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N-methyl-d -aspartate receptors in the cortex and hippocampus of baboon (Papio anubis andPapio papio)
Neuroscience
Vol. 32, No.
Printed in Great Britain
I, pp. 39-47,
1989
0306-4522/89
$3.00 + 0.00
Pergamon Press plc 0 1989 IBRO
N-METHYL-D-ASPARTATE RECEPTORS IN THE CORTEX AND HIPPOCAMPUS OF BABOON (PAP10 ANUBIS AND PAPIO PAPIO) J. W. GEDDES,*?$ S. M. COOPER,* C. W. COTMAN,* S. PATEL$ and B. S. MELDRUM$ *Department
of Psychobiology
SDepartment
of Neurology,
and tDivision of Neurosurgery, University of California, Irvine, CA 92717, U.S.A. Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 SAF, U.K.
Abstract-hoitro autoradiography
was used to examine the N-methyl-o-aspartate receptor in the brain of a baboon species, Papio anubis, and compared to that of Pupio papio which exhibits a photosensitive epilepsy. The epilepsy originates in the frontal cortex and is accompanied by an enhanced sensitivity to N-methyl-D-aspartate. In both Papio anubis and Pupio papio, the density of N-methyl-D-aspartate receptors was greatest in the hippocampus, followed by associational areas including frontal cortex, and low in primary sensory areas such as the visual cortex. The receptors were concentrated in the outer
cortical layers I-III, very low in layer IV except in primary visual cortex, and of intermediate density in layer V. The density of binding sites was approximately two-fold lower than previously observed in the rodent brain, whereas the affinity of the receptor for [‘H&-glutamate was greater in the primate versus the rodent brain. Glycine potentiated the binding of [3H]L-glutamate in both cortex and hippocampus. No significant differences in the properties of N-methyl-D-aspartate receptors were observed between the two baboon species, suggesting that the photosensitivity of Papio pupio is not due to alterations in the binding of L-glutamate to the N-methyl-D-aspartate receptor complex.
physiological abnormalities in Pupio papio might be accompanied by alterations in NMDA receptors. The frontal/rolandic zone in which the paroxysmal discharges originate2’ shows abnormal extracellular [Ca2+] decreases during sustained tonic clonic seizures.29 An increase in the calcium response has been shown to correlate with enhanced sensitivity in other seizure models (e.g. the cobal focus of rat,30 and the kindling epilepsy12) in which there was an altered laminar profile of the decrease in extracellubr [Ca2+] following the microphoretic application of NMDA. Further evidence for the possible involvement of NMDA receptors is that antagonists of excitation due to NMDA possess anticonvulsant activity and abolish the myoclonic activity in Papio pupio.” To provide a survey of the distribution of the NMDA receptor in the primate brain, and to examine the hypothesis that alterations in L-glutamate binding to the NMDA receptor might underlie the photosensitivity in Pupio pupio and account for the enhanced sensitivity to NMDA as compared to Pupio anubis, the properties of the NMDA receptor examined in the present study included the regional and laminar distribution, density, affinity, and potentiation of [3H]L-glutamate binding by glycine. We focused on the frontal cortex, the site of origin of the paroxysmal discharges,20 and on the hippocampus, a region vulnerable to sustained seizure activity.32
The N-methyl-D-aspartate (NMDA) receptor has recently been the focus of considerable interest.4.6 In addition to its role in mediating excitatory amino acid transmission, activation of the NMDA receptor appears to be required for certain types of memory and to play a pivotal role in the development of epileptic seizures and the neuronal damage resulting from seizure activity, ischemia, and hypoglycemia.‘6,‘7.‘8,34,37The distribution of the NMDA receptor has previously been characterized in the rodent brain7x’0,2s and the human hippocampus’ but not in the primate brain. Previous autoradiographic investigations of [3H]L-glutamate binding in the primate brain were performed in the presence of added Cl- ions,“.36 conditions under which the majority of the binding is to non-NMDA sites.‘,2~28,38To survey the NMDA receptors in the cortex and hippocampus of the baboon, Papio anubis, we used in vitro autoradiography and conditions designed such that the [3H]L-glutamate binding was almost entirely displaceable by NMDA. The results were compared to those obtained in a second baboon species, Papio papio, which exhibits a photosensitive epilepsy” and has been used as an experimental model of the human condition.” Previous studies have suggested that electrofTo
whom correspondence should be addressed at the Division of Neurosurgery, Rm B140, Medical Science I, University of California, Irvine, CA 92717, U.S.A. Abbreviations: NMDA, N-methyl-D-aspartate; SITS, 4-acetamido-4’-isothiocyano-2,2’-disulphonic acid.
EXPERIMENTAL Two
photosensitive
baboons
PROCEDURES (Pupio papio)
from
the
Casamance region of Senegal (one male, one female, with 39
.I. W. GEDDES et ul
40
body weights of 6.5 and 5.5 kg, the wet weight of the brains being 149.7 and 144.6g, respectively) and two control animals that did not show photosensitivity (Pupi0 unubis) (males. weirrhine 9.8 and 8.5 kg, the wet weights of the respective byainc being 183.6 and 186.7 g) were very lightly perfusion fixed with 0.1% phosphate-buffered formalin through a transcardiac cannula while under sodium pentobarbital (Nembutal) anaesthesia. In preliminary experiments, this treatment had no effect on the density or distribution of NMDA receptor binding in rat brain tissue. The baboon brains were cut into 7-mm-thick coronal blocks, frozen in isopentane at -70 to -8O’C and stored at -70°C until required. The frozen sections were processed for autoradiography using previously published methods.s.25,26 Briefly, the regions of interest were dissected from coronal brain sections and 6-pm tissue sections were thaw-mounted onto gelatincoated microscope slides. The tissues from Pupio pupio and Papio anubis were placed adjacent to each other on the same slide to ensure that any observed differences were not due to variations in experimental conditions. The slides were stored frozen for 16 h and then thawed and placed into 50 mM Tris-acetate buffer, pH 7.2, for at least 1 h at O-3°C. The tissue sections were then incubated for 10 min at 30°C in the same buffer to further remove endogenous glutamate and various ions, The slides were then incubated under conditions designed to specifically label the L-glutamate binding site of the NMDA receptor complex. Tissue sections were incubated for 10min at O-3°C in
0.9ml
of 50mM
Tris-acetate
buffer, pH 7.2, contain-
ing 100 nM [‘H]L-glutamate (50 Ci/mmol, New England Nuclear). Quisqualate (IOpM) and the chloride channel blocker 4-acetamido-4’-isothiocyano-2,2’-disulphonic acid (SITS, 100 PM) were also included in the incubation buffer to minimize binding to non-NMDA sites. Non-specific binding was determined by including 200 PM NMDA in the incubation buffer. For determination of K,, and B,,,,,, the same conditions were used except that the concentrations of [sH]L-glutamate were varied between 12.5 and 400 nM. Kinetic parameters were determined using both Scatchardj5 and EadieHofstee plots. To examine the effect of glycine on [3H]L-glutamate binding to the NMDA-sensitive site, 5 PM glycine (Sigma, St Louis, MO) was added to the preincubation, incubation, and rinse buffers. Following incubation, unbound radioactivity was removed by rinsing the slides in a series of four Coplin jars with ice-cold buffer for a total time of 30 s. Sections were then dried under a stream of cool air and placed into X-ray cassettes with tritiumsensitive film (LKB instruments, Gaithersburg MD or Amersham, Arlington Heights, IL). Autoradiograms were analysed by computer-assisted densitometry with an MCID image processing system (St Catherines, Ontario, Canada). The tissue equivalent was calibrated using [‘Hlmethacrylate tritium standards (Amersham, Arlington Heights, IL). Films were exposed for 4-6 weeks at 4 C, followed by development in Kodak D-19 at 20°C. Receptor localization was evaluated by comparison with corresponding 20-pm sections stained with Cresyl Violet.
