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The distribution of [3H]kainic acid binding sites in rat CNS as determined by autoradiography
Brain Research, 252 (1982)91-100 Elsevier Biomedical Press
91
The Distribution of [3H]Kainic Acid Binding Sites in Rat CNS as Determined by Autoradiography DANIEL T. MONAGHAN and CARL W. COTMAN* Department of Psychobiology, University of California, Irvine, CA 92717 (U.S.A.)
(Accepted May llth, 1982) Key words: kainic acid - - excitatory amino acids - - receptor autoradiography
The distribution of [aH]kainic acid (KA) binding sites in the rat CNS was determined by in vitro autoradiography. KA sites are distributed throughout the CNS gray matter in an anatomically specific pattern with telencephalic structures and the cerebellum accounting for the majority of the binding. These results, together with our previous finding that KA sites are greatly enriched at the synapse, suggest that KA binding sites are associated with select terminal fields, and hence may be involved in neurotransmission in certain CNS pathways. INTRODUCTION Kainic acid (KA) is a potent neurotoxin which appears to interact with a specific population of excitatory amino acid binding sites distinct from those which bind L-glutamate4,L K A induces prolonged depolarizations and seizure activity, resulting in lesions of select neuronal populations18,2L Recently, we reported that K A binding sites are greatly enriched in synaptic junctions and that within the rat hippocampus these sites are concentrated in the stratum lucidum 2, a region especially sensitive to the toxic actions of KA12, xS. These results suggest: (1) that K A sites might be associated with specific terminal fields, and hence may possibly be involved in excitatory neurotransmission at certain synapses, and (2) that the same K A sites may be involved in the toxic actions of this excitant. Consequently, it was of interest to determine the distribution of the K A binding sites in the CNS in order to determine the extent to which the density of these sites corresponds to specific terminal fields and to kainate-sensitive regions. Furthermore, since KA-induced lesions of the hippocampus and striatum result in pathologies similar to those occurring in * To whom correspondence should be addressed. 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
status epilepticus lz and Huntington's diseasel, 5, it may be important to understand the involvement of K A binding sites in the induced pathologies. METHODS Anatomical studies Eight male Sprague-Dawley rats (200-300 g) were decapitated, and the brains rapidly removed and frozen in powdered dry ice. In a cryostat, 20/~m horizontal or coronal sections (4 animals each) were thaw-mounted on acid-washed microscope slides. In 3 animals cervical spinal cord sections were sectioned in the transverse plane. Sections were then prepared for autoradiography by the following modification of a ligand-binding autoradiography method 21. Sections were preincubated in 50 m M H E P E S - K O H , p H 7.1, for 10 min in order to remove endogenous glutamate and N a + from the tissue sections. The slides were then transferred to coplin jars containing 30 ml of 100 nM [aH]KA (5.6 Ci/mmol, Amersham, Arlington Heights, IL) for 30 min, again on ice. Of the sections obtained (about 80/animal, coronal plane, 40/animal, horizontal plane), half were incubated in the presence of 100
92 /~M unlabeled KA to obtain measures of non-specific binding. The sections then received two 30-s. rinses in ice-cold buffer. To minimize ligand diffusion, the tissue was rapidly frozen by pressing the bottom of the slide against a slab of dry ice while the excess rinse was aspirated away. The sections were then lyophilized until fully dry (white-opaque appearance) and partially rehydrated over water vapor until translucent in appearance, without the accumulation of water on the section. Rehydration prevented tissue-photographic emulsion interactions which result in chemography. The slides with radiolabeled tissue were clamped to microscope slides which had been acid-washed and dipped into 1:1 diluted NTB-2 emulsion (Eastman Kodak), and the autoradiograms were exposed for 2 months in the dark at 4 °C. The microscope slide with the photographic emulsion was developed in D-19 and the tissue slide was stained for Nissl substance. The anatomical localization of regions differing in silver grain density was made by the comparison of autoradiograms and Nissl-stained sections with two rat brain atlases 16,20. Cortical regions were compared to those described by Krieg 6. Kinetic and pharmacological characteristics
Tissue sections were prepared as above and incubated with a range of [ZH]KA concentrations (5, 10, 25, 50, 100 and 200 nM) in 3 ml chambers. Other sections were incubated with 100 nM [3H]KA in the presence of 5 #M of one of the followingcompounds: kainic acid, L-glutamate, D-glutamate, L-aspartate (Sigma) and quisqualate (a gift from Dr. H. Shinozaki). These sections (4-5 for each condition) were then rinsed (as described above), air-dried, and prepared for the determination of radioactivity by liquid scintillation spectroscopy. Again, non-specific binding was defined as the binding in the presence of 100/~M KA. Hippocampal lesions
