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Kainate and glutamate neurotoxicity in dependence on the postnatal development with special reference to hippocampal neurons
Developmental Brain Research, 14 (1984) 15- 21 Elsevier
15
BRD 50039
Kainate and Glutamate Neurotoxicity in Dependence on the Postnatal Development with Special Reference to Hippocampal Neurons GERALD WOLF and GERBURG KEILHOFF Department of Biology, Institute of Anatomy and Biology, MedicalAcademy, 3090 Magdeburg (G. D. R.) (Accepted December 13th, 1983) Key words: kainate - - glutamate - - neurotoxicity - - hippocampal formation - - postnatal development
The particular vulnerability of hippocampal CA3/CA4 neurons to intracerebroventricularly administered kainate did not occur in rats until postnatal day 16. Glutamate applied in the same way failed to show comparable neurodegenerative signs, even when administered in large doses to adult animals. Alternatively, both kainate and glutamate exerted similar behavioral effects from postnatal day 15 onwards. No correlation appears between kainate/glutamate-induced behavioral changes and their neurodegenerative potency. INTRODUCTION Glutamate (Glu) and its structural restricted analogue kainate (kainic acid, KA) exert powerful excitatory effects on mammalian CNS neurons (for review see refs. 8, 21). Whereas Glu is the most abundant amino acid in the mammalian brain 1 and supposed to be a widespread central nervous transmitter, KA is found in plants29, and its site of action in the CNS is not fully established. Although current electrophysiologicai and pharmacological evidence 7,11,31 favors different receptor populations for the two amino acids, these populations may be partly overlapping, as KA appears to interact with Glu receptors3.16. KA has been employed as a chemical tool in neurobiology due to its striking neurodegenerative effects both when injected locally in brain structures and after systemic applicationlS, t6. To some extent, Glu also produces brain damage, so that Olney 20 initially proposed the term 'excitotoxic' amino acids for Glu and its analogues, including KA, on the assumption that in excess these compounds excited glutamoreceptive cells to death. Up to date, however, the mechanisms of Glu- and KA-induced neurodegeneration as well as the modus of interaction between KA and glutamatergic systems are largely unknown. Thus, the present study has been undertaken to examine whether the postnatal development of the vulnerability of hippocampal structures to KA is parallel 0165-3806/84/$03.00 © 1984 Elsevier Science Publishers B.V.
to the maturation of glutamatergic systems. The effects of Glu were studied for comparison. MATERIALS AND METHODS Wistar rats of our own breeding stock (derived from V E L A Z , Prague) were used. The litter size was limited to ten. Pups were housed together with their mother in individual cages until attaining the age chosen for study or until weaning at day 25 (the day of birth is taken as day 0). Animals were lightly anesthetized with sodium hexobarbital (100 mg/kg, i.p.) and mounted on a stereotaxic instrument. Craniotomy holes were drilled in the left parietal skull at 1-1.5 mm lateral to the midline, midway between bregma and lambda. A 10/~1 Hamilton microsyringe was inserted into the lateral ventricle. During 1-3 min a total of 0.005, 0.025, 0.5 and 1.0/tg of KA and of 0.5, 2 and 4 mg of sodium Glu, respectively, was injected (corresponding to the increasing ventricular lumen) in a volume of 1/A (age 0-5 days), of 3-5/A (age 6--12 days), and of 5-10/A (13 days to adult) into the left lateral ventricle. High doses of Glu (2 and 4 mg) were administered in a volume of 5 and 10/A also in animals in the first weeks postnatally. KA and Giu were dissolved in phosphate buffered (0.01 M) saline (PBS) and adjusted to pH 7.35. Control rats received PBS only. In each experimental group a minimum of 5 animals was studied. After survival periods of 4 and