RESULTS
NMDA-sensitive [3H]L-glutamate binding sites were concentrated in telencephalic regions, including cortex and hippocampus, and were low to moderate in the basal forebrain, basal ganglia, and thalamic nuclei. Within the cerebellum, receptor density was higher in the granule cell layer than in the molecular layer (Fig. 1). The highest density of NMDA receptors was in the hippocampal formation. Binding densities were
greatest in the inner molecular layer of the dentate gyrus and in stratum radiatum/pyramidale of hippocampal area CA1 (Table 1, Fig. 2) where it was difficult to distinguish between dendritic and somatic fields in the autoradiograms. Within the hilus, binding densities were moderate with a slightly greater density being observed in the outer cellular layer. The density of NMDA receptors in CA3 was very low. particularly in stratum pyramidale/lucidum, the terminal field of the mossy fibres which has a high density of the kainate subclass of excitatory amino acid receptors. 9.24A low density of NMDA receptors was also observed in the subiculum. Throughout the cortex of Pupia anubis, NMDAdisplaceable [‘H]L-glutamate binding sites were concentrated in the outer cortical layers I-III, were sparse in layer IV, and an additional band of moderate receptor density was localized to layer V (Fig. 1). In the primary visual cortex (Brodmann’s area 17). receptor density was very low in the inner cortical layers except for an additional band of NMDA receptors observed in layer IV C (Fig. 3). The cortical density of NMDA receptors was lowest in primary sensory areas including primary visual cortex and motor cortex. Higher densities were observed in secondary-sensory and associational cortices including superior frontal, insular, and cingulate cortex (Figs 1 and 3). The density of NMDA receptors in entorhinal cortex was intermediate between the values observed in frontal and visual cortices. [3H]L-Glutamate bound to the molecular layer of the dentate gyrus of Papio anubis with an affinity of 69 nM and a B,,, of 1960 fmol/mg protein (Fig. 5). In Papio pupio, the distribution and density of NMDA receptors were very similar to that observed in Pupio anubis (Table 1, Figs 1 and 2). The kinetic values observed in Pupio pupio were also very similar, with the Kd and B,,, values being 57 nM and 1640 fmol/mg protein, respectively (Fig. 5). The corresponding values obtained in CA1 of each baboon species were similar to the values obtained in the molecular layer of the dentate gyrus (data not shown). To evaluate possible differences in the allosteric regulation of the NMDA receptor in Papio papio and Papio anubis, we examined the ability of glycine to enhance the binding of [3H]L-glutamate23 in the frontal cortex (Fig. 4) and hippocampus (Fig. 2, Table 1). In frontal cortex, glycine enhanced the binding of [3H]L-glutamate in all cortical layers, with a maximal effect observed in the outer layers which possess the highest density of NMDA receptors. In both Papio anubis and Pupio pupio, glycine sharpened the laminar profile of NMDA receptors but did not alter the pattern of distribution between inner and outer cortical layers. In entorhinal cortex, the effect of the added glycine was also greatest in the superficial cortical layers (Table 1). Within the hippocampal formation itself, glycine enhanced binding by approximately 40% in all regions (Table 1, Fig. 2). The density of
NMDA receptors in baboon
41
Fig. I, Distribution of [lH]rglutamate binding in coronal sections of (A) Papio anubb (in coronal plane A 14.5B*3’) and (B) Papio papio (in coronal plane A 4.0) under conditions in which the binding is mainly to an NMDA-displaceablesite (see Experimental Procedures; for the full coronal section autoradiograms shown above, the volume of the ligand solution was increased to 3.6ml). Non-specificbinding, determined in the presence of 200,um NMDA, represented less than 10% of the total binding. Light areas represent the highest levels of binding. In both baboon species, the highest levels of binding were observed in the hippocampus. In cortical regions, binding was concentrated in outer cortical layersI-III. Low to moderate binding densities were observed in basal ganglia and thalamic nuclei. Abbreviations: C, cingulate cortex; Cb, cerebellum;Cd, caudate nucleus; E, entorhinal cortex; F, frontal cortex; H, hippocampus; I, insular cortex; Pt, putamen; Pulv, nucleus pulvinaris; V, nucleus ventralis.
[3H]L-glutamate binding sites in the presence of 5 FM glycine was not significantly different between the photosensitive and nonphotosensitive baboons (Table 1, Figs 2 and 4).