Two experimental groups were used, each containing 3 male Sprague-Dawley rats (200-300 g). In one group, hippocampal CA3 and CA4 pyramidal cells were selectively destroyed by intraventricular injections of KA as previously described 12. Dentate gyrus granule cells were removed by injecting 2.5/~g
colchicine, as described elsewhere 3. Both groups of animals were sacrificed one month following the operations, processed for autoradiography as described above, and the slides were allowed to expose for 3 months. RESULTS The binding of [~H]KA to tissue sections at concentrations between 5 and 200 nM exhibited saturation kinetics with a Ka of 69 ± 5 nM and a Bmax of 234 ± 20 fmol/mg protein (mean ~ S.E.M., n = 3, determined by Scatchard analysis). Pharmacological studies indicated that at 5 #M, quisqualate and kainate were potent displacers of the [3H]KA binding observed here; D-glutamate and L-aspartate were ineffective, and L-glutamate intermediate in displacing [3H]KA (n -- 3). Due to the similarities between the kinetic, pharmacological, and regional distribution data found here, and those found for the binding of [3H]KA to rat brain membranesT,S, 19, it is thought that the autoradiographic pattern described below represents the previously described kainate binding sites. Furthermore, KA does not exhibit uptake into rat brain slices and has a 10,000fold higher affinity for its binding sites than for the L-glutamate uptake sitesL Hence, it is unlikely that uptake or uptake site binding can account for the kainate binding observed in this study. Anatomical distribution - - telencephalon
As shown in Figs. 1-4 silver grains due to [3H]KA binding were distributed throughout the rat CNS and in a selective manner. In general, telencephalic structures had a higher density of KA binding sites than did other brain structures. Many cerebral cortical areas exhibited a trilaminar distribution with the superficial and deep layers displaying more binding than the middle layers. In much of the parietal, occipital and temporal cortices, the trilaminar appearance was not well-defined, making cortical layer assignment uncertain. In the retrohippocampal area and cingulate cortex, the trilaminar organization had sharper boundaries. The superficial dense binding layer occupied only layer I of both entorhinal and presubiculum cortices (Figs. 1 and 3), yet both I and II (and possibly III) of cingulate cortex. The deep layers containing a high density of binding sites were layers V and VI.
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Fig. 2. Dark-field autoradiographs of [3 H]KA binding sites in coronal sections. A - F were taken at approximately 2 m m intervals anterior to posterior. Arrow in B indicates the rhinal fissure immediately above which is the insular cortex. H: right, Nissl stain of cervical level spinal cord section corresponding to autoradiograph on left. I: autoradiogram of [aH]KA binding in the presence of
100/zM KA, from a section adjacent to that in E. Abbreviations: O, olfactory bulb; A, amygdala; NA, nucleus aecumbens; PO, preoptic area; GP, globus pallidus; HY hypothalamus; R, reticular nucleus of thalamus; G, cerebellar granule cell layer; M, cerebellar molecular layer; D, dentate nucleus of cerebellum; BS, brainstem; SG, substantia gelatinosa; and VH, ventral horn. Magnifications: A-F, 10x ; G and I, 11 x ; H, 20x)
96 Frontal, insular, perirhinal, and dorso-medial aspects of parietal and occipital cortices exhibited a dense distribution of KA binding sites throughout their layers, with the insular and frontal cortices showing the highest density of cortical KA binding sites (Figs. 1 and 2). The pyriform cortex, olfactory bulb, and amygdala complex had moderately high concentrations of binding sites but did not exhibit any consistant regional variations within these structures (Fig. 2B-E). Within the basal ganglia, a striking selectivity was found. The caudate nucleus (and the nucleus accumbens) had quite high levels of KA binding sites (Figs. 1 and 2B-E). In contrast, the globus pallidus had very low levels of binding (Fig. 2D). The septal and septal fimbrialis nuclei exhibited moderate levels of binding with the lateral septum having slightly higher levels than the medial septum. As we previously reported 2, the hippocampus had low levels overall, with higher levels in two distinct layers. The commissural/associational (C/A) terminal field of the dentate gyrus had moderate levels while the stratum lucidum, termination zone of the granule cell axons (mossy fibers), had the highest density of [aH]KA binding sites of all structures observed (Figs. 1, 2E, F and 3). The retrohippocampal area displayed a complex distribution of KA sites. In Fig. 3 the KA sites of this region are compared to a Timm's stain which likewise differentiates the subdivisions of this region. The presubiculum, parasubiculum, and entorhinal cortices show a trilaminar appearance, whereas the subiculum and perirhinal cortices are not laminated.
