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Different sensitivity of hippocampal neurons to kainic acid in the rabbit
DIFFERENT SENSITIVITY OF HIPPOCAMPAL NEURONS TO KAINIC ACID IN THE RABBIT A. $MIALOWXI Department
of Neuropharmacology.
Institute of Pharmacology. Krak6w. Poland
Polish Academy
of Sciences.
Abstract- The toxic effect of kainic acid. a cyclic analogue of glutamlc acid. was investigated m the rahhit hippocampus. Using histological methods it was found that intrahippocampal injection of one or IWO doses of 5 nM of kainic acid produced cell losses in different cell layers. The cells with the highest hensitlvit> to kainic acid were the cornu ammonis pyramidal cells. whereas those with the lowest \enslti\It! wcrc the dentate granular cells. These findings suggest that the sensitivity to kamic acid is different in discrete cell areas of the
hippocampus
RECEV studies have demonstrated that glutamic acid is likely to be a neurotransmitter (CURTIS & JOHNSTOL..IY74). Its action has been described in various brain regions. where it stimulates the electrical activity of nerve cells. Further studies on its action have shown. however. that glutamic acid rapidly destroys neurons in the retina or arcuate hypothalamic nucleus when administered subcutaneously or orally to infant animals of several species (OLNEY, RHEE& HO, 1974). It has also been found that analogues of glutamic acid have a similar action, the most potent destructive action being observed in the cast of kainic acid. Peripheral administration of kainic acid produced not only brum damage. characteristic for glutamic acid, but also an additional destruction of nerve cells of the hippocampus (OLNEY er ul.. 1974). Further research during which kainic acid was administered directly to the brain tissue or into cerebral ventricles pointed to its destructive activity in three other brain areas: the striatum in the rat (HATTORIMCGEER, 1977), the cerebellum in the rat and hamster (HERNDON& COLYE, 1977) and once more the hippocampus of the rat (NADLER. PERRY& COTMAN. 1978). Moreover. it has been observed that a toxic effect of kainic acid in the striatum depends upon the occurrence of glutamatecontaining nerve endings in it. and appears only when the integrity of the glutamate-containing connection is maintained (MCGEER. MCGEER & HATORI, 1978). On the basis of these results. kainic acid seems to be a substance that destroys glutamate-sensitive cells. Glutamic acid was described as an excitatory hippocampal neurotransmitter on the basis of iontophoretic injection (D~:DAR. 1974; SCH~ARTZ~OIN & ANIXRSFN. 197.5: SEGAL, 1976). Glutamic acid at a low doses excited, with short latency. hippocampal pyramidal cells. The areas responsive to glutamate are the apical and basal dendritic trees. but not the soma of pyramidal cells. In the hippocampus of the rat glutamic acid IS released from terminals of three different brain connections. The first is the commissural path, coming from the contralateral hippocampus (&GAL. 1976). the second, mossy fibers from dentate granular
cells (CRAWFORD& CONNOR. 1973). and the third. a perforant path from the ipsilateral entorhinal cortex (STORM-MATHISEN, 1977;
WHITE, NADLER, HAM-
BERGER.COTMAN & CUMMINS. 1977). [3H]glutamic
acid is accumulated selectively by nerve endings of these connections, which are found in the striatum oriens, and the stratum radiatum of cornu ammonis and in the inner part of a dentate molecular layer (IVERSEN & STORM-MATHISFK.1976: STORM-MATHISEN & IVER~EN,1979).
Up to the present there have been very few data on the occurrence and distribution of glutamergic endings in the rabbit hippocampus. In order to determine whether, and in which hippocampal regions. in the rabbit there are cells sensitive to glutamic acid we administered intrahipocampally its analogue, kainic acid. which causes a permanent loss of such cells.