16 24 h, or 7, 14, 21 and 28 days, respectively, animals were sacrified by decapitation. Brains were fixed in 10% formaldehyde or in Bouin's fluid for 1 week, in special cases (for Timm's staining) in Carnoy's solution saturated with hydrogen sulfide for 24 h. After embedding in paraffin brains were cut horizontally (10 ~m) and stained with hematoxylin and eosin or with cresyl violet. The sulfide-silver reaction according I0 30 was used for the demonstration of the hippocampal mossy fiber layer. RESULTS In adult rats both KA and Glu administration induced typical behavioral phenomena starting immediately after recovery from anesthesia. The first symptoms were 'wet dog shake' movements, trembling, reeling, tremor of the forepaws, twisting of the tail, interrupted by general convulsions. These reactions were found to be widely independent of the doses applied. Yet very low quantities of KA (within the range of 0.005-0.025 #g) produced considerably
attenuated symptoms. Some days after the injection the animals became extremely aggressive and hyperreactive to exogenous stimuli (sound, touching etc.). In young rats KA and Glu did not induce evident behavioral phenomena when injected before postnatal day 15. In some cases, however, KA and Glu administration was found to be lethal both in adult rats and in rats a few days old. Typical KA- and Glu-induced behavioral abnormalities (wet dog shake, reeling, heavy convulsions) were observed from day 17 onwards. On day 15 and 16 both KA and Giu caused rather poor signs of convulsions and trembling. Control rats showed no modification during the observation period. Histologically, intracerebroventricular injections even of very low doses of KA (0.005 and 0.025 ~g) caused in adult rats necrosis particularly in CA3 pyramidal cells and in neurons of the hilus fasciae dentatae of the ipsilateral hippocampus (Fig. 1). The contralateral hippocampus was unaffected in most cases. Four hours after KA application the neuronal perikarya were shrunken and darkly staining, Mostly the somata and the proximal parts of
Figs. 1-7. Horizontal sections through the hippocampal formation, adult rats excepting Fig. 5. Figs. 1-6 intracerebroventricular application of agents. Fig. 1. Degeneration of CA3 neurons (arrows), 4 h after application of low KA doses (a, 0.005 ~g KA; b 0.025/~g KA). Nissl staining. x 35.
17
Fig. 2. Heavy damage of CA3 neurons adjacent to the transitional zone to CA1, 0.5/~g KA, survival time 4 h. Hemalure and eosin (× 140).
the apical dendrites of CA3 pyramidal cells became edematous and showed vacuolization (Fig. 2). Higher doses of KA (0.5, 1/~g) induced a pronounced damage of hippocampal neurons. At any rate, degenerative effects appeared to be primarily less dependent on the distance from the ventricular lumen than on the cell type. Thus, neurons of the CA3 and hilus region displayed considerably stronger lesions than those of CA1 and subiculum. Ependymal cells including cells of the subcommissural organ did not display any evident signs of degeneration. At a survival time of a few days a massive proliferation of microglial cells in degenerating regions and a complete destruction of damaged material took place (Fig. 3), Possible degenerations of the hippocampal mossy fiber layer were searched using Timm's sulfide-silver technique, but in no case was an evident difference to controls found, not even after a survival period of 28 days (Fig. 4). In young rats lesions were not detected, when KA was injected before the postnatal day 16. Pathological alterations were most clearly seen in CA3/CA4 neurons of animals from day 18 onwards. In 20-day-old rats the degeneration pattern
Fig, 3. Microglialcell proliferation in CA4/CA3 14 days after KA-induced (0.5/~g) neurodegeneration. Nissl staining. × 35. Fig. 4. Parallel section to that in Fig. 3. Timm's sulfide-silver reaction demonstrating persistent mossy fibers (m.f.). × 35.
18
Fig. 5. Hippocampal formation 4 h after application of 0 5#g KA, animals aged 16 days (a), 17 days (b), and 20 days (c). Nissl staining x 35.