Diencephalon, brainstem and cerebellum Thalamus and hypothalamus had relatively low densities of silver grains with the hypothalamus and reticular nucleus of the thalamus having a higher density than the remainder of the thalamus (Fig. 2E). Midbrain and hindbrain structures were associated with quite low and uniform densities of silver grains. The only exception observed was a moderate grain density found in the central gray substance of the midbrain (Fig. 1). The cerebellum exhibited a distinctive pattern of silver grain distribution (Fig. 1 and 2G). The granule cell layer had high levels, the molecular had layer
moderate levels, and the white matter region had negligible levels of silver grains. In autoradiograms of cervical level spinal cord, the substantia gelatinosa consistently showed moderately dense binding (Fig. 2H). In all of the paired controls, the non-specific binding displayed no anatomical specificity except for a slight preference for gray matter over white matter (Fig. 21).
Hippocampal lesions The above data show that KA binding sites are localized to discrete portions of the CNS and hence are specific to select systems in the CNS. However, it has not yet been determined whether the binding sites are pre- or postsynaptic, an issue which directly concerns the mechanism of action of kainic acid. Since the mossy fiber system is the best example of a discrete pathway containing KA sites, and since it may also be selectively lesioned either pre- or postsynaptically, we chose this pathway for evaluation of the cellular localization of the KA sites. Thirty days following the selective destruction of the CA3 pyramidal cells (the postsynaptic component) the high density of silver grains associated with the stratum lucidum was entirely absent (Fig. 4A, B). The grains associated with the C/A layer were unaltered (compared to the contralateral side). Removal of the granule cells of the dentate gyrus (the presynaptic component) also removed the high density of silver grains associated with the stratum lucidum (Fig. 4C, D). However, this lesion also removed the band of grains associated with the C/A layer. Indeed, the entire dentate molecular layer appeared to have a lowered grain density. Another distinction between the two lesions was that removal of the granule cells caused a small increase in grain density within all layers of CA3, except stratum lucidum. DISCUSSION The present study demonstrates that KA binding sites are distributed throughout the CNS in well-defined anatomical zones, and suggests that these sites may be associated with specific pathways. In interpreting these autoradiographic data, it is
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Fig. 3. Horizontal sections of retrohippocampal region. Top: autoradiograph of [aH]KA binding sites. Bottom: similar section stained by the Timm's method with a Nissl counter stain. Abbreviations: MF, mossy fibers; CA, commissural/associational layer of dentate gyrus; PE, perirhinal cortex; E, entorhinal cortex; P, presubiculum and parasubiculum; S, subiculum; CA1, CA3, regions of hippocampus. Asterisks indicate location of mossy fiber and commissural/associational layers. (Magnification 35 x ):
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99 important to know whether the sites mapped are identical to those described in binding studies which exhibit both a high and low affinity. On the basis of pharmacological and kinetic characterization, and the regional distribution of KA sites obtained in this study, it is thought that the sites observed with autoradiography are identical to those described for the binding of [3H]KA to rat brain membranesT,S,19. In addition, the data suggest that the low affinity sites are included in this study. The dissociation constant obtained from tissue sections treated identically to those processed for autoradiography (69 nM) is similar to the low affinity values obtained from other studies (32--66 nM and 59 nM, respectively)S,19. The presence of a low affinity component is also suggested by the observation that the cerebellum has mostly low affinity KA sites s, and the sites observed here are found in high concentrations in the cerebellum. The presence of a high affinity component is difficult to assess due to the very low levels of radioactivity bound at low ligand concentrations. The distribution of [aH]KA binding sites within the rat CNS is anatomically selective. In agreement with studies on the regional distribution of [ZH]KA bindingS, 19, telencephalic structures and the cerebellum account for the majority of KA binding in the rat brain. In cortical structures (cerebellar cortex, hippocampus, and cerebral cortex), the density of KA binding sites exhibited variations among layers as well as among regions. In the basal ganglia there is a clear distinction between the dense distribution of KA sites in the caudate nucleus and the paucity of sites within the globus pallidus. Since KA binding sites are synaptically localized (based on cell fractionatiort studies) z, such anatomical specificity as that described here suggests that KA binding sites ate pathway-specific or transmitter-specific elements, presumably as a component of excitatory amino acid neurotrausmission. Given the select distribution of KA sites, it is predicted that certain regions would be more sensitive to the reversible depolarizing and potentiating actions of KA. This prediction is supported by the available data but, unfortunately, the data are quite limited. Within the spinal cord, interneurons receiving primary afferent innervation are more sensitive to KA than are Renshaw cells13 and, within the
