- Email: [email protected]
Brief exposure to zinc is toxic to cortical neurons
Neuroscience Letters, 71 (1986) 351-355
351
Elsevier Scientific Publishers Ireland Ltd.
NSL 04263
Brief exposure to zinc is toxic to cortical neurons M. Yokoyama, J. Koh and D.W. C h o i Department of Neurology C-338, Stanford Medical School, Stanford, CA 94305 (U.S.A.) (Received 3 April 1986; Revised version received 8 August 1986; Accepted 11 August 1986)
Key words." Zinc; Neurotoxicity; Cortex; Cell culture; Cell death; Gliotoxicity Recent evidence suggests that large amounts of releasable zinc are present in central synaptic vesicles, and that substantial (several hundred/tM) transient elevations in extracellular zinc may accompany intense neuronal excitation. Brief pulse exposure of cortical cell cultures to similar concentrations of zinc resulted in widespread dose-dependent neuronal injury; exposure to higher concentrations resulted in the addition of glial injury. The results suggest that zinc should be included on the growing list of endogenous central neurotoxins which may be involved in the pathogenesis of CNS cell loss in a variety of disease states.
The transition metal zinc (Zn) is a dietary requirement, present in large amounts (16-145/~g/g dry tissue) throughout the mammalian brain, particularly in the pineal body, the mossy fibers of the hippocampus and neocortical gray matter [9-11, 13, 26]. Recent studies have suggested that much of this Zn is primarily localized in synaptic vesicles [15, 22], and may be released from neuronal terminals during synaptic transmission [1, 3, 14]. Increased brain levels of Zn are found in Pick's disease [7] and experimental epilepsy [6, 18], leading to the speculation that excess Zn might be toxic to central neurons [1, 24], but direct evidence supporting this speculation has been sparse. We investigated the effects of defined Zn exposure on cortical neurons in the simplified model system of cell culture, and report here the novel finding that a brief exposure to Zn, at concentrations which may be realized in vivo during certain pathophysiologic states, is toxic to central neurons and glia. We therefore propose that Zn be included with glutamate [21] and quinolinic acid [23] on the growing list of endogenous neurotoxins which may play an important pathological role in diseases of the CNS. Dissociated neocortical cell cultures were prepared as previously described from neocortices removed from day 14-17 mouse embryos [4], and maintained in a medium of Eagle's MEM supplemented with 10% heat-inactivated horse serum, glucose (21 mM) and bicarbonate (38 mM). Culture dishes were selected for study after 14-24 days in vitro, by which time neurons (confirmed by Nissl stain and by intracelCorrespondence." D.W. Choi, Department of Neurology C-338, Stanford Medical School, Stanford, CA 94305, U.S.A. 0304-3940/86/$ 03,50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
352 lular recordings showing action potentials and (usually) synaptic potentials), could be unambiguously identified by extensive processes and phase-bright cell bodies [5], and glia (staining selectively with anti-glial fibrillary acidic protein (GFAP) antibody, kindly supplied by Dr. Larry Eng) had formed a confluent background mat [5]. The only Zn present in the culture media was that contributed by the sera (Hycone Defined), producing a net Zn concentration of < 6/LM in plating media, and < 2 llM in maintenance media (estimate based on the supplier's assay of Zn in the sera). Timed exposure to Zn (as ZnCI2) was carried out at room temperature in a Trisbuffered salt solution with the following composition (mM); NaC1 120, KCI 5.4, MgCI: 0.8, CaCl2 1.8, Tric-C1 25, glucose 15; the Zn solution was then removed by triple exchange with buffer solutions, media was replaced, and the cultures were returned to the incubator. Exposure of cortical cells to a pulse of 1 mM Zn for 15 min was followed within minutes by morphological changes apparent under phase contrast microscopy. The neuronal cell bodies became phase-dark, swollen and granular, and the neuronal processes became beaded. By the following day, virtually all neurons were replaced with debris (17 experiments); the glial layer remained generally intact but did in places show some patchy permeability to Trypan blue (Fig. I C). Investigation of the concentration-dependence of these 15 rain of Zn exposure revealed a sharp takeoff above 300 ~tM Zn. Cultures exposed to 100/tM Zn were