19 was similar to that of adult animals (Fig. 5). After intracerebroventricular Glu application, histological signs of neurotoxicity were usually not detectable. In some cases, however, the medial blade of the dentate fascia of animals aging 20 days or more exhibited darkly staining cells (Fig. 6), whereas the hippocampus proper was unaffected. Only in a single case of unintentional intracerebral injection of (3 mg) Glu major brain damage was observed in a circumscribed area around the injection site. The tip of the microsyringe was inserted in the lateral thalamus immediately adjacent to the hippocampai formation. Unlike the degeneration pattern induced by KA, only a portion of the CA3 region and, additionally, the medial blade of the fascia dentata were affected (Fig. 7). After administration of the vehicle solution (control animals) no evident histopathoiogical alterations occurred. DISCUSSION Striking behavioral and neurodegenerative effects
of KA administration as well as a particular vulnerability of hippocampal CA3 and CA4 neurons have been reported previously by several authorsl2,1< 19. In the present study a dependence of Glu/KA-induced behavioral phenomena on the stage of postnatal development was demonstrated. Some findings suggest that there might be a functional relation to the postnatal maturation of glutamatergic systems. Thus, a marked increase of sodium-independent Glu-binding capacity of the rat hippocampal formation 2 and the rat neocortex 23 during the first 3 weeks after birth has been reported. Such binding sites are supposed to be related to postsynaptic Glu receptors. The time course of rising Glu-binding capacity in the hippocampal formation is consistent with morphological findings on addition and maturation of (putatively Glu-ergic) synapsest726 as well as with increasing activities of Glu dehydrogenase and aspartate aminotransferase, which are supposed to be involved in Glu transmitter metabolism 22,24. In such context developmental aspects of the hippocampal mossy fiber system are of special interest, since mossy fibers synapse with neurons of the hilar
Fig. 6. Dark cells (Hemalum and eosin) in the medial blade (arrows) of the dentate fascia 24 h after application of 3 mg Glu. x 35. Fig. 7. Local injection of 3 mg Glu into the lateral thalamus (*). Parts of the dentate fascia and the CA4/CA3 area affected. Nissl staining. x 35.
2[)
(CA4) and CA3 region, both of them being particularly sensitive to KA. Using Timm's sulfide-silver technique, increasing amounts of histochemically detectable zinc were observed in the mossy fiber layer of rats up to 20 days of age 5.33, although there are some discrepancies between quantitative and histochemical data~3. Parallel to histochemical signs of maturation, the mossy fiber system becomes functional as shown by electrophysiological and electron microscopic findings 26. Though the type of transmitter used by mossy fiber endings has not been identified, Glu is a possible candidate6,27, 28. In the rat striaturn 4 and cerebellum 9 a similar parallelism between the development of Glu-ergic structures and K A sensitivity has been found (see ref. 32). On the other hand, if there should be really a functional connection between K A effects and Glu transmission processes, it is surprising that other types of glutamoreceptive neurons in the hippocampal formation, e.g. granule cells of the fascia dentata and pyramidal cells of the CA1 region, are relatively resistant to KA. As to the particular vulnerability of CA3 and hilar neurons, besides a remarkably high density of KA binding sites TM additional mechanisms might be responsible. Thus, Fuller and Olney l° reported on an enhancement of KA-induced convulsions and brain damage (with a typical degeneration pattern in the hippocampus) caused by morphine, whereas its
