the brains of aged rats. In: Cervos-Navarro, J.; Sarkander, H.-I., eds. Brain aging: Neuropathology and neuropharmacology. New York: Raven Press; 1983:363371. (Aging, vol. 21.) 28. Gramsbergen, J. B. P.; Mountjoy, C. Q.; Rossor, M. N.; Reynolds, G. P.; Roth, M.; Korf, J. A correlative study on hippocampal cation shifts and amino acids and clinico-pathological data in Alzheimer's disease. Neurobiol. Aging 8:487-494; 1987. 29. Harris, E. W.; Ganong, A. H.; Cotman, C. W. Long-term potentiation in the hippocampus involves activation of N-methyl-D-aspartate receptors. Brain Res. 323:132-137; 1984. 30. Himwich, W. A. Neurobiological aspects of maturation and ageing. In: Ford, D. H., ed. Progress in brain research. New York: Elsevier; 1973:13-25. 31. Houser, C. R.; Vaughn, J. E.; Hendry, S. H. C.; Jones, E. G.; Peters, A. GABA neurons in the cerebral cortex. In: Jones, E. G.; Peters, A., eds. Cerebral cortex, vol. 2, Functional properties of cortical cells. New York: Plenum; 1984:63-89. 32. Ingram, D. K.; London, E. D.; Goodrick, C. L. Age and neuro-
97
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34.
35. 36. 37.
38.
39.
40.
41.
42.
43.
44. 45.
46.
47.
48. 49.
50.
51.
52.
53. 54.
55.
chemical correlates of radial maze performance in rats. Neurobiol. Aging 2:41-47; 1981. Ingram, D. K.; London, E. D.; Waller, S. B.; Reynolds, M. A. Age-dependent correlation of motor performance with neurotransmitter synthetic enzyme activities in mice. Behav. Neural Biol. 39:284298; 1983. Irle, E.; Markowitsch, H. J. Basal forebrain-lesioned monkeys are severely impaired in tasks of association and recognition memory. Ann. Neurol. 22:735-743; 1987. Jones, E. G. Neurotransmitters in the cerebral cortex. J. Neurosurg. 65:135-153; 1986. Jones, E. G.; Hendry, S. H. C. Co-localization of GABA and neuropeptides in neocortical neurons. Trends Neurosci. 9:71-81; 1986. Karbon, E. W.; Duman, R.; Enna, S. J. Biochemical identification of multiple GABA-B binding sites: Association with noradrenergic terminals in rat forebrain. Brain Res. 274:393-396; 1983. Kilpatrick, G. J.; Muhyaddin, M. S.; Roberts, P. J.; Woodruff, G. N. GABA-B binding sites on rat striatal synaptic membranes. Br. J. Pharmacol. 78:(Suppl.)6P; 1983. Kleinschmidt, A.; Bear, M. F.; Singer, W. Blockade of " N M D A " receptors disrupts experience-dependent plasticity of kitten striate cortex. Science 238:355-357; 1987. Lippa, A. S.; Critchett, D. J.; Ehlert, F.; Yamamura, H. I.; Enna, S. J.; Bartus, R. T. Age-related alterations in neurotransmitter receptors: An electrophysiological and biochemical analysis. Neurobiol. Aging 2:3-8; 1981. Lowry, O. H.; Rosenbrough, N. J.; Fan-, A. L.; Randall, R. J. Protein measurement with folin phenol reagent. J. Biol. Chem. 193: 265-275; 1951. McCabe, B. J.; Horn, G. Learning and memory: Regional changes in N-methyl-D-aspartate receptors in the chick brain after imprinting. Proc. Natl. Acad. Sci. USA 85:2849-2853; 1988. McGeer, P. L.; McGeer, D. G. Enzymes associated with the metabolism of catecholamines, acetylcholine and GABA in human controls and patients with Parkinson's disease and Huntington's chorea. J. Neurochem. 26:65-79; 1976. McGeer, P. L.; McGeer, D. G.; Suzuki, J. S. Aging and extrapyramidal function. Arch. Neurol. 34:33-35; 1977. Maggi, A.; Schmidt, M. J.; Ghetti, B.; Enna, S. J. Effect of aging on neurotransmitter receptor binding in rat and human brain. Life Sci. 24:367-374; 1979. Monahan, J. B.; Michel, J. Identification and characterization of an N-methyl-D-aspartate-specificL-[3H]glutamate recognition site in synaptic plasma membranes. J. Neurochem. 48:1699-1708; 1987. Morris, R. G. M.; Anderson, E.; Lynch, G. S.; Baudry, M. Selective impairment of learning and blockade of long-term potentiation of a N-methyl-D-aspartate receptor antagonist, AP5. Nature 319:774776; 1986. Moss, M.; Rosene, D. L.; Peters, A. Effects of aging on visual recognition memory in the rhesus monkey. Primate Rep. 14:133; 1986. Olsen, R. W. The gamma-aminobutyric acid/benzodiazepine/barbiturate receptor-chloride ion channel complex of mammalian brain. In: Edelman, G. M.; Gall, W. E.; Cowan, M. W., eds. Synaptic function. New York: John Wiley & Sons; 1987:257-271. Periyasamy, S.; Masatoshi, I.; Chiu, T. H. Binding of l[3H]glutamate to repeatedly frozen-thawed rat brain membranes. Neurochem. Int. 11:69-75; 1987. Presty, S. K.; Bachevalier, J.; Walker, L. C.; Struble, R. G.; Price, D. L.; Mishkin, M.; Cork, L. C. Age differences in recognition memory of the rhesus monkey (Macaca mulatta). Neurobiol. Aging 8:435-4-440; 1987. Price, M. T.; Olney, J. W.; Haft, R. Age-related changes in glutamate concentration and synaptosomal glutamate uptake in adult rat striatum. Life Sci. 28:1365-1370; 1981. Ridley, R. M.; Baker, H. F.; Drewett, B.; Johnson, J. A. Effects of ibotenic acid lesions of the basal forebrain on serial reversal learning in marmosets. Psychopharmacology (Berlin) 86:438-443; 1985. Ridley, R. M.; Murray, T. K.; Johnson, J. A.; Baker, H. F. Learning impairment following lesion of the basal nucleus of Meynert in the marmoset: modification by cholinergic drugs. Brain Res. 376: 108-116; 1986. Roberts, P. J.; McBean, G. J.; Sharif, N. J.; Thomas, E. M. Striatal glutamatergic function: Modifications following specific lesions. Brain
