Glutamate Receptor Binding in the Human Hippocampus and Adjacent Cortex During
Development and Aging
M. JOHNSON,* R. H PERRY,J" M. A. P IGGOTT, * J. A. COURT,* D. SPURDEN,* S. LLOYD,* P. G. INCE,* A N D E. K. P E R R Y .1
*MRC Neurochemical Pathology Unit, ~'Department of Neuropathology, Newcastle General Hospital, Westgate Road Newcastle upon Tyne NE4 6BE, UK
Received 27 July 1995; Revised 12 February 1996; Accepted 7 March 1996
JOHNSON, M., R.H. PERRY, M.A. PIGGOTT, J.A. COURT, D. SPURDEN, S. LLOYD, P.G. INCE AND E. K. PERRY. Glutamate receptor binding in the human hippocampus and adjacent cortex during development and aging. NEUROBIOL AGING 17(4) 639--651, 1996.--Distinct patterns of age-related alterations in NMDA (MK801 binding) and non-NMDA, AMPA (CNQX), and kainate binding have been identified in human hippocampus and parahippocampal gyrus in normal individuals with no evidence of degenerative brain disease ranging in age from 24 gestational weeks to 94 years. Whereas MK801 binding did not alter substantially over this age range, CNQX binding rose from low levels in the fetus to maximum levels between neonate and middle age, and kainate binding declined extensively from the perinatal to adult stage. Following maturity, there were no significant changes in kainate binding, although MK801 birLding increased in CA1 and CA3 and CNQX binding declined in several regions, particularly CA2 and subiculum. For each receptor binding the timing of these fluctuations ocurring during development and aging varied within different regions of the dentate gyrus, hippocampus proper, subicular complex, and entorhinal cortex examined. The transient peaks of receptor binding are likely to reflect processes of synaptogenesis and pruning and may provide clues regarding the role of the different glutamate receptor subtypes in various pathologies of the hippocampus and adjacent cortex associated with developmental disorders (of genetic origin or due to pednatal trauma or insult). The absence of substantial changes in any subtype examined from middle to old age suggests alterations in transmitter binding to these glutamate receptors are not involved in senescent neurodegeneration.
NMDA AMPA and kainate receptors M K 8 0 1 CNQX and kainic acid binding Dentate fascia Hippocampal CA1-3 Subicular complex Parahippocampal gyrus/entorhinal cortex Synaptogenesis Synaptic pruning Perinatal and age-related neuropathology Alzheimer's disease
THE amino acid L-glutamate is considered to be the major excit- atory executive neurotransmitter in the central nervous system (8, 29). Excitatory amino acid (EAA) receptor interactions play a critical role in neuronal plasticity (17) with a major role in regu- lating the development of cytoarchitecture and connectivity (29). Abnormalities in synaptic pruning during development associated with abnormalities in glutamate receptors are likely to occur in response to toxic or traumatic insults occurring at the fetal or neonatal stage. This mechanism has been implicated in the patho- genesis of schizophrenia (11). It has been suggested that EAAs may also be involved in normal aging, associated with impair- ments of memory and learning, and in the neuropathology of Alz- heimer's disease (2,16). Excitotoxicity due to EAA receptor hy- peractivity is considered to be a major component of neuronal loss associated with hypoxia-ischemia, or prolonged seizures ocurring perinatally or later in life (4,6,12,31,32).
Ligand-gated glutamate receptor cation channels belong to three different classes (59) according to their interaction with dis-
tinct agonists: N-methyl-D-aspartate (NMDA), amino-3-hydroxy- 5-methyl-4 isoxazolepropionic acid (AMPA), and kainic acid (kainate). In addition, multiple molecular forms exist within these different classes (52). Molecular forms of the NMDA receptor (26,35) consist of the NR1 subunit combined with one of four NR2 subunits (NR2A-NR2D, 2C only being expressed in the cerebel- lum). Four forms of the AMPA receptor (52) include GLURA-D with additional diversity associated with Flip and Flop splice vari- ants of these. High-affinity kainate receptor subtypes include KA1, KA2, and GluR5-GluR7 (1). The precise pharmacology and physiological functions for these different molecular forms are currently being defined and include, for example, variability in agonist affinities, antagonist effects, and sensitivity to Mg 2÷ block- ade amongst various NMDA and AMPA receptor subunit combi- nations (18) and differences in channel conductance amongst NMDA and AMPA subunits (15,61).