REFERENCES 1 Balfizs, R., Carbohydrate metabolism. In A. Lajtha (Ed.), Handbook of Neurochemistry, VoL HI, Plenum Press, New York, 1970, pp. 1-36. 2 Baudry, M. and Lynch, G., Hippocampal glutamate receptors, Molec. Cell. Biochem.. 38 (1981) 5-18. 3 Biziere, K. and Coyle, J. T., Influence of corticostriatal afferents on striatal kainic acid neurotoxicity, Neurosci. Lett., 8 (1978) 303--310. 4 Campochiaro, P. and Coyle, J. T., Ontogenetic development of kainate neurotoxicity: correlates with glutamatergic innervation, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 2025-2029. 5 Crawford, I. L, and Connor, J. D., Zinc in maturing rat brain: hippocampal concentration and localization, J. Neurochem., 19 (1972) 1451-1458. 6 Crawford, I. L. and Connor, J. D., Localization and release of glutamic acid in relation to the hippocampal mossy fiber pathway, Nature (Lond.), 244 (1973) 442--443. 7 Davies, J., Evans, R. H., Jones, A. W. and Smith, D. A., Differential activation and blockade of excitatory amino
antagonist naloxone has been found to suppress K A neurotoxicity. Interestingly, receptors for opiates and opioid peptides (enkephalins) within the hippocampal formation are virtually restricted to the mossy fiber layerZL The mechanism of such an interaction between K A and enkephalins remains to be clarified. As to Glu, KA-like effects were observed only with regard to behavioral phenomena, including its timing. Such parallism might be conjectured as a consequence of identical or closely related target sites in the brain. However, as far as histophathological alterations were observed, the degeneration pattern differed widely from that induced by KA. Because of the extremely high concentration of the Giu solution applied, a rather unspecific effect on nerve cells must be expected. Consequently, KA and Glu may act in concert upon glutamoreceptive neurons, the neurotoxicity of KA, however, might be caused by mechanisms in addition to its Glu mimicking effect, especially since no correlation appears to exist between KA- and Glu-induced behavioral changes and, on the other hand, their neurodegenerative potencies. ACKNOWLEDGEMENTS We are grateful to Dr. Storm-Mathisen (Oslo) for helpful discussions and to Mrs. Uta Schulz for her technical assistance,
acid receptors in the mammalian and amphibian nervous systems, Comp. Biochem. Physiol., 72 (1982) 211-224. 8 Di Chiara, G. and G. U Gessa (Eds.), Glutamate as a Neurotransminer, Advanc. Biochem. Psychopharmacol., Vol. 27, Raven Press, New York, 1981. 9 Foster, G. A., Roberts, P. J., Rowlands, G. J. and Sharif, N. A., Ontogeny of neurochemical markers for glutamatergic neurons in cerebellum: implications for the development of kainate neurotoxicity, Brit. J. Pharmacol., in press. 10 Fuller, T. A., Olney, J. W., Effects of morphine or naloxone on kainic acid neurotoxicity, Life Sci., 24 (t979) 1793-1798. 11 Griesser, C. A. V., Cuenod, M. and Henke, H., Kainic acid receptor sites in the cerebellum of nervous, Purkinje cell degeneration, reeler, staggerer and weaver mice mutant strains, Brain Res, 246 (1982) 265-271. 12 Heggli, D. E., Aamodt, A. and Malthe-Screnssen, D., Kainic acid neurotoxicity; effect of systemic injection on neurotransmitter markers in different brain regions, Brain Res, 230 (1981) 253-262. 13 Kalinowski, M., Wolf, G. and Markefski, M., Concentra-