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Res. 235:83-91; 1982. 56. Scatton, B.; Bartholini, G. Gamma-aminobutyric acid (GABA) receptor stimulation. IV. Effect of progabide (SL 76002) and other GABAergic agents on acetylcholine turnover in rat brain areas. J. Pharmacol. Exp. Ther. 220:689-695; 1982. 57. Tyce, G. M.; Wong, K.-L. Conversion of glucose to neurotransmitter amino acids in the brains of young and aging rats. Exp. Gerontol. 15:527-532; 1980. 58. Vincent, S. R.; Hokfelt, T.; Skirboll, L. R.; Wu, J.-Y. Hypothalamic gamma-aminobutyric acid neurons project to the neocortex. Science 220:1309-1310; 1983. 59. Walker, L. C.; Kitt, C. A.; Struble, R. G.; Wagster, M. V.; Price, D. L.; Cork, L. C. The neural basis of memory decline in aged monkeys. Neurobiol. Aging 9:657-666; 1988. 60. Walker, L. C.; Price, D. L.; Young, W. S. GABAergic neurons in the primate basal forebrain magnocellular complex. Brain Res. 499: 188-192; 1989. 61. Watkins, J. C.; Evans, R. H. Excitatory amino acid transmitters. Annu. Rev. Pharmacol. 21:165-204; 1981. 62. Wenk, G. L. Pharmacological manipulations of the substantia innominate-cortical cholinergic pathway. Neurosci. Lett. 51:99-103; 1984.
W E N K , W A L K E R , PRICE A N D C O R K
63. Wenk, G. L.; Grey, C. M.; Ingram, D. K.; Spangler, E. L.; Olton, D. S. Retention of maze performance inversely correlates with NMDA receptor number in hippocampus and frontal neocortex in rat. Behav. Neurosci. 103:688-690; 1989. 64. Wenk, G. L.; Olton, D. S. Basal forebrain cholinergic neurons and Alzheimer's disease. In: Coyle, J. T., ed. Animal models of dementia: A synaptic neurochemical perspective, neurology and neurobiology, vol. 33. New York: Alan R. Liss; 1987:81-101. 65. Wenk, G. L.; Pierce, D. J.; Struble, R. G.; Price, D. L.; Cork, L. C. Age-related changes in multiple neurotransmitter systems in the monkey brain. Neurobiol. Aging 10:11-19; 1989. 66. Wheeler, D. D. Aging of membrane transport mechanisms in the central nervous system--high affinity glutamic acid transport in rat cortical synaptosomes. Exp. Gerontol. 15:269-284. 67. Williams, M.; Risley, E. A. Characterization of the binding of [3H]muscimol, a potent gamma-aminobutyric acid agonist, to rat brain synaptosomal membranes using a filtration assay. J. Neurochem. 32: 713-718; 1979. 68. Wood, P. L.; Richard, J. GABAergic regulation of the substantia innominata-cortical cholinergic pathway. Neuropharmacology 21:969972; 1982.
0197-4580/91 $3.00 + .00
Loss of NMDA, But Not GABA-A, Binding in the Brains of Aged Rats and Monkeys G A R Y L. W E N K , *l L A R Y C. W A L K E R , ? D O N A L D L. PRICE~: A N D L I N D A C. C O R K §
*Neuromnemonics Laboratory, Department of Psychology The Johns Hopkins University, Baltimore, MD 21218 "~Neuropathology Laboratory, Department of Pathology The Johns Hopkins University School of Medicine, Baltimore, MD 21205 ~:Neuropathology Laboratory, Departments of Pathology, Neurology and Neuroscience The Johns Hopkins University School of Medicine, Baltimore, MD 21205 #Division of Comparative Medicine and Department of Pathology The Johns Hopkins UniversiO, School of Medicine, Baltimore, MD 21205 Received 20 October 1989; Accepted 10 October 1990
WENK, G. L., L. C. WALKER, D. L. PRICE AND L. C. CORK. Loss of NMDA, but not GABA-A,bindingin the brainsof aged rats' and monkeys. NEUROBIOL AGING 12(2) 93-98, 1991.--In this quantitative neurochemical study we investigated age-related changes in the GABAergic, glutamatergic, and cholinergic neurotransmitter systems in rats and rhesus monkeys. Sixteen young (5 months) and 20 aged (24 months) rats and seven "young" (4-9 years), six "adult" (20-25 years), and five "aged" (29-34 years) monkeys were studied. NMDA-displaceable l-[3H]glutamate binding was significantly decreased in many neocortical and subcortical regions examined in aged rats and monkeys. The level of choline acetyltransferase (CHAT) activity and [3H]muscimol binding were unchanged in aged animals. Acetylcholine