EXPERIMENTAL
PROCEDURES
The research were carried out on adult rabbits with stainless steel cannulas (0.63 mm in dia.) that had been chronically implanted into the different layer of the dorsal hippocampus. The rabbits were allowed to recover for at least 2 weeks after the surgery. Kainic acid was administered unilaterally through a double-wall cannula system with an inner cannula protruding by about 0.1 mm with a Hamilton microsyringe in a concentration of 5 nM: some animals were given the same (5 nM) dose twice. with a 7 da) interval. Kainic acid was dissolved in 0.5 ~1 of phosphate buffer (pH 7.4, 0. I M) and was injected at a speed of 0. I ~1. min. Four weeks after the last kainic acid injection the rabbits were killed and after removing the brain a series of frontal sections, 20pm thick. stained with cresyl violet or hematoxyline and eosine were made.
RESULTS
Administration of kainic acid to the stratum oriens, pyramidale or radiatum of the regio superior of the cornu ammonis resulted in a loss of pyramidal cells of the cornu ammonis (fields CA I and CA 4) and the dentate polymorphic cell layer within an area of di-
1475
1476
.‘3 SMIALOWShI
ameter of 2500 firn (at a dose of 5 mM) or 4500 pm (at a dose of 2 x 5 no) (Fig. IC). The cell loss in these areas was complete. In rabbits with more laterally implanted cannula cell losses also comprised the whole fields of CA 2 and CA 3 of the cornu ammonis. Administration of kainic acid to the stratum radiaturn of field CA I caused loss of a some of cells in the dentate granular cell layer (Fig. 1C). A deeper administration, to the ventral area of the stratum radiatum induced the complete loss of granular cells situated below the cannula within an area of I 100pm (Fig. 21. Injection of kainic acid (2 x 5 nM) to the dentate granular cell layer destroyed pyramidal cells in the cornu ammonis CA I and CA 4 fields. the dentate polymorphic cell layer. but only the dentate granular cells in the immediate vicinity of the cannula. In one rabbit with a kainic acid injection mto the alveus, pyramidal cells were destroyed in the CA ! field only; pyramidal cells in field CA 1 were mdamaged in it. but a small island of glial cells devcloped in this area. The cannula itself, implanted in the hippocampus. caused destruction of the brain cells, in whole areas of the cannula track 80@ 1000 pm in diameter (Fig. IA and B). Apart from this area, in the immediate vicinity of the cannula undamaged cells of the hippocampus were stained. Similar results were obtained after control injection of phosphate buffer.
The studies carried out demonstrated permanent cell losses in the dorsal hippocampus of the rabbit. induced by administration of kainic acid. The extent of cell destruction depended upon the dose used, add place of injection. Pyramidal cells lying in all &Ids of the cornu ammonis (CA 1~CA 4) and dentate p&4morphe cells revealed the highest sendtivity to kaimc acid. The dentate granular cells were undamaged in it few rabbits, despite the loss of the CA 4 field ~rlls. situated further from the administration site: the former cells were destroyed only when the cimnula was placed near the dentate granular cell layer. Therefore It can be assumed that tn the cornu ammonia different parts of the apical and basal dendrites ;IIY sensitive to kainic acid; in the same parts glutamate excitatory nerve endings were described (JVIIKSEYX: STORM-MATHISEP;.1976). The dentate granular c&is reveal the lowest sensitivity to kainic acid. This ma) be caused b} a smaller number of gIutamatecontaining endings than m the cu[znu ammonib. Such endings were described in the molecular laler of the dentate gyrus in the rat (IVEKSEU& STORM-MAI’HISI,\, 1976). This difference of sensitivitj ti-, kainic acid indicates that in the hippocampus of the rabbit the glutamate-sensitive sites have a smiiar distribution to those in the hippocampus of the :‘&L:
REFERENCES CRAWFORDI. & CONNORJ. D. (1973) Localization and release of glutamlc acid in relation to the hqpcampal mossy tiber pathway. Nature, Lond. 244. 443-444. CURTISD. R. & JOHNSON G. (1974) Aminoacid transmitters in the mammalian central nervous sk\[em. Ku,, Phytinl. 69. 97.-188. DUDARJ. (1974) In vitro excitation of hippocampal pyramidal cell dendrites by glutamic acid. It,uropllarmcc~ok,y~ 13. 1083-1089. HATTORIT. & MCGEERE. G. (1977) Fine structural changes in the rat striatum after local injectlons ul kainic acid. Brurrl Res. 129, 174180.