indistinguishable from controls treated identically but without Zn addition (Fig. 1A) (two experiments). A 15-min pulse of 600 /IM Zn produced widespread neuronal damage but no detectable glial injury (Fig. I B) (3 experiments). Reducing the exposure time to 2-5 min also reduced the resultant neuronal injury, although a clearcut injury was reliably produced by exposure to 1 mM Zn for 5 min (7 experiments). If ZnC12 was simply added to the culture medium overnight (18 24 h), the concentration-dependence relationship remained remarkably steep but shifted to the left compared with that obtained with 15-min exposures. While no effect was seen with the overnight addition of 100/tM Zn to the medium, the overnight addition of 250/tM Zn was followed by the death and surface detachment of all cells in the dish. The observation that lower doses of Zn exposure were required to produce neuronal injury than were required to produce glial injury raised the possibility that Zn was solely neurotoxic, and that the apparent gliotoxicity was secondary to factors released by dying neurons. To study this possibility, pure glial cultures were established from day 3 postnatal mice following the same protocol used for preparing mixed cortical cell cultures. As described by others [17], we found that postnatal neurons did not survive initial plating; the surviving glial cells stained heavily with antiGFAP. Addition of 250-300/iM Zn overnight to the medium in these glial cultures was followed by the same dramatic gliotoxicity seen in the mixed cultures (4 experiments). These observations provide the first direct evidence that Zn may be a potent, rapidly acting neurotoxin, and a somewhat less potent gliotoxin, in the mammalian CNS. The net toxic effect of Zn depends on both the concentration and the duration of Zn exposure. Our finding contrasts markedly with the lack of toxicity generally
Fig. 1. An identified field of cortical neurons is shown before (top row, phase-contrast) and 1 day after (middle row, phase-contrast; lower row, brightfield following 5 min incubation in 0.4% Trypan blue) a 15-min exposure to ZnCI2 at various concentrations: I00 a M (A), 600 p M (B) and I m M (C). The 3 cultures shown were sisters of a single plating, tested concurrently. Bar = 100 a m in all parts.
.,o
354 associated with the metal. Unlike most of the other trace metals, Zn can be ingested in large amounts without ill effect [20]. There is specifically little previous evidence for Zn neurotoxicity. Massive Zn ingestion (12 g over 2 days) has been reported to be associated with lethargy in man [19], and intraventricular injection of Zn into rat brain is epileptogenic [8, 16]. In addition, implantation of Zn wires into rat brains produces after weeks some alterations in neuronal tubular structures [2], similar to those observed in organotypic cultures of dorsal root ganglia cells after 6 ~ 8 h of exposure to Zn [12]. Widespread neuronal injury was seen with transient exposure to 600/~M Zn, an event which could occur in vivo during pathological conditions characterized by intense neuroexcitation. Assaf and Chung [1] observed that 18% of the Zn contained in rat hippocampal slices (70-80/tg/g dry tissue) [11] can be released into the perfusing medium by 23.8 mM K, an amount of Zn which distributed evenly into an extracellular space representing 15% of the tissue those authors calculate could produce an extraceilular Zn concentration of 300 #M. The figure of 15% for the extracellular compartment may be high for rodent cortex [25], and furthermore, some localization of released Zn to the synaptic zone is likely; both of these considerations could act to raise the actual regional Zn concentrations achieved above the estimate of 300/~M, perhaps by a substantial amount. The recent observation that prolonged stimulation of the hippocampal perforant pathway produces both loss of Timm's stain in the mossy fibers, and damage to the neuronal targets of these fibers [24] is consistent with the hypothesis that Zn might contribute to epileptic brain damage. Many different substances are likely toxic to central neurons, but few are endogenous to the CNS in potentially toxic quantities. Zn is unquestionably important to the normal health and function of the CNS; the possibility that, in some acute or chronic disease states, endogenous CNS Zn can become a pathogenic agent warrants further investigation. We thank S. Peters for helpful discussion. This study was supported by grants from the Wills and Hereditary Disease Foundations, and by N I H Grants BRSG R R 5353 and NS 21628. D.W.C. is a Hartford Foundation Fellow. I Assaf, S.Y. and Chung, S.H., Release of endogenous Zn2+ from brain tissue during activity, Nature (London), 308 (1984) 734-736. 