NMDA-displaceable
DISCUSSION
The present study examined the anatomical distribution and kinetic properties of the NMDA-sensitive [‘H]L-glutamate binding sites in the brain of two baboon species. One notable difference between the present and previous autoradiographic studies which examined glutamate receptors in the primate primary visual cortex36and human cortical regions” is the absence of added ions in the incubation medium and the use of quisqualate and SITS to prevent [‘H]L-glutamate binding to NMDA-insensitive sites. Quisqualate blocks binding to both the quisqualate and kainate subclasses of excitatory amino acid receptors, whereas SITS blocks a recently described
W-dependent glutamate transport site.“’ These conditions were used previously to characterize NMDA receptors in rat brain autoradiograms26and result in virtually all of the [rH]L-glutamate binding being displaceable by NMDA. Under these conditions, the potencies of the phosphonic acid series of NMDA antagonists, including 2-amino-5-phosphonopentanoate and 2-amino-7-phosphonoheptanoate are similar against displacement of [3H]L-glutamatebinding, NMDA-induced depolarization, and long-term potentiation, all measured in stratum radiatum of Cal .’ Therefore, the binding conditions are such that the pharmacological profile of the binding sites appears related to the pharmacological properties of NMDA receptors, as defined using electrophysiological measures. The distribution of NMDA-displaceable ['H]Lglutamate binding sites in the brain of Pupio anubis and Papio pupio is similar but more heterogeneous than that described previously in the rodent brain. The dense band of NMDA receptors in superficial
42
J. W. GEDDES CI (11. Table 1. Distribution of N-methyl-D-aspartate-displaceable-[3H]L-glutamate binding sites in the hippocampal formation of Papio anubis and Papio papio Region ~____._~ DG-Inner DG-outer Hilus-outer Hilus-inner CA3-oriens CA3-pyr CA3-rad CA 1-oriens CA 1-pyr CA 1-rad CAl-lm Subicuium EC-inner EC-outer
Control Papio anubis Papio papio 1068 If: 176 705 + 130 567 f 147 505 + 158 541 f 131 320 f 132 427 + 116 679 _+ 163 100+243 762 +251 478 f 387 346 * 127 318+73 502 i 95
1254 k 793 + 765 f 628 + 808 t 330 * 549 + 839 f 1155 f 1088 + 723 f 389 + 369 + 627 +
151 249 95 52 206 105 188 30 141 88 130 39 99 234
Glycine (5 PM) added Papio anubir Papio papio 1408 + 112 986 k 247 713 + 173 714 f 199 714 * 141 45Ok231 600+ 130 908 ) 290 1320 + 132 1063 f 148 739 + 533 560~ 133 531 f 69 813 c42
1418 F 157 IO05 + 325 943 + 166 794 * 8 953 & 310 390 + 165 790 + 209 1136 k 105 1434 k 108 1366 i 16 916 k 52 524 + 162 597* 159 929 + 247
These values represent the amount of [3H]L-glutamatebinding (100 nM) inhibited
by the presence of 200 pM NMDA. Values are the averages obtained from two animals k standard deviation. The glycine added values were determined in the presence of 5 PM glycine included in the preincubation, incubation, and rinse buffers. Abbreviations: DC&inner, inner molecular layer of the dentate gyrus; DG-outer, outer molecular layer of the dentate gyrus; EC-inner. inner cortical layers (I-III) of the entorhinal cortex; EC-outer, outer cortical layers (IV-VI) of the entorhinal cortex; Im, stratum lacunosum-moleculare: oriens, stratum oriens: rad. stratum radiatum; pyr, stratum pyramidale.
Fig. 2. Distribution of [‘H]L-glutamate binding in coronal sections through the hippocampal formation of Papio anubis (A, C) and Papio papio (B, D). The autoradiograms in A and B respresent total [‘H]L-glutamate binding and were prepared as described in Fig. 1 and in Experimental Procedures. Autoradiograms C and D were prepared similarly except that 5 PM glycine was added to the preincubation, incubation, and rinse buffers. Representative sections are shown, and the average specific binding (displaceable by 200 PM NMDA) densities are presented in Table I. Abbreviations: H, hilus: Im, stratum lacunosum-moleculare: o. stratum oriens. p, stratum pyramidale; r. stratum radiatum; Sub, subiculurn.
NMDA receptors in baboon
Fig. 3. Dibtribution of [‘H&-glutamate binding in the primary visual cortex (Brodmann area 17) and secondary visual cortex (Brodmann area 18) of Pupiopupio. Conditions were identical to those described in Fig. 1.The arrow indicates the thin band of binding associated with layer IV C in primary visual cortex. This band is not visible in other cortical regions. A greater density of NMDA receptors was present in innner cortical layers (I-III) of secondary vs primary visual cortex.
cortical layers was apparent in all cortical regions in baboon, whereas in the rodent brain a uniform receptor density was observed in the insular, entorhinal, and perirhinal cortices.25 The greater complexity of binding patterns in the primate brain was also evident in the primary visual cortex, where an additional band of moderate receptor density was present in layer IV C. The difference in density between secondary sensory, and associprimary sensory, ational areas was more dramatic than that observed in the rodent brain. For example, the density of NMDA receptors in the frontal cortex was two-fold greater than that in primary visual cortex. Within the hippocampus, the major difference in distribution between the primate and rodent was in CA1 stratum pyramidale which contains a high density of binding sites in the primate brain but a very low density in the rat. This reflects the wider distribution of pyramidal neurons in primate CAl, and the lack of demarcation between cellular and dendritic elements. The boundary between CA1 stratum radi-
atum and pyramidale, clearly visible in the rat, was difficult to distinguish in autoradiograms of the primate hippocampus. The relative densities of NMDA-displaceable [3H]L-glutamate binding sites, determined using 100 nM [3H]L-glutamate, were similar in corresponding regions of the rodent” and primate brain. The similar relative binding densities mask differences in the affinity and B,,, of the two species, however. The B,, in the dentate gyrus molecular layer of Papio anubis and Papio papio (1960 and 1620 fmol/mg, respectively) is approximately two-fold lower than that in hippocampal area CA1 of the rodent (Bm, of 3310 fmol/mg protein), whereas the binding affinity (KJ is greater in the primate (57-69 nM) as compared to the rat brain (240 nM).25 These differences are suggestive of species differences in the L-glutamate binding properties of the NMDA receptor. No significant differences were observed between Papio papio and Papio anubis in the regional or laminal distribution, density, or affinity of NMDA
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Distance (mm) Fig. 4. (A) Distribution of NMDA receptors in the frontal/rolandic cortex of Papio pclpio. (B) Represents an enlarged view of the region contained by the white rectangle in A. Light areas represent the highest levels of binding The density proIik in (C) indicates the avcroge density of [‘I+&-ghttamate binding (fmol/mg protein) at various points across the frontal cortex of Papio ombis, beginning with the outer cortex (Layer I). The individual points represent the averag density in a singk cohmm of one pixel width (0.1 mm) as measured within a rectangle similar to that shown in A and B. The density profile for the same region in Papio papio is shown in @). In both C and D, the lower curve represents binding densities determined in the presence of IOOnM [3H]t_-ghrtamate as indicated in Fig. 1 and in Experimental Rccedures. The upper curve indicates the density of [)HJt,-glutamate binding sites in the presence of 5 pm glycine. The results were obtained from coronal sections at level A 20.“.” 44
NMDA receptors in baboon
45
1600
~H1_L-Qlumma6
C6n66ntmtbn
(nw)
Fig. 5. Specific [3H]L-glutamatebinding to the molecular layer of the dentate gyrus in Pupio unubis (open circles) and Pupio pupio (solid circles). Values were obtained from autoradiograms of coronal brain tissue sections adjacent to those shown in Fig. 2. Glutamate bound represents the specific binding (displaceable by 200 pM NMDA) under the conditions described in Experimental Procedures. The concentrations of [‘Hlrglutamate used ranged from 12.5 to 4OOnM. The inset shows Scatchard analysis of the binding data,33 with the solid line representing the curve obtained for Papio anubis and the dashed line corresponding to Pupio papio. The calculated Kd and E,,,,, values are presented in Results.