21 tion and subcellular localization of zinc in the hippocampal formation, cerebellum, and whole brain during the postnatal development of the rat, Acta histochem., 73 (1983) 33-40. 14 Kfhler, Ch., Schwarcz, R. and Fuxe, K., Perforant pathway transections protect hippocampal granule cells from kainate lesion, Neurosci. Lett., 10 (1978) 241-246. 15 McGeer, E. G., Olney, J. W. and McGeer, P. L. (Eds.), Kainic Acid as a Tool in Neurobiology, Raven Press, New York, 1978. 16 McGeer, E. G. and McGeer, P. L., Neurotoxins as tools in neurobiology, Int. Rev. NeurobioL, 22 (1981) 173-204. 17 Minkwitz, H.-G., Zur Entwicklung der Neuronenstruktur des Hippocampus w~ihrend der pr~i- und postnatalen Ontogeuese der Albinoratte, J. Hirn[orsch., 17 (1976) 213-275. 18 Monaghan, D. T. and Cotman, C. W., The distribution of [3H]kainic acid binding sites in rat CNS as determined by autoradiography, Brain Res., 252 (1982) 91-100. 19 Nadler, J. V., Perry, B. W. and Cotman, C. W., Intraventricular kainic acid preferentially destroys hippocampal pyramidal cells, Nature (Lond.), 271 (1978), 676-677. 20 Olney, J. W., Excitotoxic amino acids: research applications and safety implications. In L. J. Filer, Jr., S. Garattini, M. R. Kare and W. A. Reynolds (Eds.), GlutamicAcid: Advances in Biochemistry and Physiology, Raven Press, New York, 1979, pp. 287-319. 21 Roberts, F. J., Storm-Mathisen, J. and Johnston, G. A. R. (Eds.), Glutamate: Transmitter in the Central Nervous System, Wiley, Chichester, 1981. 22 Rothe, F., Schmidt, W. and Wolf, G., Postnatal changes in the activity of glutamate dehydrogenase and aspartate aminotransferase in the rat nervous system with special reference to the glutamate transmitter metabolism, Develop. Brain Res., 11 (1983) 67-74. 23 Sanderson, C. and Murphy, S., Glutamate binding in the rat cerebral cortex during ontogeny, Develop. Brain Res., 2 (1982) 329--339. 24 Schiinzel, G. and Wolf, G., Topographic and quantitative characteristics of glutamate dehydrogenase of the hippo-
campus formation during the postnatal development of the rat brain, Acta histochem., 71 (1982) 145-151. 25 Steengard-Pedersen, K., Fredens, K. and Larsson, L.-I., Inhibition of opiate receptor binding by zinc ions: possible physiological importance in the hippocampus, Peptides, 2 (1981) 27-35. 26 Stifling, R. V. and Bliss, T. V. P., Hippocampal mossy fiber development at the ultrastructural level. In M. A. Corner, R. E. Baker, N. E. van de Poll, D. F. Swaab and H. B. M. Uylings (Eds.), Maturation of the Nervous System, Elsevier, Amsterdam, 1978, pp. 191-198. 27 Storm-Mathisen, J. and Iverson, L. L., Uptake of [3H]glutamic acid in excitatory nerve endings: light and electronmicroscopic observations in the hippocampal formation of the rat, Neuroscience, 4 (1979) 1237-1253. 28 Storm-Mathisen, J., Leknes, A. K., Bore, A. T., Vaaland, J. L., Edminson, P., Hang, F.-M. S. and Ottersen, O. P., First visualization of glutamate and GABA in neurones by immunocytochemistry, Nature (Lond.), 301 (1983) 517-520. 29 Takemoto, T., Isolation and structural identification of naturally occurring excitatory amino acids. E. G. McGeer, J. W. Olney and P. L. McGeer (Eds.), Kainic Acid as a Tool in Neurobiology, Raven Press, New York, 1978, pp. 1-15. 30 Timm, F., Zur Histochemie der Schwermetalle. Das SulfidSilber-Verfahren, Dtsch. Z. gerichtl. Med., 46 (1958) 706-711. 31 Watkins, J. C., Pharmacology of excitatory amino acid receptors. In F. J. Roberts, J. Storm-Mathisen and G. A. R. Johnston (Eds.), Glutamate: Transmitter in the Central Nervoas System, Wiley, Chichester, 1981, pp. 1-24. 32 Wolf, G. and Keilhoff, G., Kainate resistant neurons in peripheral ganglia of the rat, Neurosci. Lett., 43 (1983) 1-5. 33 Zimmer, J., Development of the hippocampus and fascia dentata: morphological and histochemical aspects. In M. A. Corner, R. E. Baker, N. E. van de Poll, D. F. Swaab and H. B. M. Uylings (Eds.), Maturation of the Nervous System, Elsevier, Amsterdam, 1978, pp. 171-189.