2 Brosnan, C.F., Kress, Y., Gaskin, F. and Levine, S., Further studies on the inflammatory response induced by zinc wire implants in the central nervous system of rats, J. Neuropathol. Exp. Neurol., 4l (1982) 221532. 3 Charton, G., Rovira, C., Ben-Aft, Y. and Leviel, V., Spontaneous and evoked release of endogenous Zn-"~ in the hippocampal mossyfiber zone of the rat in situ, Exp. Brain Res., 58 (1985) 202-205. 4 Choi, D.W., Glutamate neurotoxicity in cortical cell culture is calcium dependent, Neurosci. Lett., 58 (1985) 293 297. 5 Choi, D.W., Maulucci-Gedde, M.A. and Kriegstein, A.K., Glutamate neurotoxicityin cortical cell culture, J. Neurosci. in press. 6 Chung,S.H. and Johnson, M.S. Divalent transition-metal ions (Cu2+ and Zn2÷) in the brains of epileptogenic and normal mice, Brain Res., 280 (1983) 323-334. 7 Constantinidis, C. and Tissot. R., Role of glutamate and zinc in the hippocampal lesions of Pick's
355
8 9 10 lI
12 13
14 15 16 17 18 19 20 21 22 23 24 25 26
disease. In G. Di Chiara and G.L. Gessa (Eds.), Glutamate as a Neurotransmitter, Raven Press, New York, 1981, pp. 413422. Donaldson, J., St. Pierre, T., Minnich, J. and Barbeau, A., Seizures in rats associated with divalent cation inhibition of Na÷-K÷-ATPase, Can. J. Biochem., 49 (1971) 1217-1224. Donaldson, J., St.Pierre, T., Minnich, J.L. and Barbeau, A., Determination of Na ÷, K ÷, Mg 2÷, Cu 2+, Zn 2+, and Mn 2+ in rat brain regions, Can. J. Biochem., 51 (1973) 87-92. Frederickson, C.J., Klitenick, M.A., Manton, W.I. and Kirkpatrick, J.B., Cytoarchitectonic distribution of zinc in the hippocampus of man and the rat, Brain Res., 273 (1983) 335-339. Frederickson, C.J., Manton, W.I., Frederickson, M.H., Howell, G.A. and Mallory, M.A., Stable-isotope dilution measurement of zinc and lead in rat hippocampus and spinal cord, Brain Res., 246 (1982) 338-341. Gaskin, F., Kress, Y., Brosnan, C. and Bornstein, M., Abnormal tubulin aggregates induced by zinc sulfate in organotypic cultures of nerve tissue, Neuroscience, 3 (1978) 1117-1128. Haug, F.M.S., Heavy metals in the brain. A light microscope study of the rat with Timm's sulphide silver method. Methodological considerations and cytological and regional staining patterns, Adv. Anat. Embryol. Cell. Biol., 47 (1973) 1-71. Howell, G.A., Welch, M.G. and Frederickson, C.J., Stimulation-induced uptake and release of zinc in hippocampal slices, Nature (London), 308 (1984) 736-738. Ibata, Y. and Otsuka, N.J., Electron microscopic demonstration of zinc in the hippocampal formation using Timm's sulfide silver technique, J. Histochem. Cytochem., 17 (1969) 17 l - 175. Itoh, M. and Ebadi, M., The selective inhibition of hippocampal glutamic acid decarboxylase in zincinduced epileptic seizures, Neurochem. Res., 7 (1982) 1287-1298. McCarthy, K.D. and deVellis, J., Preparation of separate astroglial and oligodendrogial cell cultures from rat cerebral tissue, J. Cell Biol., 85 (1980) 890-902. Mody, I. and Miller, J.J., Levels of hippocampal calcium and zinc following kindling-induced epilepsy, Can. J. Physiol. Pharmacol., 63 (1985) 159-161. Murphy, J.V., Intoxication following ingestion of elemental zinc, J.A.M.A., 212 (1970) 2119-2120. National Research Council - Subcommittee on Zinc, Zinc, Univ. Park Press, Baltimore, 1979, pp. 249268. Olney, J.W., Brain lesion, obesity and other disturbances in mice treated with monosodium glutamate, Science, 164 (1969) 719 721. Perez-Clausell, J. and Danscher, G., Intravesicular localization of zinc in rat telencephalic boutons. A histochemical study, Brain Res., 337 (1985) 91-98. Schwarcz, R., Whetsell, W.O., Mangano, R.M., Quinolinic acid: an endogenous metabolite that produces axon-sparing lesions in rat brain, Science, 219 (1982) 31 6-318. Sloviter, R.S., A selective loss of hippocampal mossy fiber Timm's stain accompanies granule cell seizure activity induced by perforant path stimulation, Brain Res., 330 (1985) 150~153. Van Harreveld, A. and Malhotra, S.K., Extracellular space in the cerebral cortex of the mouse, J. Anat., 101 (1967) 197-207. Wong, P.Y. and Fritze, K., Determination by neuron activation of copper, manganese, and zinc in the pineal body and other areas of brain tissue, J. Neurochem., 16 (1969) 1231 1234.