HERNDONR. & &LYE J. (1977) Selective destruction of neurons by a transmitter agonist. Screncu. .\. t, 198,71 72. IVERSENL. & STORM-MATHISEN J. (1976) Uptake of C3H]glutamate in excitatory nerve endmgs m the hlppocampai formation of the rat. Arta pkysiof. stand. %, 22A. MCGEER P. L., MCGEER E. G. & HATTORIT. (1978) Transmitters in the basal ganglia. In .~~IJIO .Acids (I.> Chem~al Transmitters (ed. FONNUMF.), pp. 121-130. Plenum Press, New York. NADLERJ.. PERRY B. & COTMANC. (197X) Intraventricular kainic acid preferentially destryos hlppocampal pyramIda cells. Nature, Land. 211, 67&677. OLNEY J. W., RHEEV. & Ho 0. (1974) Kainic acid: a powerful neurotoxic analogue of glutamate. &-am Rrs. 77, 507 C12. SCHWARTZKROIN P. & ANDERSENP. (1975) Glutamic acid sensitivity of dendrites in hippocampat slices in rifro .lcir Neural. 12, 45--51. SEGALM. (1976) Glutamate antagonists in rat hippocampus. Br. J. Pharmac. 58, 341 -345. STORM-MATHISEN J. (1977) Glutamic acid and excitatory nerve endings. Brain Rex 120, 37%386. STORM-MATHISEN J. & IVERSENL. L. (1979) Uptake of C3H]glutamic acid in excitatory nerve endings: light and electronmicroscopic observations in the hippocampal formation of the rat. Neuroscience 4, 1237-1253. WHITE W., NADLERJ.. HAMBERCER A.. COTMANC. & CUMMINSJ. (1977) Glutamate as transmtlter of hippocampal perforant path. Nature. Loud. 270. 356. 357.
f .4cceptrd 17 February 19801
.* .
-
FIG. 2. Losses of the granular and polymorphic ceils of the gyrus dentatus after double injection of 5 nM of kainic acid. Hematoxyline, eosine stain. For explanation see Fig. 1.
137x
of Neuropharmacology.
Institute of Pharmacology. Krak6w. Poland
Polish Academy
of Sciences.
Abstract- The toxic effect of kainic acid. a cyclic analogue of glutamlc acid. was investigated m the rahhit hippocampus. Using histological methods it was found that intrahippocampal injection of one or IWO doses of 5 nM of kainic acid produced cell losses in different cell layers. The cells with the highest hensitlvit> to kainic acid were the cornu ammonis pyramidal cells. whereas those with the lowest \enslti\It! wcrc the dentate granular cells. These findings suggest that the sensitivity to kamic acid is different in discrete cell areas of the
hippocampus
RECEV studies have demonstrated that glutamic acid is likely to be a neurotransmitter (CURTIS & JOHNSTOL..IY74). Its action has been described in various brain regions. where it stimulates the electrical activity of nerve cells. Further studies on its action have shown. however. that glutamic acid rapidly destroys neurons in the retina or arcuate hypothalamic nucleus when administered subcutaneously or orally to infant animals of several species (OLNEY, RHEE& HO, 1974). It has also been found that analogues of glutamic acid have a similar action, the most potent destructive action being observed in the cast of kainic acid. Peripheral administration of kainic acid produced not only brum damage. characteristic for glutamic acid, but also an additional destruction of nerve cells of the hippocampus (OLNEY er ul.. 1974). Further research during which kainic acid was administered directly to the brain tissue or into cerebral ventricles pointed to its destructive activity in three other brain areas: the striatum in the rat (HATTORIMCGEER, 1977), the cerebellum in the rat and hamster (HERNDON& COLYE, 1977) and once more the hippocampus of the rat (NADLER. PERRY& COTMAN. 1978). Moreover. it has been observed that a toxic effect of kainic acid in the striatum depends upon the occurrence of glutamatecontaining nerve endings in it. and appears only when the integrity of the glutamate-containing connection is maintained (MCGEER. MCGEER & HATORI, 1978). On the basis of these results. kainic acid seems to be a substance that destroys glutamate-sensitive cells. Glutamic acid was described as an excitatory hippocampal neurotransmitter on the basis of iontophoretic injection (D~:DAR. 1974; SCH~ARTZ~OIN & ANIXRSFN. 197.5: SEGAL, 1976). Glutamic acid at a low doses excited, with short latency. hippocampal pyramidal cells. The areas responsive to glutamate are the apical and basal dendritic trees. but not the soma of pyramidal cells. In the hippocampus of the rat glutamic acid IS released from terminals of three different brain connections. The first is the commissural path, coming from the contralateral hippocampus (&GAL. 1976). the second, mossy fibers from dentate granular