hippocampus, CA3 is approximately 3 orders of magnitude more sensitive than CA11L The regional distribution of the neurotoxic action of KA might likewise be expected to parallel KA site distribution. However, this correspondence is not likely to be as close since part of KA's excitotoxic action is thought to result from KA-induced epileptiform activity11. There is, however, a rough correlation between KA binding site density and sensitivity to KA toxicity. For example, CA3 regions of the hippocampus, amygdala, pyriform cortex, insular cortex and deep layers of other cortical regions are both relatively rich in KA binding sites and preferentially lesioned by low doses of KA, whereas lower brain structures have low concentrations of KA sites and are relatively insensitive to KA toxicitylS,22,. Several instances exist, however, where KA sites do not correspond to areas exceptionally sensitive to KA. As others have noted 10, the caudate nucleus has a high concentration of KA sites, and yet, is not especially sensitive to the toxic action of KA. KA injected either systemically or directly into the caudate nucleusis displays a greater toxic action on the pyriform cortex than on the caudate nucleus. In addition, from these data, it would be predicted that the nucleus accumbens, reticular nucleus of the thalamus and the granule cells of the cerebellum would be selectively destroyed by KA, which apparently is not true is,z2. (However, in the cerebellum it is difficult to know which cells should be affected by KA, given the complex synaptic arrangement at the mossy fiber terminal.) Thus, it may be that a dense distribution of KA binding sites contributes to, but is not sufficient for, KA toxicity. Selective removal of either the pre- or post-synaptic components of the hippocampal mossy fiber pathway results in a loss of the KA binding sites in the stratum lucidum. Consequently, it was not possible to determine which cell has the KA binding sites. It is likely that one of the lesions removed the cells possessing the binding sites, while the other lesion removed the binding sites via a trans-synaptic effect. However, it may be relevant to note that the removal of the the presynaptic component resulted in a modest increase in KA sites throughout the CA3-CA4 region outside of stratum lucidum (Fig. 4D). It is possible that these KA sites are binding sites which would be localized post-synaptically in
100 the mossy fiber synapse if the presynaptic compon e n t were present. Lesions induced by K A resemble the pathologies f o u n d in H u n t i n g t o n ' s diseasO, 5 a n d in status epilepticus 12. Since the results of this study suggest that
then these sites might be c o n t r i b u t i n g to the pathologies f o u n d in H u n t i n g t o n ' s disease a n d epilepsy. ACKNOWLEDGEMENTS
K A sites are largely localized to discrete systems, it
This work was supported by G r a n t M H 19691.
n o w becomes i m p o r t a n t to determine the possible f u n c t i o n o f K A sites within these select systems in n o r m a l a n d diseased states. I f K A sites n o r m a l l y
We wish to t h a n k Dr. G r a h a m E. Fagg for m a n y
mediate or potentiate excitatory neurotransmission,
helpful c o m m e n t s during the p r e p a r a t i o n of this manuscript, a n d Dr. Christine Gall for the use of the T i m m ' s stained material.
REFERENCES 1 Coyle, J. T., McGeer, E. G., McGeer, P. L. and Schwarcz, R., Neostriatal injections: a model for Huntington's chorea. In E. G. McGeer, J. W. Olney and P. L. McGeer (Eds.), Kainic Acid as a Tool in Neurobiology, Raven, New York, 1978, pp. 139-159. 2 Foster, A. C., Mena, E. E., Monaghan, D. T. and Cotman, C. W., Synaptic localization of kainic acid binding sites, Nature (Lond.), 289 (1981) 73-75. 3 Goldschmidt, R. B. and Steward, O., Preferential neurotoxicity of colchicine for granule cells of the dentate gyrus of the adult rat, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 3047-3151. 4 Hall, J. G., Hicks, T. P. and McLennan, H., Kainic acid and the glutamate receptor, Neurosei. Lett., 8 (1978) 171-175. 5 Krammer, E. B., Anterograde and transynaptic degeneration 'en cascade' in basal ganglia induced by intrastriatal injection of kainic acid: an animal analogue of Huntington's disease, Brain Research, 196 (1980) 209-221. 6 Krieg, W. J. S., Connections of the cerebral cortex, J. comp. NeuroL, 84 (1946) 221-275. 7 Johnston, G. A. R., Kennedy S. M. E., and Twitchin B., Action of the neurotoxin kainic acid on high-affinity uptake of L-glutamatic acid in rat brain slices, J. Neurochem., 32 (1979) 121-127. 8 London, E. D. and Coyle J. T., Specific binding of [aH]kainic acid to receptor sites in rat brain, Molec. Pharmacol., 15 (1979) 492-505. 9 London, E. D. and Coyle, J. T., Cooperative interactions at [aHlkainic acid binding sites in rat and human cerebellum, Europ. J. PharmacoL, 56 (1979) 287-290. 10 Nadler, J. V., Kainic acid: neurophysiological and neurotoxic actions, Life Sci., 24 (1979) 289--300. 11 Nadler, J. V., Evenson, D. A. and Smith, E. M., Evidence from lesion studies for epileptogenic and non-epileptogenic neurotoxic interactions between kainic acid and excitatory innervation, Brain Research, 205 (1981) 405-410.