Excitatory amino acid neurotransmitter systems undergo marked changes during development. It has been reported that
1 To whom requests for reprints should be addressed. Professor EK Perry, MRC Neurochemical Pathology Unit, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, NE4 6BE, UK.
639
640 JOHNSON ET AL.
there is a transient period during development in which particular populations of neurons are hypersensitive to AMPA receptor me- diated injury (30) and that a transient increased density of NMDA binding sites occurs during a restricted period of hippocampal development that may play an important role in developmental plasticity (49). Particular areas of the adult and perinatal human brain including the hippocampus, subiculum, ventral ports, and cerebellum are vulnerable to hypoxia and ischaemia, and this re- gional neuronal damage is thought to involve both NMDA and non-NMDA receptor-mediated mechanisms (31). It has been sug- gested that the pathogenesis of neuronal damage to the subiculum and CA 1 of the hippocampal formation is related to selective vul- nerability of these regions as neurons in these regions are among the first to mature (51). Represa et al. (50) reported a transiently high density of [3H] kainate binding in the human neonatal hip- pocampus, particularly dentate fascia. An early postnatal peak of NMDA sensitive and insensitive (3H) glutamate binding and of (3H) MK801 in human frontal cortex has been reported by Korn- huber et al. (22,24). Piggott et al. (46) reported a perinatal peak in human frontal cortex of non-NMDA receptors that bind to [3H] kainate and a decrease in activity of NMDA receptors in the fetus and neonate in the presence of spermidine. D'Souza et al. (10) provided evidence of an increase in glutamatergic synapses up to 20 weeks postnatally related to an increase in [3H] glycine and MK801 binding sites in the infant compared to the adult human temporal cortex.
The distribution of NMDA and non-NMDA receptors has been widely investigated in the adult human hippocampus both in nor- mal individuals and cases with Alzheimer's disease (13,19,20,23, 41). The density of [3H]MK801 binding is relatively high in the normal hippocampus and entorhinal cortex, with highest densities in CA1 and CA2 regions, where binding is mainly confined to the pyramidal layer and stratum radiatum and in the dentate granular layer (19,41). There is a marked degree of overlap in the distribu- tion of NMDA and AMPA binding in adult hippocampus. High densities of [3H]CNQX binding occur in the CA1 stratum pyrami- dale and radiatum and also in the dentate granular. Kainate binding is much lower than NMDA or AMPA binding in the adult and the pattern is distinct from that of the NMDA or AMPA. The kainate receptor is low in CA1 and while relatively high in the dentate fascia and in CA2/3 receptor is, compared with the other types, more concentrated in parahippocampal gyrus or entorhinal cortex (41,50). In Alzheimer's disease, inconsistent findings have been reported on NMDA binding in the hippocampus. A reduction in [3H]TCP binding was reported by Maragos et al. (28) and Mon- aghan et al. (33) and of (3H) MK801 binding by Penney et al. (40) and Ulas et al. (58), whereas a preservation of these or related binding sites was reported by Cowburn et al. (7) and Geddes et al. (13). Hippocampal (3H) kainate and (3H) AMPA binding have been reported to increase in the disease (14).
The developmental and aging patterns of NMDA and non- NMDA receptor binding in hippocampus and adjacent cortex have not been described in detail across the human lifespan, although we have previously reported on a developmental rise in CNQX but not MK801 binding in the CA1 region of the hippocampus and in entorhinal cortex (9). In view of the importance of the hippocam- pus and adjacent cortex in cognitive function and the vulnerability of these regions to ischaemia in infancy, adulthood and disease in old age the distribution of NMDA and non-NMDA receptor bind- ing has been investigated in over 20 different areas of this archi- cortical region from the third trimester of gestation to the tenth decade of life. Receptor analysis included autoradiography of the NMDA receptor using [3H]MK801 [dizocilpine, a noncompetitive channel antagonist with maximal binding in the presence of glu-
tamate (12)], of the AMPA receptor using [3H]CNQX a competi- tive non-NMDA receptor ligand (36) and of the kainate receptor using the selective antagonist, [3H] kainic acid.