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BRD 50039
Kainate and Glutamate Neurotoxicity in Dependence on the Postnatal Development with Special Reference to Hippocampal Neurons GERALD WOLF and GERBURG KEILHOFF Department of Biology, Institute of Anatomy and Biology, MedicalAcademy, 3090 Magdeburg (G. D. R.) (Accepted December 13th, 1983) Key words: kainate - - glutamate - - neurotoxicity - - hippocampal formation - - postnatal development
The particular vulnerability of hippocampal CA3/CA4 neurons to intracerebroventricularly administered kainate did not occur in rats until postnatal day 16. Glutamate applied in the same way failed to show comparable neurodegenerative signs, even when administered in large doses to adult animals. Alternatively, both kainate and glutamate exerted similar behavioral effects from postnatal day 15 onwards. No correlation appears between kainate/glutamate-induced behavioral changes and their neurodegenerative potency. INTRODUCTION Glutamate (Glu) and its structural restricted analogue kainate (kainic acid, KA) exert powerful excitatory effects on mammalian CNS neurons (for review see refs. 8, 21). Whereas Glu is the most abundant amino acid in the mammalian brain 1 and supposed to be a widespread central nervous transmitter, KA is found in plants29, and its site of action in the CNS is not fully established. Although current electrophysiologicai and pharmacological evidence 7,11,31 favors different receptor populations for the two amino acids, these populations may be partly overlapping, as KA appears to interact with Glu receptors3.16. KA has been employed as a chemical tool in neurobiology due to its striking neurodegenerative effects both when injected locally in brain structures and after systemic applicationlS, t6. To some extent, Glu also produces brain damage, so that Olney 20 initially proposed the term 'excitotoxic' amino acids for Glu and its analogues, including KA, on the assumption that in excess these compounds excited glutamoreceptive cells to death. Up to date, however, the mechanisms of Glu- and KA-induced neurodegeneration as well as the modus of interaction between KA and glutamatergic systems are largely unknown. Thus, the present study has been undertaken to examine whether the postnatal development of the vulnerability of hippocampal structures to KA is parallel 0165-3806/84/$03.00 © 1984 Elsevier Science Publishers B.V.
to the maturation of glutamatergic systems. The effects of Glu were studied for comparison. MATERIALS AND METHODS Wistar rats of our own breeding stock (derived from V E L A Z , Prague) were used. The litter size was limited to ten. Pups were housed together with their mother in individual cages until attaining the age chosen for study or until weaning at day 25 (the day of birth is taken as day 0). Animals were lightly anesthetized with sodium hexobarbital (100 mg/kg, i.p.) and mounted on a stereotaxic instrument. Craniotomy holes were drilled in the left parietal skull at 1-1.5 mm lateral to the midline, midway between bregma and lambda. A 10/~1 Hamilton microsyringe was inserted into the lateral ventricle. During 1-3 min a total of 0.005, 0.025, 0.5 and 1.0/tg of KA and of 0.5, 2 and 4 mg of sodium Glu, respectively, was injected (corresponding to the increasing ventricular lumen) in a volume of 1/A (age 0-5 days), of 3-5/A (age 6--12 days), and of 5-10/A (13 days to adult) into the left lateral ventricle. High doses of Glu (2 and 4 mg) were administered in a volume of 5 and 10/A also in animals in the first weeks postnatally. KA and Giu were dissolved in phosphate buffered (0.01 M) saline (PBS) and adjusted to pH 7.35. Control rats received PBS only. In each experimental group a minimum of 5 animals was studied. After survival periods of 4 and
16 24 h, or 7, 14, 21 and 28 days, respectively, animals were sacrified by decapitation. Brains were fixed in 10% formaldehyde or in Bouin's fluid for 1 week, in special cases (for Timm's staining) in Carnoy's solution saturated with hydrogen sulfide for 24 h. After embedding in paraffin brains were cut horizontally (10 ~m) and stained with hematoxylin and eosin or with cresyl violet. The sulfide-silver reaction according I0 30 was used for the demonstration of the hippocampal mossy fiber layer. RESULTS In adult rats both KA and Glu administration induced typical behavioral phenomena starting immediately after recovery from anesthesia. The first symptoms were 'wet dog shake' movements, trembling, reeling, tremor of the forepaws, twisting of the tail, interrupted by general convulsions. These reactions were found to be widely independent of the doses applied. Yet very low quantities of KA (within the range of 0.005-0.025 #g) produced considerably
attenuated symptoms. Some days after the injection the animals became extremely aggressive and hyperreactive to exogenous stimuli (sound, touching etc.). In young rats KA and Glu did not induce evident behavioral phenomena when injected before postnatal day 15. In some cases, however, KA and Glu administration was found to be lethal both in adult rats and in rats a few days old. Typical KA- and Glu-induced behavioral abnormalities (wet dog shake, reeling, heavy convulsions) were observed from day 17 onwards. On day 15 and 16 both KA and Giu caused rather poor signs of convulsions and trembling. Control rats showed no modification during the observation period. Histologically, intracerebroventricular injections even of very low doses of KA (0.005 and 0.025 ~g) caused in adult rats necrosis particularly in CA3 pyramidal cells and in neurons of the hilus fasciae dentatae of the ipsilateral hippocampus (Fig. 1). The contralateral hippocampus was unaffected in most cases. Four hours after KA application the neuronal perikarya were shrunken and darkly staining, Mostly the somata and the proximal parts of
Figs. 1-7. Horizontal sections through the hippocampal formation, adult rats excepting Fig. 5. Figs. 1-6 intracerebroventricular application of agents. Fig. 1. Degeneration of CA3 neurons (arrows), 4 h after application of low KA doses (a, 0.005 ~g KA; b 0.025/~g KA). Nissl staining. x 35.