cells (CRAWFORD& CONNOR. 1973). and the third. a perforant path from the ipsilateral entorhinal cortex (STORM-MATHISEN, 1977;
WHITE, NADLER, HAM-
BERGER.COTMAN & CUMMINS. 1977). [3H]glutamic
acid is accumulated selectively by nerve endings of these connections, which are found in the striatum oriens, and the stratum radiatum of cornu ammonis and in the inner part of a dentate molecular layer (IVERSEN & STORM-MATHISFK.1976: STORM-MATHISEN & IVER~EN,1979).
Up to the present there have been very few data on the occurrence and distribution of glutamergic endings in the rabbit hippocampus. In order to determine whether, and in which hippocampal regions. in the rabbit there are cells sensitive to glutamic acid we administered intrahipocampally its analogue, kainic acid. which causes a permanent loss of such cells.
EXPERIMENTAL
PROCEDURES
The research were carried out on adult rabbits with stainless steel cannulas (0.63 mm in dia.) that had been chronically implanted into the different layer of the dorsal hippocampus. The rabbits were allowed to recover for at least 2 weeks after the surgery. Kainic acid was administered unilaterally through a double-wall cannula system with an inner cannula protruding by about 0.1 mm with a Hamilton microsyringe in a concentration of 5 nM: some animals were given the same (5 nM) dose twice. with a 7 da) interval. Kainic acid was dissolved in 0.5 ~1 of phosphate buffer (pH 7.4, 0. I M) and was injected at a speed of 0. I ~1. min. Four weeks after the last kainic acid injection the rabbits were killed and after removing the brain a series of frontal sections, 20pm thick. stained with cresyl violet or hematoxyline and eosine were made.
RESULTS
Administration of kainic acid to the stratum oriens, pyramidale or radiatum of the regio superior of the cornu ammonis resulted in a loss of pyramidal cells of the cornu ammonis (fields CA I and CA 4) and the dentate polymorphic cell layer within an area of di-
1475
1476
.‘3 SMIALOWShI
ameter of 2500 firn (at a dose of 5 mM) or 4500 pm (at a dose of 2 x 5 no) (Fig. IC). The cell loss in these areas was complete. In rabbits with more laterally implanted cannula cell losses also comprised the whole fields of CA 2 and CA 3 of the cornu ammonis. Administration of kainic acid to the stratum radiaturn of field CA I caused loss of a some of cells in the dentate granular cell layer (Fig. 1C). A deeper administration, to the ventral area of the stratum radiatum induced the complete loss of granular cells situated below the cannula within an area of I 100pm (Fig. 21. Injection of kainic acid (2 x 5 nM) to the dentate granular cell layer destroyed pyramidal cells in the cornu ammonis CA I and CA 4 fields. the dentate polymorphic cell layer. but only the dentate granular cells in the immediate vicinity of the cannula. In one rabbit with a kainic acid injection mto the alveus, pyramidal cells were destroyed in the CA ! field only; pyramidal cells in field CA 1 were mdamaged in it. but a small island of glial cells devcloped in this area. The cannula itself, implanted in the hippocampus. caused destruction of the brain cells, in whole areas of the cannula track 80@ 1000 pm in diameter (Fig. IA and B). Apart from this area, in the immediate vicinity of the cannula undamaged cells of the hippocampus were stained. Similar results were obtained after control injection of phosphate buffer.