Glutamate
NMDA
GABA
Receptors
GLUTAMATE and gamma-aminobutyric acid (GABA) are major excitatory and inhibitory neurotransmitters, respectively, in the central nervous system, l-Glutamate is the principal neurotransmitter of the corticocortical associational pathways, and several hippocampal pathways, including the entorhinal, intrahippocampal, and hippocampal efferent pathways (16,35). N-methyl-D-aspartate (NMDA) selectively stimulates one class of glutamate receptors that mediates excitatory synaptic transmission within these pathways (61). NMDA receptors may play a critical role in many neurophysiological processes that underlie neuronal plasticity, long-term potentiation, and learning and memory (15, 29, 39, 42). GABA is a neurotransmitter in neurons of many regions of brain, including a population of intrinsic neocortical interneurons (31). Most neocortical GABA is located within intrinsic interneurons where it is frequently colocalized with specific neuropeptides (36), although a small amount of GABA may arise from subcortical neurons (23, 24, 58, 60). GABAergic neurons regulate the activity of central noradrenergic, serotonergic, dopaminergic and acetylcholinergic neurons (37, 38, 56, 62, 68) and therefore may influence many different cognitive processes and behaviors. The GABA-A receptor subtype is a major site for the inhibitory activities of GABA; this receptor is present on the ma-
Aging
Rats
Monkeys
jority of cerebral neurons in mammals (3) and is the site of action for the benzodiazepines and barbiturates (3,12). In aged rats, as compared to young controls, the concentration of glutamate in the brain (30,57), high affinity glutamate uptake in the striatum (52) and cortex (66), and the conversion of radioactively labeled glucose into glutamate (57) were decreased. In contrast, the total number of [3H]glutamate binding sites was increased in the brains of aged rats (7) and monkeys (65). The present study was designed to further investigate the nature of these changes by examination of the specific NMDA-sensitive glutamate binding site. Evidence for age-related changes in GABAergic systems is contradictory. In aging rats, the concentration of GABA was either slightly increased (30) or unchanged, the number of GABAA receptors was unchanged (12, 26, 40, 45), and the activity of glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA, was either unchanged (32) or decreased (33). In aging humans, the number of GABA receptors increased, the density of GABAergic terminals decreased, the clinical response to benzodiazepines was enhanced (2,21), and GAD activity was either decreased in selected regions (43,44) or unchanged (10). The level of choline acetyltransferase (CHAT) activity, a specific biochemical marker for cholinergic neurons, was unchanged
tRequests for reprints should be addressed to Gary L. Wenk at his present address: Division of Neural Systems, Memory and Aging, 384 Life Sciences North, University of Arizona, Tucson, AZ 85724.
93
94
WENK, WALKER, PRICE AND CORK
NMDA Receptors 700.
TABLE 1
,J "
I~
I
II
* p
/
II
5 months
I
-L
F oo
0 Ant.Ct
Post.Ct
Hippo
Cerebel
Coud
[3H]Muscimol Binding
80-
['-7 5 months B B 24. months
70-
"i 6030-
NMDA-DISPLACEABLEL-[3H]GLUTAMATEBINDING SITES§ IN MONKEYBRAIN Brain Region Gyrus Rectus Dorsal Fr. Pole Ant. Sup. Cing. Gyr. Superior Fr. Gyr. Middle Fr. Gyr. Inf. Fr. Gyr. Medial Orbital Gyr. Ant. Inf. Cing. Gyr. Precentral Gyr. Postcentral Gyr. Inf. Parietal Lobule Sup. Temporal Gyr. Middle Temporal Gyr. Parahippocampal Gyr. Hippocampus Dorsal Occipital Pole
4-9 Years
20-25 Years
29-34 Years
211.5 ~ 26.9 180.1 ± 37.6 206.5 ± 56.8 162.4 _+ 19.9 168.1 _+ 12.0 139.1 + 22.9 121.1 _+ 21.8 189.1 _+ 58.0 155.4 -+- 22.3 124.8 _+ 26.7 126.1 _+ 38.6 206.0 -+ 48.2 221.5 -+ 31.2 212.7 ___ 74.7 146.7 + 32.2 175.4 + 30.1
269.2 ___ 33.9 306.2 __+ 57.4 260.2 + 63.2 257.9 __. 33.8 205.9 _ 51.1 324.3 -+ 39.1 368.4 + 63.1 150.6 _+ 59.0 187.9 ± 37.6 230.9 ___ 15.4 183.4 _+_ 31.0 325.6 -+ 67.0 276.5 _+ 31.8 284.6 -- 44.0 436.9 _+ 39.7 338.5 _.+ 47.7
131.4 _+ 8.5* 153.8 ± 35.8 122.3 - 22.7 138.9 _+ 33.7* 61.1 _+ 15.55 80.6 -+ 9.1" 51.6 _+ 6.6t 48.3 --- 21.3 128.9 - 41.6 90.3 _+ 18.8t 93.2 --- 26.8 112.1 -+ 17.7:~ 146.1 ~ 32.5* 68.7 _+ 8.6* 70.3 _+ 20.8# 167.7 _ 30.2t
50-
*p<0.05, +p<0.01 by ANOVA. $p<0.05 vs. "Adult" levels, by t-test. Each value represents the mean - S.E.M. of seven young (4-9 years), six adult (20-25 years), and five aged (29-34 years) rhesus monkeys. §fmol/mg protein.
40-
E
20100 Ant.Ct
Post.Ct
Hippo
Cerebel (xl0)
Coud
ChATActivity 5O
I-'-I 5 months B B 24 months
40
~
3o
o.