METHOD
Cases
Human brains were obtained at autopsy from 56 individuals (Table 1) ranging in age from 24 weeks gestation to 95 years who had no clinical or, in the instances examined, pathologic evidence of neurodegenerative disease. The different age groups were gen- erally matched for autopsy delay and tissue storage time, although the delay was significantly shorter in the 41~50 year compared to some of the other groups and the storage time longer in the 21- 40-year group. Autopsy delay does not influence NMDA or non- NMDA receptor binding (24,47). In relation to the neonate group, it cannot be excluded the sudden infant death syndrome, occurring in four of seven cases, is a neurological disorder. However, recep- tor values in this subgroup did not differ from the remainder of the group, and standard deviations for the whole group were similar to those in the other age group. The single solvent abuse case in- cluded in the adolescent group was retained for the similar reason that the receptor levels were within the range of the other cases in this group.
Brain Tissue
The left hemisphere was snap frozen for neurochemical analy- sis and formalin fixed tissue from the right hemisphere was em- ployed to establish the absence of significant neuropathological abnormalities (ischemic lesions or Alzheimer-type pathology) in older (> 60 years) individuals. In patients over 2 months old 1 cm thick slices were prepared from the left hemisphere and snap fro- zen in liquid arcton (ICI) cooled in liquid nitrogen. In fetal and early perinatal cases the brain was snap frozen intact. The frozen material was stored at -70°C for 1 month to 8.5 years. Hippocam- pus and adjacent hippocampal gyms was subdissected as an intact block at -20°C. For autoradiographic analyses, 20 p,m cryostat sections obtained from the frozen blocks were thaw mounted onto Vectabond (Vector Labs)-coated slides, air dried for 1-2 h, and stored for a maximum of 4 days at -70°C prior to use. Adjacent sections were fixed and stained for microanatomical analysis using Cresyl fast violet.
Receptor Autoradiography
For [3H]MK801 binding, triplicate sections were preincubated at 4°C for 10 min in low ionic strength 5 mM Tris-HC1 (pH7.4) containing glutamic acid (50 mM), glycine (50 IxM) and spermi- dine (50 txM) to promote MK801 binding. The sections were then incubated at 20°C for 1 h in this buffer containing 3 nM [3H]MK801 (Du Pont, specific activity 28.8 Ci/mmol) in the pres- ence or absence of 10 -4 M unlabeled MK801 to define specific binding. Sections were rinsed for 30 s in three changes of ice-cold 5 mM Tris-HCl, dipped in distilled water, and rapidly dried under a stream of air. Although subsequent to this study, the molarity of the buffer has been raised and period of washing increased to promote section adherence to the slides and reduce nonspecific binding, the original protocol described here provided similar ab- solute values for specific ligand binding.
For [3H]CNQX binding, triplicate sections were preincubated in 50 mM Tris-HC1 (pH7.2) at 4°C for 30 rain and were then incubated at 4°C for 40 min in buffer containing 20 nM [3H]C- NQX (Du Pont, specific activity 22.5 Ci/mmol) in the presence or absence of 10 -3 M glutamate to define specific binding. The sec-
HUMAN HIPPOCAMPUS GLUTAMATE RECEPTORS AND AGING 641
TABLE 1
DEMOGRAPHIC CASE DETAILS
Age Group Number* Mean Age Gender (M:F) Postmortem Delay (h) Storage Period (months) Cause of Death'~
81-100 years 9 89.9 + 6.9 years 3:6 27.4 + 16.2 28.6 + 26.0 1 carcinoma breast 1 carcinoma bronchus 1 bronchopneumonia 1 peritonitis 1 pulmonary embolism 4 IHD
* In some groups, not all cases were part of the three receptor analyses. t Abbreviations: RDS = respiratory distress syndrome: SIDS = sudden infant death syndrome: RTA = road traffic accident: IHD = ischaemic heart
disease.
tions were rinsed for a total of 10 s in three changes of ice-cold buffer, dipped in distilled water, and rapidly dried under a stream of air.