receptors. The lack of change in the laminar distribution of NMDA receptors in the frontal cortex contrasts with an abnormally large decrease in extracellular [Ca2+ ] during generalized seizures in Pupio pupio, which is thought to involve the calcium channel associated with the NMDA receptor complex based on results obtained in the rat cobalt model of chronic motor seizures.29*30In control rats, the maximal decrease in extracellular [Ca*+ ] following the microphoretic application of NMDA decrease mirrors the NMDA receptor distribution, being most marked in laminae II-III and V. In epileptic tissues, this response is enhanced and shifts to become monophasic, involving layer IV. The lack of a corresponding shift in NMDA receptor density in Pupio pupio suggests that the calcium response does not simply represent an increased density of NMDA receptors and the associated ion channel. It has recently been reported that glycine potentiates NMDA responses via an allosteric mechanism.14 The distribution of strychnine-insensitive [3H]glycine binding sites3 is very similar to that observed for NMDA-sensitive [3H]L-glutamate binding sites7x2rand glycine has recently been shown to enhance NMDA-sensitive [3H]L-glutamate binding in certain regions of the rat brain.23 To examine the possibility that there may be an altered allosteric regulation of the NMDA receptor in Pupio pupio as compared to Pupio anubis, we examined the ability of glycine to potentiate NMDA-sensitive [‘H]r_-glutamate binding in the frontal/rolandic cortex and hippocampus of the two baboon species. No differences were observed in the profile or magnitude of the stimulation (Fig. 4). These results suggest that an altered allosteric regulation, via glycine, of the NMDA receptor does not underlie the photo-
sensitivity of Papio pupio or the altered calcium response to NMDA described above. A greater reduction of extracellular [Ca2+] in response to microphoretically applied NMDA is also observed in the hippocampal area CA1 of kindled as compared to control animals.” In addition, there is an enhanced inihibition by NMDA of carbacholstimulated phosphatidylinositol tumover.27 Furthermore, in kindled but not control animals, NMDA antagonists intefere in the synaptic activation of granule cells by the perforant path.21 In Pupio pupio, the hippocampus is vulnerable to sustained seizure activity. 32 In the present study, however, no differences were observed in the hippocampal formation in the density or distribution of NMDA receptors, kinetic parameters, or the potentiation by glycine of [‘H]r_-glutamate binding to the NMDA receptor (Table 1, Figs 2 and 5). Given the correspondence between the pharmacology of NMDA-sensitive [3H]L-glutamate binding and electrophysiologically-defined NMDA receptors,’ it is significant that no changes were detected in any of the binding parameters examined which might contribute to the observed changes in electrophysiological and biochemical measures. The results demonstrate that the altered sensitivity to NMDA must occur at a level distinct from the binding of L-glutamate to the NMDA receptor complex. Possibilities include alterations at another level of allosteric control, receptor phosphorylation2* regulation of the ion channel, or the coupling of the NMDA receptor complex to second messenger responses. Acknowledgements-We thank the Wellcome Trust for financial support. J.W.G. is a National Down Syndrome Society Science Scholar.
46
J.
w.
GEDDES C’f rd
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(1988) Requirement of NMDA receptor/channels for intracellular high-energy phosphates and the extent of intraneuronal calcium buffering in cultured mouse hippocampal neurons. Neurosci. Leit. 93, 73378. D. T., Olverman H. J.. Nguyen L., Watkins J. C. and Cotman C. W. (1988) Two classes of NMDA 23. Monaghan recognition sites: differential distribution and differential regulation by glycine. Proc. nutn. Acud. Sci. U.S.A. 85, 98369840. D. T. and Cotman C. W. (1982) Distribution of [3H]-kainic acid binding sites in rat CNS as determined 24. Monaghan by autoradiography. Brain Res. 252, 91~100. D. T. and Cotman C. W. (1985) Distribution of N-methyl-D-aspartate-sensitive L-[‘HIglutamate-binding 25. Monaghan sites in rat brain. J. Neurosci. 5, 2909-2919. D. T. and Cotman C. W. (1986) Identification and properties of NMDA receptors in rat brain synaptic 26. Monaghan plasma membranes. Proc. natn. Acad. Sci. U.S.A. 83, 7532-7536. 27. Morrisett R. A.. Nadler J. V. and McNamara J. 0. (1987) Evidence for enhanced N-methyl-D-aspartate receptor mediated inhibition of carbachol-stimulated phospho-inositide hydrolysis from kindled rats. Ser. Neurosci. Ahslr. 13, 946. L-[3H]ghrtamate binding, a new receptor or a 28. Pin J. P. Bockaert J. and Recasesn M. (1984) The Ca*+/Cl--dependent particular transport process? Fedn Eur. hiochem. Sots LPII. 175, 31-36. U.. Louvel J. and Silva-Barrat C. (1985) Chemical synaptic transmission is not 29. Pumain R., Menini C., Heinemann necessary for epileptic seizures to persist in the baboon Papio papio. Expl Neural. 89, 250-258. alterations in amino acid-induced ionic conductances in 30. Pumain R., Louvbel J. and Kurcewicz I. (1986) Long-term chronic epilepsy. In E.xcitatory Amino Acids and Epiiepsv (eds Schwartz R. and Ben-Ari Y.), pp. 439447. Plenum. New York. A., Bert J. and Naquet R. (1968) Atlas stereotaxique du ceroeuu de bubouin (Papio papio). 31. Riche D.. Christolomme Travail real& avec’ lu collaboration de J. M. Huguenin, J. Lavielle. Editions du Centre national de la recherche scientifique, Paris. D. and Naquet R. (1971) Frequent attacks and anatomical lesions in the photosensitive 32. Riche D., Gambarelli-Dubois Pupio papio. Electroenceph. clin. Neurophysiol. 30, 371. 33. Ross D. and Huffman R. D. (1968) A Sfereotaxic Atlas of the Brain of the Baboon (Papio). University of Texas Press, TX.