: Ant.Ct
:All Post.Ct
Hippo
Cerebel
fll
(,lo)
Coud
FIG. 1. The number of NMDA-displaceable 1-[3H]glutamate and [3H]muscimol binding sites, and the level of ChAT activity in five brain regions of rats. Each value represents the mean--+S.E.M. of 16 young (5 months) or 20 aged (24 months) Fisher 344 rats. *Indicates a significant difference, p<0.05, as compared to levels in young rats. ( x 10) Indicates that the unit of measurement for this region should be multiplied by 10. Ant.C, sensory-motor cortex; Post.Ct, parietal-occipital cortex; Hippo, hippocampus; Cerebel, cerebellum; Caud, caudate nucleus.
basalis of Meynert was not changed (14). The level of ChAT activity is usually unchanged in the brains of healthy elderly humans (5). Experimental destruction of basal forebrain cholinergic cells in rats and monkeys produces memory deficits similar to those associated with normal aging and Alzheimer's disease (AD) (1, 34, 53, 54, 64). In AD, the amount of glutamate and GABA, the number of glutamate terminals (17,28), and various biochemical markers of cholinergic function (18) are decreased in many cortical and subcortical brain regions. The decline in cholinergic function in AD correlates somewhat with the degeneration of acetylcholine-containing cells in the basal forebrain, the density of neuropathological changes in the cortex and hippocampus, and the degree of dementia (9,64). An investigation of age-associated changes in glutamatergic and GABAergic systems in nonhuman primates would provide valuable information that cannot be reliably obtained from aged humans. In humans, extensive postmortem delays and antemortem treatments with various pharmacotherapies can significantly alter many neurochemical markers. The present study investigated ChAT activity, as a measure of the integrity of cholinergic presynaptic terminals, and NMDA-type glutamate and bicuculline-sensitive GABA-A binding in the brains of young and aged rats and monkeys. If neurons giving rise to long corticocortical pathways are most vulnerable to aging, then there should be a decline in glutamatergic, but not GABAergic, markers in the neocortex of aged animals. METHOD
throughout the neocortex and hippocampus in aged rats (19,22) and decreased only in the frontal pole and motor cortex of aged monkeys (65). The cholinergic cells in the basal forebrain that project to these cortical regions may decrease in size and number with aging in rats (22); measures of neuronal size have not been reported in aged humans, but the number of cells in the nucleus
Subjects The subjects were 16 young (5 months) and 20 aged (24 months) male F344 rats. Each rat was housed in a metal hanging cage and maintained on a 16:8 h light/dark cycle with lights on at 07:00 h. Eighteen rhesus monkeys (Macaca mulatta) were studied, in-
NMDA BINDING AND AGING
95
TABLE 2 ChAT ACTIVITY* IN MONKEY BRAIN
Brain Region Gyrus Rectus Dorsal Fr. Pole Ant. Sup. Cing. Gyr. Superior Fr. Gyr. Middle Ft. Gyr. Inf. Fr. Gyr. Medial OrbitalGyr. Ant. Inf. Cing. Gyr. Caudate Nuc., Head Rostral Putamen Precentral Gyr. Postcentral Gyr. Inf. Parietal Lobule Sup. Temporal Gyr. Middle Temporal Gyr. Parahippocampal Gyr. Hippocampus Dorsal Occipital Pole
4-9 Years 8.71 8.89 12.2 7.64 10.7 9.60 11.6 14.4 112.8 141.5 11.7 8.27 9.26 10.3 7.98 13.9 20.9 5.26
± 0.84 ± 1.10 ± 2.2 ± 1.51 ± 1.9 _+ 1.50 --- 1.8 ± 2.6 ± 11.5 ± 24.5 ± 1.4 _+ 0.86 ± 1.42 --- 0.6 ± 0.71 ± 1.6 ± 1.9 ± 0.79
20-25 Years 10.7 8.08 17.2 10.9 11.8 12.9 13.6 14.9 140.1 232.9 13.5 10.8 11.5 9.04 6.70 12.7 20.1 6.01
± 0.7 ± 0.52 ± 0.6 ± 0.4 ± 0.7 z 1.7 ± 1.1 +-- 2.8 +_. 43.1 + 85.3 --- 1.4 --- 0.6 ± 0.9 ± 0.59 ± 0.26 ± 2.0 ± 1.7 -+ 0.50
29-34 Years 7.61 8.48 11.0 9.13 7.73 7.26 14.1 13.9 76.2 150.1 8.45 7.87 8.69 9.01 6.89 12.8 20.7 3.53
+-- 0.42 ± 1.18 -4- 0.7 ± 2.19 ± 1.32 ± 0.84 ± 2.3 ± 1.4 ± 12.2 --- 26.1 --- 0.49 - 0.91 ± 0.67 ± 1.35 -+ 0.52 ± 3.4 ± 4.2 ± 0.77
Each value represents the mean ± S.E.M. of seven young (4-9 years), six adult (20-25 years), and five aged (29-34 years) rhesus monkeys. *nmol acetylcholine/mg protein/h.
cluding seven " y o u n g " (4-9 years), six " a d u l t " (20-25 years), and five " a g e d " monkeys (29-34 years). All of the young and adult monkeys, and two of the aged monkeys, had known birthdates. The ages of the other two aged monkeys were minimum estimates based on their years in captivity and their sexual maturity when captured. Because rhesus monkeys reach sexual maturity between 3--4 years of age (3-4 kg body weight), animals weighing more than 3 - 4 kg, or which were sexually mature at capture, were assumed to be at least 3 years of age.