For [3H] kainate binding, triplicate sections were preincubated in 50 mM Tris citrate (pH 7.1) for 1 h at 4°C followed by 20 min at 20°C. Sections were then incubated in the same buffer with 5 nM vinylidine-[3H] kainic acid (Du Pont, specific activity 58 Ci/ mmol) in the presence and absence of 0.5 mM glutamic acid for 60 rain at 4°C. Sections were rinsed for 15 s in three changes of the cold buffer, dipped in distilled water, and rapidly dried under a stream of air.
Autoradiographic images were generated using tritium sensi- tive film (Hyperfilm, Amersham) apposed with the tissue sections and autoradiographic [3H] standards (Amersham) for 2-3 weeks (MK801 and CNQX) or ,$--8 weeks (kainic acid). Following a standardized development procedure (Kodak D19) the autoradio- graphs were quantified, using a Joyce Loebl Magiscan 2A image analysis system, in the areas illustrated in Fig. 1.
The average percentage specific binding in grey matter regions ranged from maximal 80% to minimal 22% for MK801; from maximal 89% to minimal 24% for CNQX and was 100% (i.e., no nonspecific binding) for kalnic acid throughout the age groups.
Statistical Analysis
This receptor analysis in the aging human brain is primarily descriptive and conventional statistical analysis of small numbers in each age group may not be ideal. Nevertheless, to focus on potentially important changes, the following analyses were carried out using MINITAB for Windows (Version 10).
Analysis of variance (ANOVA) was applied to individual re- ceptors in each of the areas examined. When F ratios were statis- tically significant, Fisher 's test with the individual error rate set to 0.05 was performed to compare means (Table 2). Where group sizes were three or less, statistical comparisons were not per- formed.
RESULTS
Areas in the hippocampus and adjacent gyms that were sub- jected to image analysis are illustrated in Fig. 1. Representative photomicrographs derived from autoradiograms from individual cases across the age range are illustrated in Figs. 2-4. Patterns of binding during development and aging in select areas, including those with highest binding and with the most distinct alterations with age, are illustrated quantitatively in Figs. 5-7. Statistically
642 JOHNSON ET AL.
STRATUM ORIENS
STRATUM LACUNOSUM
(SLM)
FIMBRIA
STRATUM . . . . . (R) . .,
STRATUM J PYRAMIDALE (P)
DENTATE MOLECULAR LAYER (DML)
DENTATE GRANULAR LAYER (DGL)
PRESUBICULUM (PRE
WHITE MATTER
PARA SUBICULUM (PAS)
' D , i J
S PARAHIPPOCAMPAL c B GYRUS .
FIG. 1. Diagram of hippocampus and adjacent parahippocampal gyrus depicting areas analysed and abbreviations used in Figs. 5-7. Abbreviations as follows: DF---dentate fascia or gyms; DML---dentate molecular layer; DGL---dentate granular layer, (o, outer, i, inner); CA1, CA2, CA3-0, P, R, and SLM--stratum oriens, pyramidal layer, stratum radiatum, and stratum lacunosum moleculare of Comu Ammonis; S--subiculum (M and P--rnolecular and pyramidal layers); PreS--presubiculum; PaS--parasubiculum; PHG--parahippocampal gyrus (A-D---outer, outer middle, inner middle, and inner layers).
significant changes are summarized in Table 2. Previous measure- ments of AMPA and NMDA receptor binding in frontal cortex of the same series (45,46) indicate that alterations in binding during development and aging are unlikely to be associated with changes in Kd values accross this age span.
AMPA Receptor
The pattern of [3H]CNQX-binding from prenatal to old age was broadly similar in all areas quantified. An initial peak was apparent between birth and middle age, the maximum occurring at different ages for different areas: at around 10 years in dentate fascia, den- tate granular layer, strata oriens, radiatum, and pyramidale of CA1 and CA2, and in subiculum molecular layer; at 20-40 years in the subicular and parasubicular pyramidal layers and in parahippo- campal gyms; plateauing across a broad age range of 1-40 years in the dentate molecular layer and granular layer, CA3 pyramidal layer, and molecular layers of the pre- and parasubiculum. On either side of this peak, binding was relatively low in the fetal/ neonatal and middle aged to elderly group.