The studies carried out demonstrated permanent cell losses in the dorsal hippocampus of the rabbit. induced by administration of kainic acid. The extent of cell destruction depended upon the dose used, add place of injection. Pyramidal cells lying in all &Ids of the cornu ammonis (CA 1~CA 4) and dentate p&4morphe cells revealed the highest sendtivity to kaimc acid. The dentate granular cells were undamaged in it few rabbits, despite the loss of the CA 4 field ~rlls. situated further from the administration site: the former cells were destroyed only when the cimnula was placed near the dentate granular cell layer. Therefore It can be assumed that tn the cornu ammonia different parts of the apical and basal dendrites ;IIY sensitive to kainic acid; in the same parts glutamate excitatory nerve endings were described (JVIIKSEYX: STORM-MATHISEP;.1976). The dentate granular c&is reveal the lowest sensitivity to kainic acid. This ma) be caused b} a smaller number of gIutamatecontaining endings than m the cu[znu ammonib. Such endings were described in the molecular laler of the dentate gyrus in the rat (IVEKSEU& STORM-MAI’HISI,\, 1976). This difference of sensitivitj ti-, kainic acid indicates that in the hippocampus of the rabbit the glutamate-sensitive sites have a smiiar distribution to those in the hippocampus of the :‘&L:
REFERENCES CRAWFORDI. & CONNORJ. D. (1973) Localization and release of glutamlc acid in relation to the hqpcampal mossy tiber pathway. Nature, Lond. 244. 443-444. CURTISD. R. & JOHNSON G. (1974) Aminoacid transmitters in the mammalian central nervous sk\[em. Ku,, Phytinl. 69. 97.-188. DUDARJ. (1974) In vitro excitation of hippocampal pyramidal cell dendrites by glutamic acid. It,uropllarmcc~ok,y~ 13. 1083-1089. HATTORIT. & MCGEERE. G. (1977) Fine structural changes in the rat striatum after local injectlons ul kainic acid. Brurrl Res. 129, 174180.
HERNDONR. & &LYE J. (1977) Selective destruction of neurons by a transmitter agonist. Screncu. .\. t, 198,71 72. IVERSENL. & STORM-MATHISEN J. (1976) Uptake of C3H]glutamate in excitatory nerve endmgs m the hlppocampai formation of the rat. Arta pkysiof. stand. %, 22A. MCGEER P. L., MCGEER E. G. & HATTORIT. (1978) Transmitters in the basal ganglia. In .~~IJIO .Acids (I.> Chem~al Transmitters (ed. FONNUMF.), pp. 121-130. Plenum Press, New York. NADLERJ.. PERRY B. & COTMANC. (197X) Intraventricular kainic acid preferentially destryos hlppocampal pyramIda cells. Nature, Land. 211, 67&677. OLNEY J. W., RHEEV. & Ho 0. (1974) Kainic acid: a powerful neurotoxic analogue of glutamate. &-am Rrs. 77, 507 C12. SCHWARTZKROIN P. & ANDERSENP. (1975) Glutamic acid sensitivity of dendrites in hippocampat slices in rifro .lcir Neural. 12, 45--51. SEGALM. (1976) Glutamate antagonists in rat hippocampus. Br. J. Pharmac. 58, 341 -345. STORM-MATHISEN J. (1977) Glutamic acid and excitatory nerve endings. Brain Rex 120, 37%386. STORM-MATHISEN J. & IVERSENL. L. (1979) Uptake of C3H]glutamic acid in excitatory nerve endings: light and electronmicroscopic observations in the hippocampal formation of the rat. Neuroscience 4, 1237-1253. WHITE W., NADLERJ.. HAMBERCER A.. COTMANC. & CUMMINSJ. (1977) Glutamate as transmtlter of hippocampal perforant path. Nature. Loud. 270. 356. 357.
f .4cceptrd 17 February 19801
.* .
-
FIG. 2. Losses of the granular and polymorphic ceils of the gyrus dentatus after double injection of 5 nM of kainic acid. Hematoxyline, eosine stain. For explanation see Fig. 1.
137x