17
Fig. 2. Heavy damage of CA3 neurons adjacent to the transitional zone to CA1, 0.5/~g KA, survival time 4 h. Hemalure and eosin (× 140).
the apical dendrites of CA3 pyramidal cells became edematous and showed vacuolization (Fig. 2). Higher doses of KA (0.5, 1/~g) induced a pronounced damage of hippocampal neurons. At any rate, degenerative effects appeared to be primarily less dependent on the distance from the ventricular lumen than on the cell type. Thus, neurons of the CA3 and hilus region displayed considerably stronger lesions than those of CA1 and subiculum. Ependymal cells including cells of the subcommissural organ did not display any evident signs of degeneration. At a survival time of a few days a massive proliferation of microglial cells in degenerating regions and a complete destruction of damaged material took place (Fig. 3), Possible degenerations of the hippocampal mossy fiber layer were searched using Timm's sulfide-silver technique, but in no case was an evident difference to controls found, not even after a survival period of 28 days (Fig. 4). In young rats lesions were not detected, when KA was injected before the postnatal day 16. Pathological alterations were most clearly seen in CA3/CA4 neurons of animals from day 18 onwards. In 20-day-old rats the degeneration pattern
Fig, 3. Microglialcell proliferation in CA4/CA3 14 days after KA-induced (0.5/~g) neurodegeneration. Nissl staining. × 35. Fig. 4. Parallel section to that in Fig. 3. Timm's sulfide-silver reaction demonstrating persistent mossy fibers (m.f.). × 35.
18
Fig. 5. Hippocampal formation 4 h after application of 0 5#g KA, animals aged 16 days (a), 17 days (b), and 20 days (c). Nissl staining x 35.
19 was similar to that of adult animals (Fig. 5). After intracerebroventricular Glu application, histological signs of neurotoxicity were usually not detectable. In some cases, however, the medial blade of the dentate fascia of animals aging 20 days or more exhibited darkly staining cells (Fig. 6), whereas the hippocampus proper was unaffected. Only in a single case of unintentional intracerebral injection of (3 mg) Glu major brain damage was observed in a circumscribed area around the injection site. The tip of the microsyringe was inserted in the lateral thalamus immediately adjacent to the hippocampai formation. Unlike the degeneration pattern induced by KA, only a portion of the CA3 region and, additionally, the medial blade of the fascia dentata were affected (Fig. 7). After administration of the vehicle solution (control animals) no evident histopathoiogical alterations occurred. DISCUSSION Striking behavioral and neurodegenerative effects
of KA administration as well as a particular vulnerability of hippocampal CA3 and CA4 neurons have been reported previously by several authorsl2,1< 19. In the present study a dependence of Glu/KA-induced behavioral phenomena on the stage of postnatal development was demonstrated. Some findings suggest that there might be a functional relation to the postnatal maturation of glutamatergic systems. Thus, a marked increase of sodium-independent Glu-binding capacity of the rat hippocampal formation 2 and the rat neocortex 23 during the first 3 weeks after birth has been reported. Such binding sites are supposed to be related to postsynaptic Glu receptors. The time course of rising Glu-binding capacity in the hippocampal formation is consistent with morphological findings on addition and maturation of (putatively Glu-ergic) synapsest726 as well as with increasing activities of Glu dehydrogenase and aspartate aminotransferase, which are supposed to be involved in Glu transmitter metabolism 22,24. In such context developmental aspects of the hippocampal mossy fiber system are of special interest, since mossy fibers synapse with neurons of the hilar
Fig. 6. Dark cells (Hemalum and eosin) in the medial blade (arrows) of the dentate fascia 24 h after application of 3 mg Glu. x 35. Fig. 7. Local injection of 3 mg Glu into the lateral thalamus (*). Parts of the dentate fascia and the CA4/CA3 area affected. Nissl staining. x 35.