Tissue Preparation Each rat was sacrificed by decapitation (between 09:00 and 11:00 h) and the brain was quickly removed and dissected on ice. Five areas were sampled from each rat brain, including the anterior (sensory-motor) and posterior (parietal-occipital) cerebral cortex, the entire hippocampus, caudate nucleus and cerebellum. Each monkey was restrained with ketamine, euthanized with a lethal dose of sodium pentobarbital, and then perfused transcardially for 3-5 minutes with cold (4°C) isotonic saline. The brain was quickly blocked stereotaxically in a cold room (4°C) and removed; the blocks were frozen on dry ice and stored at - 8 0 ° C .
TABLE 3 [3H]MUSCIMOL BINDING SITES* IN MONKEY BRAIN
Brain Region Frontal Pole Middle Temporal Gyrus Occipital Pole Cerebellum
4-6 Years 171.1 103.1 147.6 278.5
_± 17.4 ± 27.7 --. 23.5 ± 39.9
31-34 Years 145.1 125.1 147.5 250.2
± 17.7 +-- 23.7 ± 20.7 ± 57.8
Each value represents the mean __. S.E.M. of 4 young (4-6 years) or 4 aged (31-34 years) rhesus monkeys. *fmol/mg protein.
Samples of specific regions were later taken from the frozen blocks at - 2 0 ° C and then returned to - 8 0 ° C until assayed. Each monkey had a complete autopsy after tissues were taken for analysis. No significant pathological c h a n g e s , e . g . , brain tumors, cerebrovascular or metabolic diseases, were found that might affect the interpretation of the neurochemical data. Tissues from all rats and monkeys were processed identically. Each sample was homogenized in 0.32 M sucrose and stored at - 8 0 ° C until assayed. Repeated freeze-thawing of the tissues does not inactivate, and may improve, the NMDA-sensitive 1[3H]glutamate (50) and [3H]muscimol (49) binding. For these binding assays, the membranes were extensively washed in order to remove any endogenous ligands present in the tissue. In order to perform all three biochemical analyses on each small tissue sample, only single-point determinations were made for the two receptor sites. Therefore, changes in binding could reflect either a change in the number and/or affinity of the receptors. Proteins were measured by standard methods (41).
GABA Receptor Binding Studies [3H]Muscimol binding to the bicuculline-sensitive G A B A - A binding site was performed according to the method of Williams and Risley (67). The incubation was conducted in a sodium-free medium in order to inactivate high affinity G A B A uptake sites. Membrane preparations were incubated in 50 mM Tris-citrate buffer (pH 7.1) with [3H]muscimol (2 nM) in the presence or absence of GABA (10 txM) to define nonspecific binding.
NMDA Receptor Binding Studies NMDA-displaceable 1-[3H]glutamate binding sites were defined according to a modified method of Monahan and Michel (46). These sites are probably associated with postsynaptic, Na+-independent, NMDA-type glutamate binding sites (8,46). The tissue preparation has been described previously (63). Mem-
96
WENK, WALKER, PRICE AND CORK
brane preparations were incubated in 50 mM Tris-acetate buffer (pH 7.4) with l-[3H]-glutamate (10 nM) in the presence or absence of NMDA (0.5 raM) to define nonspecific binding.
ChAT Activity' A 40-1xl sucrose aliquot was combined with 10 ~1 of a solution containing 2% Triton X-100 and 50 mM EDTA (pH 7.4). ChAT activity was determined (25) using [~4C]acetyl Coenzyme-A. RESULTS NMDA-displaceable 1-[3H]glutamate binding was decreased in many brain regions in aged rats, and particularly in the frontal and temporal lobes of the aged monkeys (see Fig. 1 and Table 1). NMDA binding tended to increase, but not significantly, between ages 4-25 years; in the middle frontal gyrus and superior temporal gyrus the decline in NMDA binding sites was only significant as compared to these elevated levels. [3H]Muscimol binding and the level of ChAT activity in rats and monkeys did not change with age (see Fig. 1 and Tables 2 and 3). DISCUSSION The primary finding of this investigation was a widespread decline in NMDA-displaceable 1-[3H]glutamate binding in the brains of aged rats and monkeys. The decline in NMDA binding in the aged rat and monkey brain is similar to that reported in aged humans with AD (17,28) and may be due to the loss of neurons that give rise to corticocortical associational pathways (11,35), the entorhinal pathways into the hippocampus (16), cortical afferents (20), and/or the cortical projections to the caudate nucleus (55). Two recent reports determined that the total number of [3H]glutamate binding sites, including both NMDA and nonNMDA sites, was increased in the brains of aged rats (7) and monkeys (65). In the present study, a general widespread increase in NMDA-displaceable 1-[3H]glutamate binding was observed between ages 4 and 25 years, although the increase was not statistically significant. Taken together, these results suggest that future investigations should examine this age group more closely, with particular emphasis on the number and distribution of non-NMDA type glutamate binding sites. NMDA-displaceable 1-[3H]glutamate binding in hippocampus and neocortex of young rats correlated significantly with performance in tasks that require learning and memory (63), and pharmacological antagonism of NMDA receptors, using AP5, impaired the performance of rats in tasks that require learning and memory (47). Similar to the effects of AP5, the loss of NMDA bind-