NMDA receptors in baboon
47
34. Rothman S. M. and Olney J. W. (1987) Excitotoxicity and the NMDA receptor. Trenris Neurosci. 10, 299-302. 35. Scatchard G. (1949) The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 6-72. 36. Shaw C. and Cynader M. (1986) Laminar distribution of receptors in monkey (Macaca fisicularis) geniculostriate system. J. camp. Neurol. 248, 301-312. 37. Simon R. P., Schmidley J. W., Meldrum B. S., Swan J. H. and Chapman A. G. (1986) Excitotoxic mechanisms in hypoglycaemic hippocampal injury. Neuropaih. appl. Neurobiol. 12, 567-576. 38. Zazczek R., Arlis S., Mark1 A., Murphy T., Drucker H. and Coyle J. T. (1987) Characteristics of chloride-dependent incorporation of glutamate into brain membranes argue against a receptor binding site. Neuropharmacology 26, 281-287. (Accepted
15 March 1989)
Vol. 32, No.
Printed in Great Britain
I, pp. 39-47,
1989
0306-4522/89
$3.00 + 0.00
Pergamon Press plc 0 1989 IBRO
N-METHYL-D-ASPARTATE RECEPTORS IN THE CORTEX AND HIPPOCAMPUS OF BABOON (PAP10 ANUBIS AND PAPIO PAPIO) J. W. GEDDES,*?$ S. M. COOPER,* C. W. COTMAN,* S. PATEL$ and B. S. MELDRUM$ *Department
of Psychobiology
SDepartment
of Neurology,
and tDivision of Neurosurgery, University of California, Irvine, CA 92717, U.S.A. Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 SAF, U.K.
Abstract-hoitro autoradiography
was used to examine the N-methyl-o-aspartate receptor in the brain of a baboon species, Papio anubis, and compared to that of Pupio papio which exhibits a photosensitive epilepsy. The epilepsy originates in the frontal cortex and is accompanied by an enhanced sensitivity to N-methyl-D-aspartate. In both Papio anubis and Pupio papio, the density of N-methyl-D-aspartate receptors was greatest in the hippocampus, followed by associational areas including frontal cortex, and low in primary sensory areas such as the visual cortex. The receptors were concentrated in the outer
cortical layers I-III, very low in layer IV except in primary visual cortex, and of intermediate density in layer V. The density of binding sites was approximately two-fold lower than previously observed in the rodent brain, whereas the affinity of the receptor for [‘H&-glutamate was greater in the primate versus the rodent brain. Glycine potentiated the binding of [3H]L-glutamate in both cortex and hippocampus. No significant differences in the properties of N-methyl-D-aspartate receptors were observed between the two baboon species, suggesting that the photosensitivity of Papio pupio is not due to alterations in the binding of L-glutamate to the N-methyl-D-aspartate receptor complex.
physiological abnormalities in Pupio papio might be accompanied by alterations in NMDA receptors. The frontal/rolandic zone in which the paroxysmal discharges originate2’ shows abnormal extracellular [Ca2+] decreases during sustained tonic clonic seizures.29 An increase in the calcium response has been shown to correlate with enhanced sensitivity in other seizure models (e.g. the cobal focus of rat,30 and the kindling epilepsy12) in which there was an altered laminar profile of the decrease in extracellubr [Ca2+] following the microphoretic application of NMDA. Further evidence for the possible involvement of NMDA receptors is that antagonists of excitation due to NMDA possess anticonvulsant activity and abolish the myoclonic activity in Papio pupio.” To provide a survey of the distribution of the NMDA receptor in the primate brain, and to examine the hypothesis that alterations in L-glutamate binding to the NMDA receptor might underlie the photosensitivity in Pupio pupio and account for the enhanced sensitivity to NMDA as compared to Pupio anubis, the properties of the NMDA receptor examined in the present study included the regional and laminar distribution, density, affinity, and potentiation of [3H]L-glutamate binding by glycine. We focused on the frontal cortex, the site of origin of the paroxysmal discharges,20 and on the hippocampus, a region vulnerable to sustained seizure activity.32
The N-methyl-D-aspartate (NMDA) receptor has recently been the focus of considerable interest.4.6 In addition to its role in mediating excitatory amino acid transmission, activation of the NMDA receptor appears to be required for certain types of memory and to play a pivotal role in the development of epileptic seizures and the neuronal damage resulting from seizure activity, ischemia, and hypoglycemia.‘6,‘7.‘8,34,37The distribution of the NMDA receptor has previously been characterized in the rodent brain7x’0,2s and the human hippocampus’ but not in the primate brain. Previous autoradiographic investigations of [3H]L-glutamate binding in the primate brain were performed in the presence of added Cl- ions,“.36 conditions under which the majority of the binding is to non-NMDA sites.‘,2~28,38To survey the NMDA receptors in the cortex and hippocampus of the baboon, Papio anubis, we used in vitro autoradiography and conditions designed such that the [3H]L-glutamate binding was almost entirely displaceable by NMDA. The results were compared to those obtained in a second baboon species, Papio papio, which exhibits a photosensitive epilepsy” and has been used as an experimental model of the human condition.” Previous studies have suggested that electrofTo
whom correspondence should be addressed at the Division of Neurosurgery, Rm B140, Medical Science I, University of California, Irvine, CA 92717, U.S.A. Abbreviations: NMDA, N-methyl-D-aspartate; SITS, 4-acetamido-4’-isothiocyano-2,2’-disulphonic acid.