Compared with the maxima, the level of fetal binding was lower by between one-third and one-tenth. Binding was signifi- cantly lower in fetal and neonate compared with older cases in most areas, although fetal and neonate did not differ from each other. The lowest fetal level and most extensive increases during
development occurring in the inner layer of the dentate granule layer, the pyramidal layer of the subiculum and presubiculum, and in the upper layers of the parahippocampal gyms. Unlike other areas in CA1 and CA2, the stratum lacunosum moleculare did not show any binding in the fetal group, despite being structurally developed at this stage. Stratum lacunosum binding was first ap- parent in the neonate with levels then progressively decreasing from childhood into old age.
Compared with these developmental rises, there were fewer significant decreases with age following young adulthood. These occurred in most layers of CA1 and 2, in dentate fascia, inner granular layer, subicular molecular and pyramidal layers, and up- per middle parahippocampal gyms. In addition to differences in the timing of the decline in CNQX binding from maximal levels in the infant or adult brain, there were also marked differences in the extent of this reduction. This was most extensive (up to a threefold loss) in the stratum lacunosum of CA2 and in the subicular com- plex and least extensive in the parahippocampal gyms where there were no significant reductions between the ages of 20 and 100 years. There was little evidence of any decline between middle to old age.
White matter binding did not change significantly with age, there being relatively low binding levels (0--80 fmol per mg) for all age groups. This suggests that 13 particle quenching variations due to changes in white matter density did not significantly affect
TABLE 2 STATIS'HCAL ANALYSIS OF RF_L-'EV~OR BINDING (SIGNIFICANT DWt~ERENCES BETWEEN AGE GROUPS)
Area Age Neonate (1-10) ( I I -20) (21--40) (41-60) (6t-80) (81-I00)
C N O X
C A 1---Stra tum m d i a t u m Feta l * *
Neona ta l * *
( 1 1 - 2 0 )
C A 1 - - P y r a m i d a l Feta l * *
Neona ta l *
( 11 -20 )
C A 1 - - L a c u n o s u r n molecu la re ( 11 -20 )
( 2 1 - 4 0 )
C A 2 - - O r i e n s Fe ta l * *
Neona ta l * *
CA2. R a d i a t u m Feta l * *
Neona ta l * *
(11-20) (21--40)
Denta te fasc ia Neona ta l * *
( 1 1 - 2 0 )
( 2 1 - 4 0 )
Denta te g ranu le (inner) Neona ta l * *
( 1 1 - 2 0 )
S u b i c u l u m - - M o l e c u l a r ( 1 1 - 2 0 )
S u b i c u l u m - - P y r a m i d a l Feta l *
( 1 1 - 2 0 )
Pa ra -h ippocampa l G y m s A Neona ta l *
Pa ra -h ippocampa l G y r u s B Neona ta l *
( I 1 -20 )
Pa ra -h ippocampa l G y r u s C Neona ta l *
M K 8 0 1
C A l - - P y r a m i d a l * * * *
C A 3 - - R a d i a t m n
Kaina te
Denta te fasc ia
Denta te g ranu le
C A 3 - - P y r a m i d a l
C A 2 - - P y r a m i d a l
C A 1 - - P y r a m i d a l
S u b i c u l u m
P r e s u b i c u l u m - - M o l e c u l a r
P r e s u b i c u l u m - - P y r a m i d a l
Pa ra -h ippocampa l G y r u s A
Pa ra -h ippocampa l G y r u s B
Pa ra -h ippocampa l Gyrus C
Pa ra -h ippocampa l G y r u s 1)
( 8 1 - 1 0 0 )
Neona ta l
( 11 -20 )
( 41 -60 )
Fe ta l * * * * *
Neona ta l * *
Feta l * * * * * *
Neona ta l * * * * *
Feta l * * * *
Neona ta l * * * * *
Fe ta l * * *
Neona ta l * * * * *
Feta l * * * * *
Neona te * * * * *
(1 -10) * * * * *
Neona ta l * * * * * *
(1 -10) * * * * *
Feta l * * * * *
Neonata l * * * * *
Feta l * * *
Neona ta l * * *
Fe ta l * * *
Neona ta l * * * * *
(1 -10) * * * * *
( 6 1 - - 8 0 ) *
Fe ta l * * * * * *
Neona ta l * * * * * *
( 1 - 1 0 ) * *
Feta l * * * * * * *
Neona ta l * * * * *
( 1 - 1 0 ) *
Fe ta l * * *
Neona ta l * * * * * *
(1 -10) * * * * *
* p < 0.05.
