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Research report
Regional changes in the hippocampal density of AMPA and NMDA
receptors across the lifespan of the rat
*
Gary L. Wenk , Carol A. Barnes
Arizona Research Laboratories, Division of Neural Systems, Memory & Aging, University of Arizona, Tucson, AZ, USA
Accepted 2 August 2000
Abstract
The current study dissected the fascia dentata (FD) and hilar region from the CA and subicular cell fields of the rat and conducted in vitro determinations of the number of binding sites for N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazole
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(AMPA) glutamate receptors across the lifespan. We determined the density of binding of [ H]-glutamate or [ H]-AMPA to NMDA or AMPA receptor sites, respectively. The changes reported might be due to either a change in receptor number or an alteration in the binding characteristics of the receptor site with aging. We found an age-related decline in the number of NMDA receptors in the CA1, CA3 and subicular cell regions of the hippocampus, but not in the FD/ hilar region, and an age-related decline in the number of AMPA receptors in the FD/ hilar region, but not in the CA fields. The decline in the number of NMDA or AMPA receptors that occurs with aging was not a continuous or homogeneous process. These changes in receptor number might underlie selected age-associated changes in sensitivity to drugs that influence hippocampal function as well as to changes in NMDA-dependent long-term potentiation. A thorough understanding of the mechanisms underlying changes in glutamate receptor function in discrete brain regions, using combined neurochemical and electrophysiological methods, may ultimately provide insight into the fundamental substrates of age-associated memory disorders related to hippocampal dysfunction. 2000 Elsevier Science B.V. All rights reserved.
Theme: Neural basis of behavior
Topic: Aging
Keywords: NMDA; AMPA; Receptor; Aging; Rodents; Hippocampus
1. Introduction these glutamate receptor subtypes impairs learning and
memory performance [17] and can block the induction of Normal aging is associated with a decline in hippocam- long-term potentiation (LTP) in the hippocampus [19]. pal-dependent mnemonic abilities in humans [1], non- While facilitation of the function of these receptor sub-human primates [2] and rodents [4]. Glutamate is an types can enhance memory [24], hyperactivity of these important neurotransmitter at hippocampal synapses and is neurotransmitter receptors may underlie the neurode-known to be critical for normal learning and memory generative changes and decline in hippocampal function processes [8]. Glutamate receptors that are sensitive to associated with aging [20]. Investigations of changes in the either N-methyl-D-aspartate (NMDA) or a-amino-3-hy- number of these receptors in aging populations have
droxy-5-methyl-4-isoxazole (AMPA) are involved in produced conflicting results. Laboratories have reported synaptic modification that can be induced in this structure either no change [23] or a decline [7,15,21,25,26] in [7,8] that has been hypothesized to reflect a mechanism of numbers of these receptors within the hippocampus. Much information storage [6,16]. Pharmacological blockade of of this variation may be related to differences in the species or strain investigated [13,15] or the particular ligand used in the binding assays. In addition, the apparent
*Corresponding author. Tel.: 11-520-626-2617; fax: 1
1-520-626-discrepancy might also be related to regional changes in
2618.
E-mail address: [email protected] (G.L. Wenk). receptor density within the hippocampus. In the present
2 G.L. Wenk, C.A. Barnes / Brain Research 885 (2000) 1 –5
study, we dissected the fascia dentata (FD), and hilar ice-cold 50 mM Tris–acetate / EGTA buffer, pH 7.2, region from the CA and subicular cell fields and conducted followed immediately by filtration through GF / C filters in vitro determinations of the number of binding sites for that had been presoaked in 0.03% polyethylenimine. AMPA and NMDA glutamate receptors across the lifespan. AMPA receptor binding assays were performed
accord-ing to the method of Hoffman et al. [12]. The P2 fractions were resuspended in buffer containing 500 mM Tris–
2. Materials and methods acetate / 50 mM EGTA / 50 mM KSCN. The assays were
conducted in an incubation volume of 500 ml containing
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2.1. Subjects [ H]-AMPA (4.8hM) and 15–20mg of membrane protein.
Non-specific binding was defined by the addition of 5 mM Thirty-eight male Sprague–Dawley rats (National Insti- glutamate. Incubation was carried out at 08C for 60 min tute on Aging, Bethesda, MD) were fed ad libitum and and terminated by dilution with 4 ml of ice-cold 50 mM housed under 12 / 12 h light conditions until sacrificed. The Tris–KSCN buffer, pH 7.4, followed immediately by age (in months) and number (in parentheses) of rats used filtration through GF / C filters that had been presoaked in in this study were as follows: 3 (3), 6(3), 9(3), 12(2), 0.03% polyethylenimine. Age-related differences in the 14(1), 15(2), 16(1), 17(1), 18(2), 23(2), 24(2), 25(4), number of receptor binding sites were analyzed by
26(2), 28(2), 29(7) and 31(1). ANOVA.
2.2. Tissue dissection
3. Results The rats were briefly anesthetized using metophane gas
and then quickly sacrificed by decapitation. The brains 3.1. NMDA receptor changes were quickly removed and the hippocampus was dissected
out, placed ventral side up (Fig. 1A) on a microdissection There was a significant main effect of Age [F11,3452.89, tray on ice. Using a microspatula, the FD was dissected P50.0016] for the number of NMDA receptors in the CA free of the CA and subicular fields by gently teasing it region of the hippocampus. Post-hoc comparisons using away along the hippocampal fissure. Further transection Student–Newman–Keuls method found that levels of along the margin of the free blade produced a block NMDA receptors in the 25 and 29 month groups differed consisting of subiculum, CA1 and most of CA3 (Fig. 1B) significantly (P,0.05) from the 3 month levels. In con-with no granule cells attached, and a block consisting of trast, there was no significant main effect of Age [F11,345
primarily granule cells of the FD hilar neurons and some 1.14, P50.38] for the number of NMDA receptors in the CA3c pyramidal cells (Fig. 1C). The tissues were quickly FD region. The results are shown in the top frame of Fig. frozen on dry ice and then stored (at 2708C) until all of 2: the data have been grouped as follows: 3–9 month,
the samples were collected from all rats. 12–17 month, 18–24 month, 25–28 month and 29–31
month.