EXPERIMENTAL Two
photosensitive
baboons
PROCEDURES (Pupio papio)
from
the
Casamance region of Senegal (one male, one female, with 39
.I. W. GEDDES et ul
40
body weights of 6.5 and 5.5 kg, the wet weight of the brains being 149.7 and 144.6g, respectively) and two control animals that did not show photosensitivity (Pupi0 unubis) (males. weirrhine 9.8 and 8.5 kg, the wet weights of the respective byainc being 183.6 and 186.7 g) were very lightly perfusion fixed with 0.1% phosphate-buffered formalin through a transcardiac cannula while under sodium pentobarbital (Nembutal) anaesthesia. In preliminary experiments, this treatment had no effect on the density or distribution of NMDA receptor binding in rat brain tissue. The baboon brains were cut into 7-mm-thick coronal blocks, frozen in isopentane at -70 to -8O’C and stored at -70°C until required. The frozen sections were processed for autoradiography using previously published methods.s.25,26 Briefly, the regions of interest were dissected from coronal brain sections and 6-pm tissue sections were thaw-mounted onto gelatincoated microscope slides. The tissues from Pupio pupio and Papio anubis were placed adjacent to each other on the same slide to ensure that any observed differences were not due to variations in experimental conditions. The slides were stored frozen for 16 h and then thawed and placed into 50 mM Tris-acetate buffer, pH 7.2, for at least 1 h at O-3°C. The tissue sections were then incubated for 10 min at 30°C in the same buffer to further remove endogenous glutamate and various ions, The slides were then incubated under conditions designed to specifically label the L-glutamate binding site of the NMDA receptor complex. Tissue sections were incubated for 10min at O-3°C in
0.9ml
of 50mM
Tris-acetate
buffer, pH 7.2, contain-
ing 100 nM [‘H]L-glutamate (50 Ci/mmol, New England Nuclear). Quisqualate (IOpM) and the chloride channel blocker 4-acetamido-4’-isothiocyano-2,2’-disulphonic acid (SITS, 100 PM) were also included in the incubation buffer to minimize binding to non-NMDA sites. Non-specific binding was determined by including 200 PM NMDA in the incubation buffer. For determination of K,, and B,,,,,, the same conditions were used except that the concentrations of [sH]L-glutamate were varied between 12.5 and 400 nM. Kinetic parameters were determined using both Scatchardj5 and EadieHofstee plots. To examine the effect of glycine on [3H]L-glutamate binding to the NMDA-sensitive site, 5 PM glycine (Sigma, St Louis, MO) was added to the preincubation, incubation, and rinse buffers. Following incubation, unbound radioactivity was removed by rinsing the slides in a series of four Coplin jars with ice-cold buffer for a total time of 30 s. Sections were then dried under a stream of cool air and placed into X-ray cassettes with tritiumsensitive film (LKB instruments, Gaithersburg MD or Amersham, Arlington Heights, IL). Autoradiograms were analysed by computer-assisted densitometry with an MCID image processing system (St Catherines, Ontario, Canada). The tissue equivalent was calibrated using [‘Hlmethacrylate tritium standards (Amersham, Arlington Heights, IL). Films were exposed for 4-6 weeks at 4 C, followed by development in Kodak D-19 at 20°C. Receptor localization was evaluated by comparison with corresponding 20-pm sections stained with Cresyl Violet.
RESULTS
NMDA-sensitive [3H]L-glutamate binding sites were concentrated in telencephalic regions, including cortex and hippocampus, and were low to moderate in the basal forebrain, basal ganglia, and thalamic nuclei. Within the cerebellum, receptor density was higher in the granule cell layer than in the molecular layer (Fig. 1). The highest density of NMDA receptors was in the hippocampal formation. Binding densities were
greatest in the inner molecular layer of the dentate gyrus and in stratum radiatum/pyramidale of hippocampal area CA1 (Table 1, Fig. 2) where it was difficult to distinguish between dendritic and somatic fields in the autoradiograms. Within the hilus, binding densities were moderate with a slightly greater density being observed in the outer cellular layer. The density of NMDA receptors in CA3 was very low. particularly in stratum pyramidale/lucidum, the terminal field of the mossy fibres which has a high density of the kainate subclass of excitatory amino acid receptors. 9.24A low density of NMDA receptors was also observed in the subiculum. Throughout the cortex of Pupia anubis, NMDAdisplaceable [‘H]L-glutamate binding sites were concentrated in the outer cortical layers I-III, were sparse in layer IV, and an additional band of moderate receptor density was localized to layer V (Fig. 1). In the primary visual cortex (Brodmann’s area 17). receptor density was very low in the inner cortical layers except for an additional band of NMDA receptors observed in layer IV C (Fig. 3). The cortical density of NMDA receptors was lowest in primary sensory areas including primary visual cortex and motor cortex. Higher densities were observed in secondary-sensory and associational cortices including superior frontal, insular, and cingulate cortex (Figs 1 and 3). The density of NMDA receptors in entorhinal cortex was intermediate between the values observed in frontal and visual cortices. [3H]L-Glutamate bound to the molecular layer of the dentate gyrus of Papio anubis with an affinity of 69 nM and a B,,, of 1960 fmol/mg protein (Fig. 5). In Papio pupio, the distribution and density of NMDA receptors were very similar to that observed in Pupio anubis (Table 1, Figs 1 and 2). The kinetic values observed in Pupio pupio were also very similar, with the Kd and B,,, values being 57 nM and 1640 fmol/mg protein, respectively (Fig. 5). The corresponding values obtained in CA1 of each baboon species were similar to the values obtained in the molecular layer of the dentate gyrus (data not shown). To evaluate possible differences in the allosteric regulation of the NMDA receptor in Papio papio and Papio anubis, we examined the ability of glycine to enhance the binding of [3H]L-glutamate23 in the frontal cortex (Fig. 4) and hippocampus (Fig. 2, Table 1). In frontal cortex, glycine enhanced the binding of [3H]L-glutamate in all cortical layers, with a maximal effect observed in the outer layers which possess the highest density of NMDA receptors. In both Papio anubis and Pupio pupio, glycine sharpened the laminar profile of NMDA receptors but did not alter the pattern of distribution between inner and outer cortical layers. In entorhinal cortex, the effect of the added glycine was also greatest in the superficial cortical layers (Table 1). Within the hippocampal formation itself, glycine enhanced binding by approximately 40% in all regions (Table 1, Fig. 2). The density of
NMDA receptors in baboon
41
Fig. I, Distribution of [lH]rglutamate binding in coronal sections of (A) Papio anubb (in coronal plane A 14.5B*3’) and (B) Papio papio (in coronal plane A 4.0) under conditions in which the binding is mainly to an NMDA-displaceablesite (see Experimental Procedures; for the full coronal section autoradiograms shown above, the volume of the ligand solution was increased to 3.6ml). Non-specificbinding, determined in the presence of 200,um NMDA, represented less than 10% of the total binding. Light areas represent the highest levels of binding. In both baboon species, the highest levels of binding were observed in the hippocampus. In cortical regions, binding was concentrated in outer cortical layersI-III. Low to moderate binding densities were observed in basal ganglia and thalamic nuclei. Abbreviations: C, cingulate cortex; Cb, cerebellum;Cd, caudate nucleus; E, entorhinal cortex; F, frontal cortex; H, hippocampus; I, insular cortex; Pt, putamen; Pulv, nucleus pulvinaris; V, nucleus ventralis.