644 JOHNSON ET AL.
FIG. 2. Autoradiographic distribution of [3H]CNQX binding in human hippocampus and parahippocampal gyrus in representative individuals at different ages. F and N = fetal and neonate. Magnification xl.83.
;ii~!
FIG. 3. Autoradiographic distribution of [3H]MK801 acid binding in hu- man hippocampus and parahippocampal gyrus at different ages. Magnifi- cation xl.83.
binding levels (unless increased axonal binding during develop- ment was paralleled by decreased binding due to quenching with the reverse occurring in old age).
NMDA Receptor
In contrast to CNQX-binding there were less marked changes with age in MK801 binding. There was generally no significant increase between fetal and adolescent periods, although there were trends in the dentate granule and molecular layers towards increas- ing binding (around twofold) that just failed to reach significance between fetal and adult stages. In the stratum radiatum of CA1 and
CA3 of the hippocampus binding tended to decrease from the fetal to elderly stage, although in CA1 there was a significant increase in the 80-100-year group. In the subiculum and parasubiculum, this decline up to middle age did not reach significance.
Binding did not alter from adulthood (over 20 years) to old age in most areas, although there was a significant increase in CA1 stratum radiatum in cases over, compared with under, the age of 60 years, which did not reach significance for other layers of CA1 or the stratum radiatum in CA2 or CA3. The pyramidal layer of the subiculum showed an increase in binding in the 80-100- compared to 61-80-year group. (Although ANOVA across this area was not significant, the two age groups differed in Fisher's pair-wise corn-
HUMAN HIPPOCAMPUS GLUTAMATE RECEPTORS AND AGING 645
FIG. 4. Autoradiographic distribution of [3I-I] kainic acid binding in human hippocampus and parahippocampal gyrus at different ages. Magnification xl.83.
parisons.) In the pyramidal layer of the para subiculum there was also a similar though not statistically significant increase in bind- ing from middle (41-60) to old (80-100) age. In contrast, binding in the CA3 stratum oriens, pyramidale and radiatum all showed a similar binding pattern, with age decreasing significantly from 40 to 100 years (in this insl~nce Spearman rank correlations were analyzed, being significant for all layers and p < 0.01 in the py- ramidal layers). As with AMPA binding, there was no apparent binding of MK801 in the, stratum lacunosum in the fetal group; binding appeared in the neonate group and remained largely un- altered accross the lifespan.
In white matter binding showed a significant difference be-
tween the fetal and other groups. This difference disappeared if one fetal case with a high value was omitted.
Kainate Receptor
Significant alterations in kalnate binding were evident in al- most all areas examined due to a reduction in binding from the fetal and neonatal compared to adult stage. This was most marked (fivefold reduction) in the dentate granular layer and also extensive in the pyramidal layer of CA1 and CA2, in the subiculum, presu- biculum, and in the lower layers of the parahippocampal gyms. In some areas such as the pyramidal layer and stratum lacunosum moleculare of CA1, the pyramidal layers of CA2, CA3, subiculum, and presubiculum and lower layer of parahippocampal gyrus bind- ing in the neonates was the same as or higher than that in the fetus, indicative of peak activity in late gestation. In the lower parahip- pocampal gyral layer binding in neonate was signifcantly higher than in the fetus. In contrast to MK801 and CNQX, kainate bind- ing was evident at the fetal stage in the stratum lacunosum mo- leculare, although this was relatively low and did not fall so dra- matically as in other regions after birth. In most areas binding stabilized at low levels by the age of 20 years. There were no significant alterations in binding from adulthood to old age and no significant changes from middle to old age in any area examined.