351
Elsevier Scientific Publishers Ireland Ltd.
NSL 04263
Brief exposure to zinc is toxic to cortical neurons M. Yokoyama, J. Koh and D.W. C h o i Department of Neurology C-338, Stanford Medical School, Stanford, CA 94305 (U.S.A.) (Received 3 April 1986; Revised version received 8 August 1986; Accepted 11 August 1986)
Key words." Zinc; Neurotoxicity; Cortex; Cell culture; Cell death; Gliotoxicity Recent evidence suggests that large amounts of releasable zinc are present in central synaptic vesicles, and that substantial (several hundred/tM) transient elevations in extracellular zinc may accompany intense neuronal excitation. Brief pulse exposure of cortical cell cultures to similar concentrations of zinc resulted in widespread dose-dependent neuronal injury; exposure to higher concentrations resulted in the addition of glial injury. The results suggest that zinc should be included on the growing list of endogenous central neurotoxins which may be involved in the pathogenesis of CNS cell loss in a variety of disease states.
The transition metal zinc (Zn) is a dietary requirement, present in large amounts (16-145/~g/g dry tissue) throughout the mammalian brain, particularly in the pineal body, the mossy fibers of the hippocampus and neocortical gray matter [9-11, 13, 26]. Recent studies have suggested that much of this Zn is primarily localized in synaptic vesicles [15, 22], and may be released from neuronal terminals during synaptic transmission [1, 3, 14]. Increased brain levels of Zn are found in Pick's disease [7] and experimental epilepsy [6, 18], leading to the speculation that excess Zn might be toxic to central neurons [1, 24], but direct evidence supporting this speculation has been sparse. We investigated the effects of defined Zn exposure on cortical neurons in the simplified model system of cell culture, and report here the novel finding that a brief exposure to Zn, at concentrations which may be realized in vivo during certain pathophysiologic states, is toxic to central neurons and glia. We therefore propose that Zn be included with glutamate [21] and quinolinic acid [23] on the growing list of endogenous neurotoxins which may play an important pathological role in diseases of the CNS. Dissociated neocortical cell cultures were prepared as previously described from neocortices removed from day 14-17 mouse embryos [4], and maintained in a medium of Eagle's MEM supplemented with 10% heat-inactivated horse serum, glucose (21 mM) and bicarbonate (38 mM). Culture dishes were selected for study after 14-24 days in vitro, by which time neurons (confirmed by Nissl stain and by intracelCorrespondence." D.W. Choi, Department of Neurology C-338, Stanford Medical School, Stanford, CA 94305, U.S.A. 0304-3940/86/$ 03,50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
352 lular recordings showing action potentials and (usually) synaptic potentials), could be unambiguously identified by extensive processes and phase-bright cell bodies [5], and glia (staining selectively with anti-glial fibrillary acidic protein (GFAP) antibody, kindly supplied by Dr. Larry Eng) had formed a confluent background mat [5]. The only Zn present in the culture media was that contributed by the sera (Hycone Defined), producing a net Zn concentration of < 6/LM in plating media, and < 2 llM in maintenance media (estimate based on the supplier's assay of Zn in the sera). Timed exposure to Zn (as ZnCI2) was carried out at room temperature in a Trisbuffered salt solution with the following composition (mM); NaC1 120, KCI 5.4, MgCI: 0.8, CaCl2 1.8, Tric-C1 25, glucose 15; the Zn solution was then removed by triple exchange with buffer solutions, media was replaced, and the cultures were returned to the incubator. Exposure of cortical cells to a pulse of 1 mM Zn for 15 min was followed within minutes by morphological changes apparent under phase contrast microscopy. The neuronal cell bodies became phase-dark, swollen and granular, and the neuronal processes became beaded. By the following day, virtually all neurons were replaced with debris (17 experiments); the glial layer remained generally intact but did in places show some patchy permeability to Trypan blue (Fig. I C). Investigation of the concentration-dependence of these 15 rain of Zn exposure revealed a sharp takeoff above 300 ~tM Zn. Cultures exposed to 100/tM Zn were indistinguishable from controls treated identically but without Zn addition (Fig. 