12 Nadler, J. V., Perry, B. W. and Cotman, C. W., Intraventricular kainic acid preferentially destroys hippocampal pyramidal cells, Nature (Lond.), 271 0978) 676-677. 13 McCulloch, R. M., Johnston, G. A. R., Game, C. J. A. and Curtis, D. R., The differential sensitivity of spinal interneurons and renshaw cells to kainate and N-methylo-aspartate, Exp. Brain Res., 2l (1974) 515-518. 14 Olney, J. W., Neurotoxicity of excitatory amino acids. In E. G. McGeer J. W. Olney and P. L. McGeer (Eds.), Kainic Acid as a Tool in Neurobiology, Raven, New York, 1978, pp. 95-121. 15 Olney, J. W., Fuller, T. and De Gubareff, T., Acute dendrotoxic changes in the hippocampus of kainic acid treated rats, Brain Research, 176 (1979) 91-100. 16 Pellegrino, L. J., Pelligrino, A. S. and Cushmann, A. J., A Stereotaxic Atlas o f the Rat Brain, 2nd edn., Plenum, New York, 1979. 17 Robertson, J. H. and Deadwyler, S. A., Kainic acid produces depolarization of CA3 pyramidal cells in the in vitro hippocampal slice, Brain Research, 221 (1981) 117-127. 18 Schwob, J. E., Fuller, T., Price, J. L. and Olney, J. W., Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: a histological study, Neuroscience, 5 (1980)991-1014. 19 Simon, J. R., Contrera, J. F. and Kuhar, M. J., Binding of [aHlkainic acid, an analogue of L-glutamate, to brain membranes, J. Neurochem., 26 (1976) 141-147. 20 Simon, E. L., Jones, A. P. and Gold, R. M., Horizontal stereotaxic atlas of the albino rat brain, Brain Res. Bull., 6 (1981) 297-326. 21 Young, W. S. and Kuhar, M. J., A new method for receptor autoradiography: [aH]opioid receptors in rat brain, Brain Research, 179 (1979) 255-270. 22 Zaczek, R., Simonton, S. and Coyle, J. T., Local and distant neuronal degeneration following intrastriatal injection of kainic acid, J. NeuropathL exp. Neurol., 39 (1980) 245-264.
91
The Distribution of [3H]Kainic Acid Binding Sites in Rat CNS as Determined by Autoradiography DANIEL T. MONAGHAN and CARL W. COTMAN* Department of Psychobiology, University of California, Irvine, CA 92717 (U.S.A.)
(Accepted May llth, 1982) Key words: kainic acid - - excitatory amino acids - - receptor autoradiography
The distribution of [aH]kainic acid (KA) binding sites in the rat CNS was determined by in vitro autoradiography. KA sites are distributed throughout the CNS gray matter in an anatomically specific pattern with telencephalic structures and the cerebellum accounting for the majority of the binding. These results, together with our previous finding that KA sites are greatly enriched at the synapse, suggest that KA binding sites are associated with select terminal fields, and hence may be involved in neurotransmission in certain CNS pathways. INTRODUCTION Kainic acid (KA) is a potent neurotoxin which appears to interact with a specific population of excitatory amino acid binding sites distinct from those which bind L-glutamate4,L K A induces prolonged depolarizations and seizure activity, resulting in lesions of select neuronal populations18,2L Recently, we reported that K A binding sites are greatly enriched in synaptic junctions and that within the rat hippocampus these sites are concentrated in the stratum lucidum 2, a region especially sensitive to the toxic actions of KA12, xS. These results suggest: (1) that K A sites might be associated with specific terminal fields, and hence may possibly be involved in excitatory neurotransmission at certain synapses, and (2) that the same K A sites may be involved in the toxic actions of this excitant. Consequently, it was of interest to determine the distribution of the K A binding sites in the CNS in order to determine the extent to which the density of these sites corresponds to specific terminal fields and to kainate-sensitive regions. Furthermore, since KA-induced lesions of the hippocampus and striatum result in pathologies similar to those occurring in * To whom correspondence should be addressed. 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
status epilepticus lz and Huntington's diseasel, 5, it may be important to understand the involvement of K A binding sites in the induced pathologies. METHODS Anatomical studies Eight male Sprague-Dawley rats (200-300 g) were decapitated, and the brains rapidly removed and frozen in powdered dry ice. In a cryostat, 20/~m horizontal or coronal sections (4 animals each) were thaw-mounted on acid-washed microscope slides. In 3 animals cervical spinal cord sections were sectioned in the transverse plane. Sections were then prepared for autoradiography by the following modification of a ligand-binding autoradiography method 21. Sections were preincubated in 50 m M H E P E S - K O H , p H 7.1, for 10 min in order to remove endogenous glutamate and N a + from the tissue sections. The slides were then transferred to coplin jars containing 30 ml of 100 nM [aH]KA (5.6 Ci/mmol, Amersham, Arlington Heights, IL) for 30 min, again on ice. Of the sections obtained (about 80/animal, coronal plane, 40/animal, horizontal plane), half were incubated in the presence of 100