ing sites, coupled with a decrease of glutamate synthesis (57), could contribute to the cognitive decline observed in aged rats (32,33), aged monkeys (6, 48, 51, 59) and humans with AD. The overall stability of the levels of ChAT activity with age confirms previous reports on aged rats (19), monkeys (65), and humans (10). We had previously shown that there is considerable variability in the individual levels of ChAT activity in monkey brain, and that the selection of cases might influence whether an age-related statistical decline in ChAT activity is observed (65). Fischer et al. (22) report a degeneration of cholinergic basal forebrain neurons without the loss of cortical or hippocampal levels of ChAT activity, suggesting that cholinergic cells that survive the age-associated neurodegenerative processes may augment ChAT activity to compensate for the loss of cholinergic terminals within the cortex. The elevated levels of cholinergic synthetic enzyme may underlie the finding that selected cholinergic agents can facilitate performance in aged, memory-impaired monkeys (4). Increased levels of neuronal ChAT would allow the cholinergic cell to respond to increased levels of precursors, provided by choline or lecithin in the diet, and thereby produce more acetylcholine for release. GABA may provide postsynaptic inhibition of interneurons via GABA-A receptors (37). GABA may also inhibit transmitter release presynaptically via GABA-A or GABA-B receptors on the terminals of glutamatergic, noradrenergic or serotonergic cortical afferent pathways (37,38). In the present study, [3H]muscimol binding to GABA-A sites was unchanged in aged rats and rhesus monkeys. In rats, aging was associated with a decreased number of [3H]GABA receptors in the substantia nigra, but not in the cerebral cortex (26,27). In humans, aging was associated with an increased number of [3H]muscimol receptors in the temporal lobe, decreased GAD activity in the temporal cortex, and decreased [3H]GABA uptake in the frontal neocortex (2). These results are consistent with the loss of a selected population of GABAergic terminals with normal aging in humans. In patients with AD, the number of GABA-A receptors was unchanged, while the density of GABA-B receptors was decreased by 68% (13). The apparent sparing of GABA-A sites, the loss of GABAergic terminals, and the significant loss of NMDA sites are all consistent with the hypothesis that smaller intracortical interneurons are spared with normal aging in rats, monkeys, and aged humans with or without AD, and that corticocortical associational and selected cortical afferent pathways, many of which may be glutamatergic (36), are lost (11, 13, 20). ACKNOWLEDGEMENTS We thank Anu Durr for typing the manuscript and Irene Malak for excellent technical assistance. This work was supported by PHS grants (NIH NS 20471 and AG 07735).
REFERENCES 1. Aigner, T. G.; Mitchell, S. J.; Aggleton, J. P.; DeLong, M. R.; Struble, R. G.; Price, D. L.; Wenk, G. L,; Mishkin, M. Effects of scopolamine and physostigmine on recognition memory in monkeys with ibotenic acid lesions of the nucleus basalis of Meynert. Psychopharmacology (Berlin) 92:292-300; 1987. 2. Allen, S. J.; Benton, J. S.; Goodhardt, M. J.; Haan, E. A.; Sims, N. R.; Smith, C. C. T.; Spillane, J. A.; Bowen, D. M.; Davison, A. N. Biochemical evidence of selective nerve cell changes in the normal ageing human and rat brain. J. Neurochem. 41:256-265; 1983. 3. Barnard, E. A.; Darlison, M. G.; Seeburg, P. Molecular biology of the GABA-A receptor: the receptor/channel superfamily. Trends Neurosci. 10:502-509; 1987. 4. Bartus, R. T.; Dean, R. L.; Beer, B. Memory deficits in aged Cebus monkeys and facilitation with central cholinomimetics. Neurobiol. Aging 1:145-152; 1980.
5. Bartus, R. T.; Dean, R. L.; Beer, B.; Lippa, A. S. The cholinergic hypothesis of geriatric memory dysfunction. Science 217:408-417; 1982. 6. Bartus, R. T.; Fleming, D.; Johnson, H. R. Aging in the rhesus monkey: debilitating effects on short-term memory. J. Gerontol. 33: 858-871; 1978. 7. Baudry, M.; Arst, D. S.; Lynch, G. Increased [3H]glutamate receptor binding in aged rats. Brain Res. 223:195-198; 1981. 8. Baudry, M.; Lynch, G. Characterization of two [3H]-glutamate binding sites in rat hippocampal membranes. J. Neurochem. 36:811-820; 1981. 9. Blessed, G.; Tomlimson, B. E.; Roth, M. The association between quantitative measures of dementia and of senile change in the cerebral grey matter of elderly subjects. Br. J. Psychiatry 14:797-811; 1968.
NMDA BINDING AND AGING