[3H]L-glutamate binding sites in the presence of 5 FM glycine was not significantly different between the photosensitive and nonphotosensitive baboons (Table 1, Figs 2 and 4).
NMDA-displaceable
DISCUSSION
The present study examined the anatomical distribution and kinetic properties of the NMDA-sensitive [‘H]L-glutamate binding sites in the brain of two baboon species. One notable difference between the present and previous autoradiographic studies which examined glutamate receptors in the primate primary visual cortex36and human cortical regions” is the absence of added ions in the incubation medium and the use of quisqualate and SITS to prevent [‘H]L-glutamate binding to NMDA-insensitive sites. Quisqualate blocks binding to both the quisqualate and kainate subclasses of excitatory amino acid receptors, whereas SITS blocks a recently described
W-dependent glutamate transport site.“’ These conditions were used previously to characterize NMDA receptors in rat brain autoradiograms26and result in virtually all of the [rH]L-glutamate binding being displaceable by NMDA. Under these conditions, the potencies of the phosphonic acid series of NMDA antagonists, including 2-amino-5-phosphonopentanoate and 2-amino-7-phosphonoheptanoate are similar against displacement of [3H]L-glutamatebinding, NMDA-induced depolarization, and long-term potentiation, all measured in stratum radiatum of Cal .’ Therefore, the binding conditions are such that the pharmacological profile of the binding sites appears related to the pharmacological properties of NMDA receptors, as defined using electrophysiological measures. The distribution of NMDA-displaceable ['H]Lglutamate binding sites in the brain of Pupio anubis and Papio pupio is similar but more heterogeneous than that described previously in the rodent brain. The dense band of NMDA receptors in superficial
42
J. W. GEDDES CI (11. Table 1. Distribution of N-methyl-D-aspartate-displaceable-[3H]L-glutamate binding sites in the hippocampal formation of Papio anubis and Papio papio Region ~____._~ DG-Inner DG-outer Hilus-outer Hilus-inner CA3-oriens CA3-pyr CA3-rad CA 1-oriens CA 1-pyr CA 1-rad CAl-lm Subicuium EC-inner EC-outer
Control Papio anubis Papio papio 1068 If: 176 705 + 130 567 f 147 505 + 158 541 f 131 320 f 132 427 + 116 679 _+ 163 100+243 762 +251 478 f 387 346 * 127 318+73 502 i 95
1254 k 793 + 765 f 628 + 808 t 330 * 549 + 839 f 1155 f 1088 + 723 f 389 + 369 + 627 +
151 249 95 52 206 105 188 30 141 88 130 39 99 234
Glycine (5 PM) added Papio anubir Papio papio 1408 + 112 986 k 247 713 + 173 714 f 199 714 * 141 45Ok231 600+ 130 908 ) 290 1320 + 132 1063 f 148 739 + 533 560~ 133 531 f 69 813 c42
1418 F 157 IO05 + 325 943 + 166 794 * 8 953 & 310 390 + 165 790 + 209 1136 k 105 1434 k 108 1366 i 16 916 k 52 524 + 162 597* 159 929 + 247
These values represent the amount of [3H]L-glutamatebinding (100 nM) inhibited
by the presence of 200 pM NMDA. Values are the averages obtained from two animals k standard deviation. The glycine added values were determined in the presence of 5 PM glycine included in the preincubation, incubation, and rinse buffers. Abbreviations: DC&inner, inner molecular layer of the dentate gyrus; DG-outer, outer molecular layer of the dentate gyrus; EC-inner. inner cortical layers (I-III) of the entorhinal cortex; EC-outer, outer cortical layers (IV-VI) of the entorhinal cortex; Im, stratum lacunosum-moleculare: oriens, stratum oriens: rad. stratum radiatum; pyr, stratum pyramidale.
Fig. 2. Distribution of [‘H]L-glutamate binding in coronal sections through the hippocampal formation of Papio anubis (A, C) and Papio papio (B, D). The autoradiograms in A and B respresent total [‘H]L-glutamate binding and were prepared as described in Fig. 1 and in Experimental Procedures. Autoradiograms C and D were prepared similarly except that 5 PM glycine was added to the preincubation, incubation, and rinse buffers. Representative sections are shown, and the average specific binding (displaceable by 200 PM NMDA) densities are presented in Table I. Abbreviations: H, hilus: Im, stratum lacunosum-moleculare: o. stratum oriens. p, stratum pyramidale; r. stratum radiatum; Sub, subiculurn.