92 /~M unlabeled KA to obtain measures of non-specific binding. The sections then received two 30-s. rinses in ice-cold buffer. To minimize ligand diffusion, the tissue was rapidly frozen by pressing the bottom of the slide against a slab of dry ice while the excess rinse was aspirated away. The sections were then lyophilized until fully dry (white-opaque appearance) and partially rehydrated over water vapor until translucent in appearance, without the accumulation of water on the section. Rehydration prevented tissue-photographic emulsion interactions which result in chemography. The slides with radiolabeled tissue were clamped to microscope slides which had been acid-washed and dipped into 1:1 diluted NTB-2 emulsion (Eastman Kodak), and the autoradiograms were exposed for 2 months in the dark at 4 °C. The microscope slide with the photographic emulsion was developed in D-19 and the tissue slide was stained for Nissl substance. The anatomical localization of regions differing in silver grain density was made by the comparison of autoradiograms and Nissl-stained sections with two rat brain atlases 16,20. Cortical regions were compared to those described by Krieg 6. Kinetic and pharmacological characteristics
Tissue sections were prepared as above and incubated with a range of [ZH]KA concentrations (5, 10, 25, 50, 100 and 200 nM) in 3 ml chambers. Other sections were incubated with 100 nM [3H]KA in the presence of 5 #M of one of the followingcompounds: kainic acid, L-glutamate, D-glutamate, L-aspartate (Sigma) and quisqualate (a gift from Dr. H. Shinozaki). These sections (4-5 for each condition) were then rinsed (as described above), air-dried, and prepared for the determination of radioactivity by liquid scintillation spectroscopy. Again, non-specific binding was defined as the binding in the presence of 100/~M KA. Hippocampal lesions
Two experimental groups were used, each containing 3 male Sprague-Dawley rats (200-300 g). In one group, hippocampal CA3 and CA4 pyramidal cells were selectively destroyed by intraventricular injections of KA as previously described 12. Dentate gyrus granule cells were removed by injecting 2.5/~g
colchicine, as described elsewhere 3. Both groups of animals were sacrificed one month following the operations, processed for autoradiography as described above, and the slides were allowed to expose for 3 months. RESULTS The binding of [~H]KA to tissue sections at concentrations between 5 and 200 nM exhibited saturation kinetics with a Ka of 69 ± 5 nM and a Bmax of 234 ± 20 fmol/mg protein (mean ~ S.E.M., n = 3, determined by Scatchard analysis). Pharmacological studies indicated that at 5 #M, quisqualate and kainate were potent displacers of the [3H]KA binding observed here; D-glutamate and L-aspartate were ineffective, and L-glutamate intermediate in displacing [3H]KA (n -- 3). Due to the similarities between the kinetic, pharmacological, and regional distribution data found here, and those found for the binding of [3H]KA to rat brain membranesT,S, 19, it is thought that the autoradiographic pattern described below represents the previously described kainate binding sites. Furthermore, KA does not exhibit uptake into rat brain slices and has a 10,000fold higher affinity for its binding sites than for the L-glutamate uptake sitesL Hence, it is unlikely that uptake or uptake site binding can account for the kainate binding observed in this study. Anatomical distribution - - telencephalon
As shown in Figs. 1-4 silver grains due to [3H]KA binding were distributed throughout the rat CNS and in a selective manner. In general, telencephalic structures had a higher density of KA binding sites than did other brain structures. Many cerebral cortical areas exhibited a trilaminar distribution with the superficial and deep layers displaying more binding than the middle layers. In much of the parietal, occipital and temporal cortices, the trilaminar appearance was not well-defined, making cortical layer assignment uncertain. In the retrohippocampal area and cingulate cortex, the trilaminar organization had sharper boundaries. The superficial dense binding layer occupied only layer I of both entorhinal and presubiculum cortices (Figs. 1 and 3), yet both I and II (and possibly III) of cingulate cortex. The deep layers containing a high density of binding sites were layers V and VI.