10. Bowen, D. M.; Smith, C. B., White, P.; Davison, A. N. Neurotransmitter-related indices of hypoxia in senile dementia and other abiotrophies. Brain 99:459-496; 1976. 11. Brizzee, K. R.; Ordy, J. M.; Bartus, R. T. Localization of cellular changes within multimodal sensory regions in aged monkey brain: Possible implications for age-related cognitive loss. Neurobiol. Aging 1:45-52; 1980. 12. Calderini, G.; Bonetti, A. C.; Aldinio, A.; Savoini, G.; DiPerri, B.; Biggio, G.; Toffano, G. Functional interaction between benzodiazepine and GABA recognition sites in aged rats. Neurobiol. Aging 2: 309-323; 1981. 13. Chu, D. C. M.; Penney, J. B.; Young, A. B. Cortical GABA-B and GABA-A receptors in Alzheimer's disease: A quantitative autoradiographic study. Neurology 37:1454-1459; 1987. 14. Chui, H. C.; Bondareff, W.; Zarow, C.; Slager, U. Stability of neuronal number in the human nucleus basalis of Meynert with age. Neurobiol. Aging 5:83-88; 1984. 15. Collingridge, G. L. The role of NMDA receptors in learning and memory. Nature 330:604-605; 1987. 16. Collingridge, G. L.; Kehl, S. J.; McLennan, H. Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus. J. Physiol. 334:33-46; 1983. 17. Cowburn, R.; Hardy, J.; Roberts, P.; Briggs, R. Presynaptic and postsynaptic glutamatergic function in Alzheimer's disease. Neurosci. Lett. 86:109-113; 1988. 18. Coyle, J. T.; Price, D. L.; DeLong, M. R. Alzheimer's disease: A disorder of cortical cholinergic innervation. Science 219:1184-1190; 1983. 19. Decker, M. W. The effects of aging on hippocampal and cortical projections of the forebrain cholinergic system. Brain Res. Rev. 12: 423-438; 1987. 20. DeKosky, S. T.; Bass, N. H. Aging, senile dementia, and the intralaminar microchemistry of cerebral cortex. Neurology 32:12271233; 1982. 21. Epstein, J. L. Anxiolytics, antidepressants, and neuroleptics in the treatment of geriatric patients. In: Lipton, M. A.; DiMascio, A.; Killam, K. F., eds. Psychopharmacology: A generation of progress. New York: Raven Press; 1978:1517-1523. 22. Fischer, W.; Gage, F. H.; Bjorklund, A. Degenerative changes in forebrain nuclei correlate with cognitive impairments in aged rats. Eur. J. Neurosci. 1:34-45; 1989. 23. Fisher, R. S.; Levine, M. S. Transmitter cosynthesis by corticopetal basal forebrain neurons. Brain Res. 491:163-168; 1989. 24. Fisher, R. S.; Buchwald, N. A.; Hull, C. D.; Levine, M. S. GABAergic basal forebrain neurons project to the neocortex: The localization of glutamic acid decarboxylase and choline acetyltransferase in feline corticopetal neurons. J. Comp. Neurol. 272:489-502; 1988. 25. Fonnum, F. Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities. Biochem. J. 115:465---472; 1969. 26. Govoni, S.; Memo, M.; Saiani, L.; Spano, P. F.; Trabucchi, M. Impairment of brain neurotransmitter receptors in aged rats. Mech. Ageing Dev. 12:39-46; 1980. 27. Govoni, S.; Trabucchi, M.; Bosio, A.; Spano, P. F. Functional changes of neurotransmitter receptors in the brains of aged rats. In: Cervos-Navarro, J.; Sarkander, H.-I., eds. Brain aging: Neuropathology and neuropharmacology. New York: Raven Press; 1983:363371. (Aging, vol. 21.) 28. Gramsbergen, J. B. P.; Mountjoy, C. Q.; Rossor, M. N.; Reynolds, G. P.; Roth, M.; Korf, J. A correlative study on hippocampal cation shifts and amino acids and clinico-pathological data in Alzheimer's disease. Neurobiol. Aging 8:487-494; 1987. 29. Harris, E. W.; Ganong, A. H.; Cotman, C. W. Long-term potentiation in the hippocampus involves activation of N-methyl-D-aspartate receptors. Brain Res. 323:132-137; 1984. 30. Himwich, W. A. Neurobiological aspects of maturation and ageing. In: Ford, D. H., ed. Progress in brain research. New York: Elsevier; 1973:13-25. 31. Houser, C. R.; Vaughn, J. E.; Hendry, S. H. C.; Jones, E. G.; Peters, A. GABA neurons in the cerebral cortex. In: Jones, E. G.; Peters, A., eds. Cerebral cortex, vol. 2, Functional properties of cortical cells. New York: Plenum; 1984:63-89. 32. Ingram, D. K.; London, E. D.; Goodrick, C. L. Age and neuro-
97
33.
34.
35. 36. 37.
38.
39.
40.
41.
42.
43.
44. 45.
46.
47.
48. 49.
50.
51.
52.
53. 54.
55.
chemical correlates of radial maze performance in rats. Neurobiol. Aging 2:41-47; 1981. Ingram, D. K.; London, E. D.; Waller, S. B.; Reynolds, M. A. Age-dependent correlation of motor performance with neurotransmitter synthetic enzyme activities in mice. Behav. Neural Biol. 39:284298; 1983. Irle, E.; Markowitsch, H. J. Basal forebrain-lesioned monkeys are severely impaired in tasks of association and recognition memory. Ann. Neurol. 22:735-743; 1987. Jones, E. G. Neurotransmitters in the cerebral cortex. J. Neurosurg. 65:135-153; 1986. Jones, E. G.; Hendry, S. H. C. Co-localization of GABA and neuropeptides in neocortical neurons. Trends Neurosci. 9:71-81; 1986. Karbon, E. W.; Duman, R.; Enna, S. J. Biochemical identification of multiple GABA-B binding sites: Association with noradrenergic terminals in rat forebrain. Brain Res. 274:393-396; 1983. Kilpatrick, G. J.; Muhyaddin, M. S.; Roberts, P. J.; Woodruff, G. N. GABA-B binding sites on rat striatal synaptic membranes. Br. J. Pharmacol. 