1A) (two experiments). A 15-min pulse of 600 /IM Zn produced widespread neuronal damage but no detectable glial injury (Fig. I B) (3 experiments). Reducing the exposure time to 2-5 min also reduced the resultant neuronal injury, although a clearcut injury was reliably produced by exposure to 1 mM Zn for 5 min (7 experiments). If ZnC12 was simply added to the culture medium overnight (18 24 h), the concentration-dependence relationship remained remarkably steep but shifted to the left compared with that obtained with 15-min exposures. While no effect was seen with the overnight addition of 100/tM Zn to the medium, the overnight addition of 250/tM Zn was followed by the death and surface detachment of all cells in the dish. The observation that lower doses of Zn exposure were required to produce neuronal injury than were required to produce glial injury raised the possibility that Zn was solely neurotoxic, and that the apparent gliotoxicity was secondary to factors released by dying neurons. To study this possibility, pure glial cultures were established from day 3 postnatal mice following the same protocol used for preparing mixed cortical cell cultures. As described by others [17], we found that postnatal neurons did not survive initial plating; the surviving glial cells stained heavily with antiGFAP. Addition of 250-300/iM Zn overnight to the medium in these glial cultures was followed by the same dramatic gliotoxicity seen in the mixed cultures (4 experiments). These observations provide the first direct evidence that Zn may be a potent, rapidly acting neurotoxin, and a somewhat less potent gliotoxin, in the mammalian CNS. The net toxic effect of Zn depends on both the concentration and the duration of Zn exposure. Our finding contrasts markedly with the lack of toxicity generally
Fig. 1. An identified field of cortical neurons is shown before (top row, phase-contrast) and 1 day after (middle row, phase-contrast; lower row, brightfield following 5 min incubation in 0.4% Trypan blue) a 15-min exposure to ZnCI2 at various concentrations: I00 a M (A), 600 p M (B) and I m M (C). The 3 cultures shown were sisters of a single plating, tested concurrently. Bar = 100 a m in all parts.
.,o
354 associated with the metal. Unlike most of the other trace metals, Zn can be ingested in large amounts without ill effect [20]. There is specifically little previous evidence for Zn neurotoxicity. Massive Zn ingestion (12 g over 2 days) has been reported to be associated with lethargy in man [19], and intraventricular injection of Zn into rat brain is epileptogenic [8, 16]. In addition, implantation of Zn wires into rat brains produces after weeks some alterations in neuronal tubular structures [2], similar to those observed in organotypic cultures of dorsal root ganglia cells after 6 ~ 8 h of exposure to Zn [12]. Widespread neuronal injury was seen with transient exposure to 600/~M Zn, an event which could occur in vivo during pathological conditions characterized by intense neuroexcitation. Assaf and Chung [1] observed that 18% of the Zn contained in rat hippocampal slices (70-80/tg/g dry tissue) [11] can be released into the perfusing medium by 23.8 mM K, an amount of Zn which distributed evenly into an extracellular space representing 15% of the tissue those authors calculate could produce an extraceilular Zn concentration of 300 #M. The figure of 15% for the extracellular compartment may be high for rodent cortex [25], and furthermore, some localization of released Zn to the synaptic zone is likely; both of these considerations could act to raise the actual regional Zn concentrations achieved above the estimate of 300/~M, perhaps by a substantial amount. The recent observation that prolonged stimulation of the hippocampal perforant pathway produces both loss of Timm's stain in the mossy fibers, and damage to the neuronal targets of these fibers [24] is consistent with the hypothesis that Zn might contribute to epileptic brain damage. Many different substances are likely toxic to central neurons, but few are endogenous to the CNS in potentially toxic quantities. Zn is unquestionably important to the normal health and function of the CNS; the possibility that, in some acute or chronic disease states, endogenous CNS Zn can become a pathogenic agent warrants further investigation. We thank S. Peters for helpful discussion. This study was supported by grants from the Wills and Hereditary Disease Foundations, and by N I H Grants BRSG R R 5353 and NS 21628. D.W.C. is a Hartford Foundation Fellow. I Assaf, S.Y. and Chung, S.H., Release of endogenous Zn2+ from brain tissue during activity, Nature (London), 308 (1984) 734-736. 