DISCUSSION
Each of the binding sites investigated demonstrated a totally different trend during development. MKS01 binding to the NMDA receptor complex in most areas apparently reached adult levels by 24 gestational weeks. The transient peaks in receptor densities, reported in developing rodent brain (3,57), are evident perinatally for the other subtypes examined in the present human brain analy- sis and presumably reflect subunit synthesis at an even earlier fetal stage. In certain regions, particularly stratum radiatum of CA1- CA3, there was a loss of binding between the fetal and young adult group, consistent with the report of Represa et al. (49) that the density of NMDA displaced glutumate binding decreases between fetus (23-27 gestational weeks) and adult in stratum lucidum of CA3. In contrast, the present analysis suggests that in other areas, particularly the dentate granular layer, density of MKS01 binding may increase perinatally. The overall trends in human hippocam- pus and adjacent cortex, reported here, contrast with that in human cerebellum (21), where MK801 binding rises from low levels in the fetus to reach adult levels in the neonate and early childhood. Evidence that polyamines such as spermidine inhibit MK801 bind- ing in fetus have little effect in neonate and are stimulatory in the adult (44) complicate interpretation of these data because it is not known if such developmental changes in the NMDA modulatory site are regionally specific. Another modulatory change occurring during development that may complicate interpretation of ligand binding in human temporal cortex is the fourfold stimulation of MK801 binding by glycine (and glutamate) evident in the 6-month neonate compared to adult (55).
Because kainate binding declined dramatically from the fetal period to childhood, it can be presumed that peak activity occurred during late gestation. This observation is consistent with that of Represa et al. (50) of a transient high density of kalnate binding in the supragranular layer of the dentate fascia of the human neonate considered to correspond with maturation of mossy fibers. In ro- dent brain, kainate binding did not peak until after postnatal day 14 (37,50) in keeping with the later developmental stage of rat com- pared to human neonatal brain. Binding to the AMPA receptor did not peak until after birth--in some areas not until middle age; a similar trend has been observed in human cerebellum (21). As- suming that periods of elevated binding coincide with transient
646 JOHNSON ET AL.
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HUMAN HIPPOCAMPUS GLUTAMATE RECEPTORS AND AGING 647
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FIG. 6. NMDA (3H-MK801) binding in human hippocampus and parahippocampal gyms in normal individuals grouped according to age. Presentation as in Fig. 2; abbreviations as in Fig. 1.
synaptic overexpression, then the late staging of the AMPA maxi- mum is of particular interest because it suggests this glutamate receptor type is associatext with synaptogenesis occurring during adolescence and in some areas during adulthood, presumably in conjunction with environmental input.
If periods of synaptogenesis and synaptic exuberance are asso- ciated with heightened sensitivity to toxicological or traumatic/ ischaemic damage, then deanage to the hippocampus incurred dur- ing fetal development is mast likely related to the NMDA subtype. Perinatally induced insult~; are more likely related to the kainate
subtype and damage during childhood to the AMPA subtype. These differential sequences in the hippocampus may be relevant to developmentally related disorders including mental handicap, associated with learning difficulties. Because temporal regions such as the entorhinal cortex have been implicated in schizophre- nia, it would be logical on the basis of the present data and possible role of glutamate to implicate the NMDA or kainate but not AMPA subtypes in any hypothesis of this disorder based on fetal trauma and AMPA or kainate subtypes in perinatal or childhood insults.
648 JOHNSON ET AL.
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FIG. 7. Kainate (3H-kainic acid) binding in human hippocampus and parahippocampal gyms in normal individuals grouped according to age. Presentation and abbreviations as in Figs. 1 and 2.
HUMAN I-IIPPOCAMPUS GLUTAMATE RECEPTORS AND AGING 649
In relation to senescence, the CA1 sector of the hippocampus and the entorhinal cortex are particularly vulnerable to Alzheimer- type pathologic changes such as neurofibrillary tangle formation, neuron loss, and [3-amyloid plaques. In the hippocampus and ad- jacent cortex there were no significant reductions in MK801 or kainate binding between the ages of 20 and 100 years. This con- trasts with some reports in rat and monkey hippocampus that NMDA receptor binding is reduced with age (5,56,60), although more recently no change in [aH]CGS 19755 was reported (25). In the present analysis of hum~tn brain there was, particularly in CA1 and the subicular complex, evidence of elevated binding in the elderly (over 80 years) possibly related to compensatory, axonal sprouting. Although there were significant reductions in AMPA binding between the ages of 20 and 100 years that were particu- larly marked in CA1--CA3 and subicular complex, there was little evidence of changes over the age of 40 years.