78:(Suppl.)6P; 1983. Kleinschmidt, A.; Bear, M. F.; Singer, W. Blockade of " N M D A " receptors disrupts experience-dependent plasticity of kitten striate cortex. Science 238:355-357; 1987. Lippa, A. S.; Critchett, D. J.; Ehlert, F.; Yamamura, H. I.; Enna, S. J.; Bartus, R. T. Age-related alterations in neurotransmitter receptors: An electrophysiological and biochemical analysis. Neurobiol. Aging 2:3-8; 1981. Lowry, O. H.; Rosenbrough, N. J.; Fan-, A. L.; Randall, R. J. Protein measurement with folin phenol reagent. J. Biol. Chem. 193: 265-275; 1951. McCabe, B. J.; Horn, G. Learning and memory: Regional changes in N-methyl-D-aspartate receptors in the chick brain after imprinting. Proc. Natl. Acad. Sci. USA 85:2849-2853; 1988. McGeer, P. L.; McGeer, D. G. Enzymes associated with the metabolism of catecholamines, acetylcholine and GABA in human controls and patients with Parkinson's disease and Huntington's chorea. J. Neurochem. 26:65-79; 1976. McGeer, P. L.; McGeer, D. G.; Suzuki, J. S. Aging and extrapyramidal function. Arch. Neurol. 34:33-35; 1977. Maggi, A.; Schmidt, M. J.; Ghetti, B.; Enna, S. J. Effect of aging on neurotransmitter receptor binding in rat and human brain. Life Sci. 24:367-374; 1979. Monahan, J. B.; Michel, J. Identification and characterization of an N-methyl-D-aspartate-specificL-[3H]glutamate recognition site in synaptic plasma membranes. J. Neurochem. 48:1699-1708; 1987. Morris, R. G. M.; Anderson, E.; Lynch, G. S.; Baudry, M. Selective impairment of learning and blockade of long-term potentiation of a N-methyl-D-aspartate receptor antagonist, AP5. Nature 319:774776; 1986. Moss, M.; Rosene, D. L.; Peters, A. Effects of aging on visual recognition memory in the rhesus monkey. Primate Rep. 14:133; 1986. Olsen, R. W. The gamma-aminobutyric acid/benzodiazepine/barbiturate receptor-chloride ion channel complex of mammalian brain. In: Edelman, G. M.; Gall, W. E.; Cowan, M. W., eds. Synaptic function. New York: John Wiley & Sons; 1987:257-271. Periyasamy, S.; Masatoshi, I.; Chiu, T. H. Binding of l[3H]glutamate to repeatedly frozen-thawed rat brain membranes. Neurochem. Int. 11:69-75; 1987. Presty, S. K.; Bachevalier, J.; Walker, L. C.; Struble, R. G.; Price, D. L.; Mishkin, M.; Cork, L. C. Age differences in recognition memory of the rhesus monkey (Macaca mulatta). Neurobiol. Aging 8:435-4-440; 1987. Price, M. T.; Olney, J. W.; Haft, R. Age-related changes in glutamate concentration and synaptosomal glutamate uptake in adult rat striatum. Life Sci. 28:1365-1370; 1981. Ridley, R. M.; Baker, H. F.; Drewett, B.; Johnson, J. A. Effects of ibotenic acid lesions of the basal forebrain on serial reversal learning in marmosets. Psychopharmacology (Berlin) 86:438-443; 1985. Ridley, R. M.; Murray, T. K.; Johnson, J. A.; Baker, H. F. Learning impairment following lesion of the basal nucleus of Meynert in the marmoset: modification by cholinergic drugs. Brain Res. 376: 108-116; 1986. Roberts, P. J.; McBean, G. J.; Sharif, N. J.; Thomas, E. M. Striatal glutamatergic function: Modifications following specific lesions. Brain
98
Res. 235:83-91; 1982. 56. Scatton, B.; Bartholini, G. Gamma-aminobutyric acid (GABA) receptor stimulation. IV. Effect of progabide (SL 76002) and other GABAergic agents on acetylcholine turnover in rat brain areas. J. Pharmacol. Exp. Ther. 220:689-695; 1982. 57. Tyce, G. M.; Wong, K.-L. Conversion of glucose to neurotransmitter amino acids in the brains of young and aging rats. Exp. Gerontol. 15:527-532; 1980. 58. Vincent, S. R.; Hokfelt, T.; Skirboll, L. R.; Wu, J.-Y. Hypothalamic gamma-aminobutyric acid neurons project to the neocortex. Science 220:1309-1310; 1983. 59. Walker, L. C.; Kitt, C. A.; Struble, R. G.; Wagster, M. V.; Price, D. L.; Cork, L. C. The neural basis of memory decline in aged monkeys. Neurobiol. Aging 9:657-666; 1988. 60. Walker, L. C.; Price, D. L.; Young, W. S. GABAergic neurons in the primate basal forebrain magnocellular complex. Brain Res. 499: 188-192; 1989. 61. Watkins, J. C.; Evans, R. H. Excitatory amino acid transmitters. Annu. Rev. Pharmacol. 21:165-204; 1981. 62. Wenk, G. L. Pharmacological manipulations of the substantia innominate-cortical cholinergic pathway. Neurosci. Lett. 51:99-103; 1984.
W E N K , W A L K E R , PRICE A N D C O R K
63. Wenk, G. L.; Grey, C. M.; Ingram, D. K.; Spangler, E. L.; Olton, D. S. Retention of maze performance inversely correlates with NMDA receptor number in hippocampus and frontal neocortex in rat. Behav. Neurosci. 103:688-690; 1989. 64. Wenk, G. L.; Olton, D. S. Basal forebrain cholinergic neurons and Alzheimer's disease. In: Coyle, J. T., ed. Animal models of dementia: A synaptic neurochemical perspective, neurology and neurobiology, vol. 33. New York: Alan R. Liss; 1987:81-101. 65. Wenk, G. L.; Pierce, D. J.; Struble, R. G.; Price, D. L.; Cork, L. C. Age-related changes in multiple neurotransmitter systems in the monkey brain. Neurobiol. Aging 10:11-19; 1989. 66. Wheeler, D. D. Aging of membrane transport mechanisms in the central nervous system--high affinity glutamic acid transport in rat cortical synaptosomes. Exp. Gerontol. 15:269-284. 67. Williams, M.; Risley, E. A. Characterization of the binding of [3H]muscimol, a potent gamma-aminobutyric acid agonist, to rat brain synaptosomal membranes using a filtration assay. J. Neurochem. 32: 713-718; 1979. 68. Wood, P. L.; Richard, J. GABAergic regulation of the substantia innominata-cortical cholinergic pathway. Neuropharmacology 21:969972; 1982.