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Fig. 2. Dark-field autoradiographs of [3 H]KA binding sites in coronal sections. A - F were taken at approximately 2 m m intervals anterior to posterior. Arrow in B indicates the rhinal fissure immediately above which is the insular cortex. H: right, Nissl stain of cervical level spinal cord section corresponding to autoradiograph on left. I: autoradiogram of [aH]KA binding in the presence of
100/zM KA, from a section adjacent to that in E. Abbreviations: O, olfactory bulb; A, amygdala; NA, nucleus aecumbens; PO, preoptic area; GP, globus pallidus; HY hypothalamus; R, reticular nucleus of thalamus; G, cerebellar granule cell layer; M, cerebellar molecular layer; D, dentate nucleus of cerebellum; BS, brainstem; SG, substantia gelatinosa; and VH, ventral horn. Magnifications: A-F, 10x ; G and I, 11 x ; H, 20x)
96 Frontal, insular, perirhinal, and dorso-medial aspects of parietal and occipital cortices exhibited a dense distribution of KA binding sites throughout their layers, with the insular and frontal cortices showing the highest density of cortical KA binding sites (Figs. 1 and 2). The pyriform cortex, olfactory bulb, and amygdala complex had moderately high concentrations of binding sites but did not exhibit any consistant regional variations within these structures (Fig. 2B-E). Within the basal ganglia, a striking selectivity was found. The caudate nucleus (and the nucleus accumbens) had quite high levels of KA binding sites (Figs. 1 and 2B-E). In contrast, the globus pallidus had very low levels of binding (Fig. 2D). The septal and septal fimbrialis nuclei exhibited moderate levels of binding with the lateral septum having slightly higher levels than the medial septum. As we previously reported 2, the hippocampus had low levels overall, with higher levels in two distinct layers. The commissural/associational (C/A) terminal field of the dentate gyrus had moderate levels while the stratum lucidum, termination zone of the granule cell axons (mossy fibers), had the highest density of [aH]KA binding sites of all structures observed (Figs. 1, 2E, F and 3). The retrohippocampal area displayed a complex distribution of KA sites. In Fig. 3 the KA sites of this region are compared to a Timm's stain which likewise differentiates the subdivisions of this region. The presubiculum, parasubiculum, and entorhinal cortices show a trilaminar appearance, whereas the subiculum and perirhinal cortices are not laminated.
Diencephalon, brainstem and cerebellum Thalamus and hypothalamus had relatively low densities of silver grains with the hypothalamus and reticular nucleus of the thalamus having a higher density than the remainder of the thalamus (Fig. 2E). Midbrain and hindbrain structures were associated with quite low and uniform densities of silver grains. The only exception observed was a moderate grain density found in the central gray substance of the midbrain (Fig. 1). The cerebellum exhibited a distinctive pattern of silver grain distribution (Fig. 1 and 2G). The granule cell layer had high levels, the molecular had layer
moderate levels, and the white matter region had negligible levels of silver grains. In autoradiograms of cervical level spinal cord, the substantia gelatinosa consistently showed moderately dense binding (Fig. 2H). In all of the paired controls, the non-specific binding displayed no anatomical specificity except for a slight preference for gray matter over white matter (Fig. 21).
Hippocampal lesions The above data show that KA binding sites are localized to discrete portions of the CNS and hence are specific to select systems in the CNS. However, it has not yet been determined whether the binding sites are pre- or postsynaptic, an issue which directly concerns the mechanism of action of kainic acid. Since the mossy fiber system is the best example of a discrete pathway containing KA sites, and since it may also be selectively lesioned either pre- or postsynaptically, we chose this pathway for evaluation of the cellular localization of the KA sites. Thirty days following the selective destruction of the CA3 pyramidal cells (the postsynaptic component) the high density of silver grains associated with the stratum lucidum was entirely absent (Fig. 4A, B). The grains associated with the C/A layer were unaltered (compared to the contralateral side). Removal of the granule cells of the dentate gyrus (the presynaptic component) also removed the high density of silver grains associated with the stratum lucidum (Fig. 4C, D). However, this lesion also removed the band of grains associated with the C/A layer. Indeed, the entire dentate molecular layer appeared to have a lowered grain density. Another distinction between the two lesions was that removal of the granule cells caused a small increase in grain density within all layers of CA3, except stratum lucidum. DISCUSSION The present study demonstrates that KA binding sites are distributed throughout the CNS in well-defined anatomical zones, and suggests that these sites may be associated with specific pathways. In interpreting these autoradiographic data, it is
97
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Fig. 3. Horizontal sections of retrohippocampal region. Top: autoradiograph of [aH]KA binding sites. Bottom: similar section stained by the Timm's method with a Nissl counter stain. Abbreviations: MF, mossy fibers; CA, commissural/associational layer of dentate gyrus; PE, perirhinal cortex; E, entorhinal cortex; P, presubiculum and parasubiculum; S, subiculum; CA1, CA3, regions of hippocampus. Asterisks indicate location of mossy fiber and commissural/associational layers. (Magnification 35 x ):