NMDA receptors in baboon
Fig. 3. Dibtribution of [‘H&-glutamate binding in the primary visual cortex (Brodmann area 17) and secondary visual cortex (Brodmann area 18) of Pupiopupio. Conditions were identical to those described in Fig. 1.The arrow indicates the thin band of binding associated with layer IV C in primary visual cortex. This band is not visible in other cortical regions. A greater density of NMDA receptors was present in innner cortical layers (I-III) of secondary vs primary visual cortex.
cortical layers was apparent in all cortical regions in baboon, whereas in the rodent brain a uniform receptor density was observed in the insular, entorhinal, and perirhinal cortices.25 The greater complexity of binding patterns in the primate brain was also evident in the primary visual cortex, where an additional band of moderate receptor density was present in layer IV C. The difference in density between secondary sensory, and associprimary sensory, ational areas was more dramatic than that observed in the rodent brain. For example, the density of NMDA receptors in the frontal cortex was two-fold greater than that in primary visual cortex. Within the hippocampus, the major difference in distribution between the primate and rodent was in CA1 stratum pyramidale which contains a high density of binding sites in the primate brain but a very low density in the rat. This reflects the wider distribution of pyramidal neurons in primate CAl, and the lack of demarcation between cellular and dendritic elements. The boundary between CA1 stratum radi-
atum and pyramidale, clearly visible in the rat, was difficult to distinguish in autoradiograms of the primate hippocampus. The relative densities of NMDA-displaceable [3H]L-glutamate binding sites, determined using 100 nM [3H]L-glutamate, were similar in corresponding regions of the rodent” and primate brain. The similar relative binding densities mask differences in the affinity and B,,, of the two species, however. The B,, in the dentate gyrus molecular layer of Papio anubis and Papio papio (1960 and 1620 fmol/mg, respectively) is approximately two-fold lower than that in hippocampal area CA1 of the rodent (Bm, of 3310 fmol/mg protein), whereas the binding affinity (KJ is greater in the primate (57-69 nM) as compared to the rat brain (240 nM).25 These differences are suggestive of species differences in the L-glutamate binding properties of the NMDA receptor. No significant differences were observed between Papio papio and Papio anubis in the regional or laminal distribution, density, or affinity of NMDA
I-..!
0
.i
0.25
0.50
,.--r---1
O.i5
1.00
1125
&
1.75
2.00
2.25
Distance (mm)
I--r--.-,-0
0.25
0.50
.,...I‘
0.75
1.00
.I-’
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1.60
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1.75
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Distance (mm) Fig. 4. (A) Distribution of NMDA receptors in the frontal/rolandic cortex of Papio pclpio. (B) Represents an enlarged view of the region contained by the white rectangle in A. Light areas represent the highest levels of binding The density proIik in (C) indicates the avcroge density of [‘I+&-ghttamate binding (fmol/mg protein) at various points across the frontal cortex of Papio ombis, beginning with the outer cortex (Layer I). The individual points represent the averag density in a singk cohmm of one pixel width (0.1 mm) as measured within a rectangle similar to that shown in A and B. The density profile for the same region in Papio papio is shown in @). In both C and D, the lower curve represents binding densities determined in the presence of IOOnM [3H]t_-ghrtamate as indicated in Fig. 1 and in Experimental Rccedures. The upper curve indicates the density of [)HJt,-glutamate binding sites in the presence of 5 pm glycine. The results were obtained from coronal sections at level A 20.“.” 44
NMDA receptors in baboon
45
1600
~H1_L-Qlumma6
C6n66ntmtbn
(nw)
Fig. 5. Specific [3H]L-glutamatebinding to the molecular layer of the dentate gyrus in Pupio unubis (open circles) and Pupio pupio (solid circles). Values were obtained from autoradiograms of coronal brain tissue sections adjacent to those shown in Fig. 2. Glutamate bound represents the specific binding (displaceable by 200 pM NMDA) under the conditions described in Experimental Procedures. The concentrations of [‘Hlrglutamate used ranged from 12.5 to 4OOnM. The inset shows Scatchard analysis of the binding data,33 with the solid line representing the curve obtained for Papio anubis and the dashed line corresponding to Pupio papio. The calculated Kd and E,,,,, values are presented in Results.
receptors. The lack of change in the laminar distribution of NMDA receptors in the frontal cortex contrasts with an abnormally large decrease in extracellular [Ca2+ ] during generalized seizures in Pupio pupio, which is thought to involve the calcium channel associated with the NMDA receptor complex based on results obtained in the rat cobalt model of chronic motor seizures.29*30In control rats, the maximal decrease in extracellular [Ca*+ ] following the microphoretic application of NMDA decrease mirrors the NMDA receptor distribution, being most marked in laminae II-III and V. In epileptic tissues, this response is enhanced and shifts to become monophasic, involving layer IV. The lack of a corresponding shift in NMDA receptor density in Pupio pupio suggests that the calcium response does not simply represent an increased density of NMDA receptors and the associated ion channel. It has recently been reported that glycine potentiates NMDA responses via an allosteric mechanism.14 The distribution of strychnine-insensitive [3H]glycine binding sites3 is very similar to that observed for NMDA-sensitive [3H]L-glutamate binding sites7x2rand glycine has recently been shown to enhance NMDA-sensitive [3H]L-glutamate binding in certain regions of the rat brain.23 To examine the possibility that there may be an altered allosteric regulation of the NMDA receptor in Pupio pupio as compared to Pupio anubis, we examined the ability of glycine to potentiate NMDA-sensitive [‘H]r_-glutamate binding in the frontal/rolandic cortex and hippocampus of the two baboon species. No differences were observed in the profile or magnitude of the stimulation (Fig. 4). These results suggest that an altered allosteric regulation, via glycine, of the NMDA receptor does not underlie the photo-
sensitivity of Papio pupio or the altered calcium response to NMDA described above. A greater reduction of extracellular [Ca2+] in response to microphoretically applied NMDA is also observed in the hippocampal area CA1 of kindled as compared to control animals.” In addition, there is an enhanced inihibition by NMDA of carbacholstimulated phosphatidylinositol tumover.27 Furthermore, in kindled but not control animals, NMDA antagonists intefere in the synaptic activation of granule cells by the perforant path.21 In Pupio pupio, the hippocampus is vulnerable to sustained seizure activity. 32 In the present study, however, no differences were observed in the hippocampal formation in the density or distribution of NMDA receptors, kinetic parameters, or the potentiation by glycine of [‘H]r_-glutamate binding to the NMDA receptor (Table 1, Figs 2 and 5). Given the correspondence between the pharmacology of NMDA-sensitive [3H]L-glutamate binding and electrophysiologically-defined NMDA receptors,’ it is significant that no changes were detected in any of the binding parameters examined which might contribute to the observed changes in electrophysiological and biochemical measures. The results demonstrate that the altered sensitivity to NMDA must occur at a level distinct from the binding of L-glutamate to the NMDA receptor complex. Possibilities include alterations at another level of allosteric control, receptor phosphorylation2* regulation of the ion channel, or the coupling of the NMDA receptor complex to second messenger responses. Acknowledgements-We thank the Wellcome Trust for financial support. J.W.G. is a National Down Syndrome Society Science Scholar.
46
J.
w.
GEDDES C’f rd
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