2 Brosnan, C.F., Kress, Y., Gaskin, F. and Levine, S., Further studies on the inflammatory response induced by zinc wire implants in the central nervous system of rats, J. Neuropathol. Exp. Neurol., 4l (1982) 221532. 3 Charton, G., Rovira, C., Ben-Aft, Y. and Leviel, V., Spontaneous and evoked release of endogenous Zn-"~ in the hippocampal mossyfiber zone of the rat in situ, Exp. Brain Res., 58 (1985) 202-205. 4 Choi, D.W., Glutamate neurotoxicity in cortical cell culture is calcium dependent, Neurosci. Lett., 58 (1985) 293 297. 5 Choi, D.W., Maulucci-Gedde, M.A. and Kriegstein, A.K., Glutamate neurotoxicityin cortical cell culture, J. Neurosci. in press. 6 Chung,S.H. and Johnson, M.S. Divalent transition-metal ions (Cu2+ and Zn2÷) in the brains of epileptogenic and normal mice, Brain Res., 280 (1983) 323-334. 7 Constantinidis, C. and Tissot. R., Role of glutamate and zinc in the hippocampal lesions of Pick's
355
8 9 10 lI
12 13
14 15 16 17 18 19 20 21 22 23 24 25 26
disease. In G. Di Chiara and G.L. Gessa (Eds.), Glutamate as a Neurotransmitter, Raven Press, New York, 1981, pp. 413422. Donaldson, J., St. Pierre, T., Minnich, J. and Barbeau, A., Seizures in rats associated with divalent cation inhibition of Na÷-K÷-ATPase, Can. J. Biochem., 49 (1971) 1217-1224. Donaldson, J., St.Pierre, T., Minnich, J.L. and Barbeau, A., Determination of Na ÷, K ÷, Mg 2÷, Cu 2+, Zn 2+, and Mn 2+ in rat brain regions, Can. J. Biochem., 51 (1973) 87-92. Frederickson, C.J., Klitenick, M.A., Manton, W.I. and Kirkpatrick, J.B., Cytoarchitectonic distribution of zinc in the hippocampus of man and the rat, Brain Res., 273 (1983) 335-339. Frederickson, C.J., Manton, W.I., Frederickson, M.H., Howell, G.A. and Mallory, M.A., Stable-isotope dilution measurement of zinc and lead in rat hippocampus and spinal cord, Brain Res., 246 (1982) 338-341. Gaskin, F., Kress, Y., Brosnan, C. and Bornstein, M., Abnormal tubulin aggregates induced by zinc sulfate in organotypic cultures of nerve tissue, Neuroscience, 3 (1978) 1117-1128. Haug, F.M.S., Heavy metals in the brain. A light microscope study of the rat with Timm's sulphide silver method. Methodological considerations and cytological and regional staining patterns, Adv. Anat. Embryol. Cell. Biol., 47 (1973) 1-71. Howell, G.A., Welch, M.G. and Frederickson, C.J., Stimulation-induced uptake and release of zinc in hippocampal slices, Nature (London), 308 (1984) 736-738. Ibata, Y. and Otsuka, N.J., Electron microscopic demonstration of zinc in the hippocampal formation using Timm's sulfide silver technique, J. Histochem. Cytochem., 17 (1969) 17 l - 175. Itoh, M. and Ebadi, M., The selective inhibition of hippocampal glutamic acid decarboxylase in zincinduced epileptic seizures, Neurochem. Res., 7 (1982) 1287-1298. McCarthy, K.D. and deVellis, J., Preparation of separate astroglial and oligodendrogial cell cultures from rat cerebral tissue, J. Cell Biol., 85 (1980) 890-902. Mody, I. and Miller, J.J., Levels of hippocampal calcium and zinc following kindling-induced epilepsy, Can. J. Physiol. Pharmacol., 63 (1985) 159-161. Murphy, J.V., Intoxication following ingestion of elemental zinc, J.A.M.A., 212 (1970) 2119-2120. National Research Council - Subcommittee on Zinc, Zinc, Univ. Park Press, Baltimore, 1979, pp. 249268. Olney, J.W., Brain lesion, obesity and other disturbances in mice treated with monosodium glutamate, Science, 164 (1969) 719 721. Perez-Clausell, J. and Danscher, G., Intravesicular localization of zinc in rat telencephalic boutons. A histochemical study, Brain Res., 337 (1985) 91-98. Schwarcz, R., Whetsell, W.O., Mangano, R.M., Quinolinic acid: an endogenous metabolite that produces axon-sparing lesions in rat brain, Science, 219 (1982) 31 6-318. Sloviter, R.S., A selective loss of hippocampal mossy fiber Timm's stain accompanies granule cell seizure activity induced by perforant path stimulation, Brain Res., 330 (1985) 150~153. Van Harreveld, A. and Malhotra, S.K., Extracellular space in the cerebral cortex of the mouse, J. Anat., 101 (1967) 197-207. Wong, P.Y. and Fritze, K., Determination by neuron activation of copper, manganese, and zinc in the pineal body and other areas of brain tissue, J. Neurochem., 16 (1969) 1231 1234.