In the present series of individuals examined over the age of 50 years parallel investigations of the extent of ~3-amyloidosis as- sessed on the basis of 13-A4 immunoreactivity indicated the pres- ence of 13-amyloidosis in the entorhinal cortex or parahippocampal gyms in eight cases [(42); Griffiths et al., in preparation]. How- ever, there was no significant difference in the binding levels in parahippocampal gyms of any of the three glutamate receptor sub- types between those with and without 13-amyloidosis. In contrast, the nicotinic cholinergic receptor undergoes a significant decline with age in entorhinal cortex after maturity and differentiates nor- mal individuals with and without 13-amyloidosis (42). Together with the absence of any marked changes in the three glutamate receptor subtypes from midLdle to old age in entorhinal cortex this comparison suggests that ligand binding to the glutumate receptor subtypes examined are unlikely to be closely involved in the early stages of Alzheimer type pathology. This important conclusion is compatible with the absence of any concrete evidence for a direct involvement of excitatory amino acids in Alzheimer's disease, despite the attraction of the original glutamate hypothesis (16). The exact linkage of glutamate receptors to diseases such as Alzhei- mer's disease in terms of events downstream from receptor acti- vation remains, however, to be explored.
There are intriguing regional variations in aging within the different areas examined :[or each of the receptor types. These include the absence of CNQX or MK801 binding but presence of kainate binding in the stratum lacunosum moleculare in the fetal compared to neonatal brain. Also, the fact that CNQX binding continues to rise up to middle age in some areas--notably in certain layers of the subicular complex--suggests that attempts to separate developmental from adult periods may have to incorpo- rate the notion that the adtflt brain is in some respects still devel-
oping, in the course of adult life. A similar observation was pre- viously made in relation to the pattern of choline acetyltransferase in the human hippocampus that does not reach a maximum until middle age (43). The continued development of EAA synapses throughout adult life is not apparent for the kainate subtype, which remains relatively constant from late childhood to old age.
In future analyses of alterations in glutamate receptors in the developing and aging human brain, expression of mRNA species of the various molecular forms needs to be examined (53). In rodent brain, various developmental changes have been reported including increased mRNA for NRI and NR2A in hippocampus between 7 and 20 postnatal days (27,62); transient expression of NR2 in hippocampus (48); progressive alterations in the subunit composition of heteromeric NMDA receptors in cortex (54); in- creases in AMPA GluR-A to -D Flop but not Flip variants post- natally (34); peak GLUR-5 at birth in hippocampal CA1 interneu- rons and KA1 in CA3 and dentate fascia, KA2 expression occur- ring much earlier and remaining constant throughout life (1); and a decline in GLURI and GLUR2 mRNA in aged compared to young hippocampus (37). It is difficult to make direct comparisons between perinatal changes in glutamate receptor mRNA in rodent and receptor binding in human brain because the two species differ in their developmental staging at birth and receptor mRNA and synaptic binding are not necessarily colocalized nor does the level of the one invariably reflect the other. Nevertheless, it is intriguing to consider the relation between the peri- and postnatal increase and subsequent decline in AMPA and kainate binding in the hu- man hippocampus observed in the present study and the increase in Glu RI and Glu R3 to Gin R2 gene expression in the developing rat hippocampus between postnatal day 7-21, following by a decline. A high value in this subunits ratio is associated with increased Ca 2÷ permeability and there may be a developmental switch in which Ca 2÷ permeable glutamate receptors are turned off follow- ing early developmental events (38). Postmortem human brain is not ideal for mRNA analyses due to the susceptibility of RNA to effects of perimortem factors (39). Examination of the different molecular species using specific antibodies in appropriately fixed material is likely to be a more productive approach. Ultimately, however, it is the pharmacology and physiology of the molecules that most directly governs their role in development and aging.
ACKNOWLEDGEMENTS
We are most grateful to Dr. Chris Wright for providing access to paediatric autopsy material, to Andrew Brown for preparing the graphs, to Arthur Oakley for help with the photographs, and to Lorraine Hood for preparing the manuscript.
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