2[)
(CA4) and CA3 region, both of them being particularly sensitive to KA. Using Timm's sulfide-silver technique, increasing amounts of histochemically detectable zinc were observed in the mossy fiber layer of rats up to 20 days of age 5.33, although there are some discrepancies between quantitative and histochemical data~3. Parallel to histochemical signs of maturation, the mossy fiber system becomes functional as shown by electrophysiological and electron microscopic findings 26. Though the type of transmitter used by mossy fiber endings has not been identified, Glu is a possible candidate6,27, 28. In the rat striaturn 4 and cerebellum 9 a similar parallelism between the development of Glu-ergic structures and K A sensitivity has been found (see ref. 32). On the other hand, if there should be really a functional connection between K A effects and Glu transmission processes, it is surprising that other types of glutamoreceptive neurons in the hippocampal formation, e.g. granule cells of the fascia dentata and pyramidal cells of the CA1 region, are relatively resistant to KA. As to the particular vulnerability of CA3 and hilar neurons, besides a remarkably high density of KA binding sites TM additional mechanisms might be responsible. Thus, Fuller and Olney l° reported on an enhancement of KA-induced convulsions and brain damage (with a typical degeneration pattern in the hippocampus) caused by morphine, whereas its
REFERENCES 1 Balfizs, R., Carbohydrate metabolism. In A. Lajtha (Ed.), Handbook of Neurochemistry, VoL HI, Plenum Press, New York, 1970, pp. 1-36. 2 Baudry, M. and Lynch, G., Hippocampal glutamate receptors, Molec. Cell. Biochem.. 38 (1981) 5-18. 3 Biziere, K. and Coyle, J. T., Influence of corticostriatal afferents on striatal kainic acid neurotoxicity, Neurosci. Lett., 8 (1978) 303--310. 4 Campochiaro, P. and Coyle, J. T., Ontogenetic development of kainate neurotoxicity: correlates with glutamatergic innervation, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 2025-2029. 5 Crawford, I. L, and Connor, J. D., Zinc in maturing rat brain: hippocampal concentration and localization, J. Neurochem., 19 (1972) 1451-1458. 6 Crawford, I. L. and Connor, J. D., Localization and release of glutamic acid in relation to the hippocampal mossy fiber pathway, Nature (Lond.), 244 (1973) 442--443. 7 Davies, J., Evans, R. H., Jones, A. W. and Smith, D. A., Differential activation and blockade of excitatory amino
antagonist naloxone has been found to suppress K A neurotoxicity. Interestingly, receptors for opiates and opioid peptides (enkephalins) within the hippocampal formation are virtually restricted to the mossy fiber layerZL The mechanism of such an interaction between K A and enkephalins remains to be clarified. As to Glu, KA-like effects were observed only with regard to behavioral phenomena, including its timing. Such parallism might be conjectured as a consequence of identical or closely related target sites in the brain. However, as far as histophathological alterations were observed, the degeneration pattern differed widely from that induced by KA. Because of the extremely high concentration of the Giu solution applied, a rather unspecific effect on nerve cells must be expected. Consequently, KA and Glu may act in concert upon glutamoreceptive neurons, the neurotoxicity of KA, however, might be caused by mechanisms in addition to its Glu mimicking effect, especially since no correlation appears to exist between KA- and Glu-induced behavioral changes and, on the other hand, their neurodegenerative potencies. ACKNOWLEDGEMENTS We are grateful to Dr. Storm-Mathisen (Oslo) for helpful discussions and to Mrs. Uta Schulz for her technical assistance,
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