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99 important to know whether the sites mapped are identical to those described in binding studies which exhibit both a high and low affinity. On the basis of pharmacological and kinetic characterization, and the regional distribution of KA sites obtained in this study, it is thought that the sites observed with autoradiography are identical to those described for the binding of [3H]KA to rat brain membranesT,S,19. In addition, the data suggest that the low affinity sites are included in this study. The dissociation constant obtained from tissue sections treated identically to those processed for autoradiography (69 nM) is similar to the low affinity values obtained from other studies (32--66 nM and 59 nM, respectively)S,19. The presence of a low affinity component is also suggested by the observation that the cerebellum has mostly low affinity KA sites s, and the sites observed here are found in high concentrations in the cerebellum. The presence of a high affinity component is difficult to assess due to the very low levels of radioactivity bound at low ligand concentrations. The distribution of [aH]KA binding sites within the rat CNS is anatomically selective. In agreement with studies on the regional distribution of [ZH]KA bindingS, 19, telencephalic structures and the cerebellum account for the majority of KA binding in the rat brain. In cortical structures (cerebellar cortex, hippocampus, and cerebral cortex), the density of KA binding sites exhibited variations among layers as well as among regions. In the basal ganglia there is a clear distinction between the dense distribution of KA sites in the caudate nucleus and the paucity of sites within the globus pallidus. Since KA binding sites are synaptically localized (based on cell fractionatiort studies) z, such anatomical specificity as that described here suggests that KA binding sites ate pathway-specific or transmitter-specific elements, presumably as a component of excitatory amino acid neurotrausmission. Given the select distribution of KA sites, it is predicted that certain regions would be more sensitive to the reversible depolarizing and potentiating actions of KA. This prediction is supported by the available data but, unfortunately, the data are quite limited. Within the spinal cord, interneurons receiving primary afferent innervation are more sensitive to KA than are Renshaw cells13 and, within the
hippocampus, CA3 is approximately 3 orders of magnitude more sensitive than CA11L The regional distribution of the neurotoxic action of KA might likewise be expected to parallel KA site distribution. However, this correspondence is not likely to be as close since part of KA's excitotoxic action is thought to result from KA-induced epileptiform activity11. There is, however, a rough correlation between KA binding site density and sensitivity to KA toxicity. For example, CA3 regions of the hippocampus, amygdala, pyriform cortex, insular cortex and deep layers of other cortical regions are both relatively rich in KA binding sites and preferentially lesioned by low doses of KA, whereas lower brain structures have low concentrations of KA sites and are relatively insensitive to KA toxicitylS,22,. Several instances exist, however, where KA sites do not correspond to areas exceptionally sensitive to KA. As others have noted 10, the caudate nucleus has a high concentration of KA sites, and yet, is not especially sensitive to the toxic action of KA. KA injected either systemically or directly into the caudate nucleusis displays a greater toxic action on the pyriform cortex than on the caudate nucleus. In addition, from these data, it would be predicted that the nucleus accumbens, reticular nucleus of the thalamus and the granule cells of the cerebellum would be selectively destroyed by KA, which apparently is not true is,z2. (However, in the cerebellum it is difficult to know which cells should be affected by KA, given the complex synaptic arrangement at the mossy fiber terminal.) Thus, it may be that a dense distribution of KA binding sites contributes to, but is not sufficient for, KA toxicity. Selective removal of either the pre- or post-synaptic components of the hippocampal mossy fiber pathway results in a loss of the KA binding sites in the stratum lucidum. Consequently, it was not possible to determine which cell has the KA binding sites. It is likely that one of the lesions removed the cells possessing the binding sites, while the other lesion removed the binding sites via a trans-synaptic effect. However, it may be relevant to note that the removal of the the presynaptic component resulted in a modest increase in KA sites throughout the CA3-CA4 region outside of stratum lucidum (Fig. 4D). It is possible that these KA sites are binding sites which would be localized post-synaptically in
100 the mossy fiber synapse if the presynaptic compon e n t were present. Lesions induced by K A resemble the pathologies f o u n d in H u n t i n g t o n ' s diseasO, 5 a n d in status epilepticus 12. Since the results of this study suggest that
then these sites might be c o n t r i b u t i n g to the pathologies f o u n d in H u n t i n g t o n ' s disease a n d epilepsy. ACKNOWLEDGEMENTS
K A sites are largely localized to discrete systems, it
This work was supported by G r a n t M H 19691.
n o w becomes i m p o r t a n t to determine the possible f u n c t i o n o f K A sites within these select systems in n o r m a l a n d diseased states. I f K A sites n o r m a l l y
We wish to t h a n k Dr. G r a h a m E. Fagg for m a n y
mediate or potentiate excitatory neurotransmission,
helpful c o m m e n t s during the p r e p a r a t i o n of this manuscript, a n d Dr. Christine Gall for the use of the T i m m ' s stained material.
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