2.3. Receptor binding assays There was a significant main effect of Age [F11,3452.69,
P50.049] for the number of AMPA receptors in the FD Tissues from all age groups were processed at the same region. Post-hoc comparisons found that levels of AMPA time. Membrane fractions were prepared from the tissues receptors in the 18 month group differed significantly by homogenization and repeated centrifugation to produce (P,0.05) from the 3 month levels. In contrast, there was the P2 fraction; these were followed by hypo-osmotic lysis no main effect of Age [F11,3451.06, P50.39] for the and repeated washings to remove endogenous neurotrans- number of AMPA receptors in the CA1 region of the mitters. The left hemisphere’s hippocampus was used for hippocampus. The results are shown in the bottom frame the AMPA assays while the right side was used for the of Fig. 2.
NMDA receptor determinations. Due to the small size of the samples and the desire to avoid pooling tissues only
single-point determinations were made. 4. Discussion
The NMDA receptor binding assays were performed
according to the method of Hall et al. [11]. The P2 The current investigation provides a converging line of fractions were resuspended in buffer containing 500 mM evidence with earlier autoradiographic studies of age-re-Tris–acetate / 50mM EGTA. The assays were conducted in lated changes in hippocampal glutamatergic receptors
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Fig. 1. Hippocampal dissection method (dorsal view) used to provide tissue for the glutamate receptor binding study of hippocampal subregions. (A) Hippocampus from one hemisphere dissected away from the neocortex, thalamic and other regions to which it is attached. The hippocampus of the other hemisphere is dissected into two parts. What remains in (B) are the CA fields and subiculum, and the portion shown in (C) contains the granule cells of the fascia dentata and the hilar region. These are the two regions compared across the lifespan of the F344 rat. Scale bar51.1 mm.
decline in the number of AMPA receptors in the FD/ hilar data are consistent, however, with the findings in which the region, but not in the CA fields. The changes reported numbers of AMPA receptors were reported to decline might be due to either a change in receptor number or an within the FD region, the number of NMDA receptors alteration in the binding characteristics of the receptor site were reported to decline within the CA1 region, and with with aging. The results also suggest that changes in the the observation of no decline in AMPA receptors within number of NMDA and AMPA receptors that occur with CA1. In addition, consistent with a previous analysis of aging are not a continuous or homogeneous process from glutamate receptors in hippocampal subfields [21], we youth to old age. These results are partially consistent with found that the overall numbers of AMPA-selective gluta-previous findings, using different methods [7,15], although mate receptors was higher in the FD as compared to the we did not detect a change in the number of NMDA CA1 region in young rats.
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re-4 G.L. Wenk, C.A. Barnes / Brain Research 885 (2000) 1 –5
and CA1 during aging, a different pattern of results might have been predicted from what was observed in the present study or in previous experiments in which glutamate receptor binding was measured. For example, old, memory-impaired rats show smaller AMPA- and NMDA-mediated responses at the Schaffer collateral-CA1 synapse [3]. This is consistent with the observed decrease in NMDA receptor protein, but not with the finding of a stability in AMPA receptor protein that was observed across the lifespan in the present study. For FD, the AMPA-mediated responses at the medial perforant path-granule cell synapse are larger in old rats, while the NMDA-mediated synaptic responses are reduced in old versus middle-aged rats [5,22]. Part of the explanation for the apparent discrepancy may reside in the fact that the electrophysiological studies assessed specific synaptic con-nections in CA1 and FD, whereas the receptor binding studies assessed the entire CA1 field, plus CA3 and subicular areas, and the entire FD and hilar regions. Alternatively, the methods used in the present study to assess the existence or absence of receptors, plus the autoradiographic methods used in other studies, may not directly reflect the functional state of those receptors at their synaptic sites. A thorough understanding of the mechanisms underlying the changes in glutamate receptor function in discrete brain regions, using combined neuro-chemical and electrophysiological methods, may ultimately provide insight into the fundamental substrates of age-associated memory disorders related to hippocampal dysfunction.
Fig. 2. Age-related changes in NMDA and AMPA receptor number in
two discrete regions of the hippocampus of rats. Thirty-eight rats, ranging Acknowledgements in age from 3 to 31 months were used in this study. NMDA receptor
number declined with aging in the CA and subicular fields region (CA)
Supported by the U.S. Public Health Service, Contract
but not in the fascia dentate (FD). AMPA receptor number declined with
grant number AG10546 and an Alzheimer’s Association,
aging in the FD but not in the CA regions. Values are expressed as
IIRG-95-004 award to GLW and AG03376 to CAB. We
mean1S.D.
thank Bruce McNaughton for developing the microdissec-tion procedure used in these experiments, and Geeta Rao ceptors might suggest a decrease in the number of these
and Frank Houston for assistance with the dissections. receptor sites. On the other hand, the age-related changes
could also be due to changes in the binding characteristics of the glutamate receptor complex. Recently, similar
age-related changes in receptor binding that occurred in the References
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