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A selective loss of hippocampal mossy fiber Timm stain accompanies granule cell seizure activity induced by perforant path stimulation
150
Brain Research, 330 (1985) 150-153 Elsevier
BRE 20668
A selective loss of hippocampal mossy fiber Timm stain accompanies granule cell seizure activity induced by perforant path stimulation ROBERT S. SLOVITER Neurology Center, Helen Hayes Hospital, West Haverstraw, NY 10993 and Departments of Pharrnacology and Neurology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (U.S.A.) (Accepted October 23rd, 1984) Key words: hippocampus - - zinc - - epilepsy - - Timm stain
Perforant path stimulation for 24 h evoked hippocampal granule cell spikes and epileptiform discharges throughout the stimulation period. After stimulation, hippocampal neurons that receive granule cell output were irreversibly damaged and the stimulated hippocampus of each rat revealed a selective loss of Timm stain in the mossy fiber pathway. These results provide evidence in vivo for a role for synaptic zinc in neurotransmission and/or seizure mechanisms.
Although the function of synaptic metals present in the normal brain is unclear, considerable evidence suggests a role for metal ions in neurotransmission2,7,t3,15,26,29,30. We have used a recently developed focal seizure model in rats 25 to address the possible role of zinc in hippocampal function or dysfunction and report that the electrical stimulation that evokes hippocampal granule cell discharges also causes a selective, nearly total loss of histochemically stainable transitional metal in the zinc-containing a0,H hippocampal mossy fiber pathway. Eleven male S p r a g u e - D a w l e y descendant rats (300-450 g) were allowed free access to food and water and were housed on a 12-h light/dark cycle. Rats were anesthetized with urethane (Sigma Chemicals; 1.25 g/kg i.p.) and placed in a stereotaxic instrument. Rectal temperature was monitored and maintained at 37 + 1 °C for the 24 h duration of the experiment. Hippocampal granule cell firing was evoked in vivo by electrical stimulation of the perforant path 1, the main excitatory afferent to the hippocampus 23. Evoked granule cell firing was maintained and recorded for 24 h as described in detail previously25. The stimulation paradigm used to evoke granule cell spikes and epileptiform discharges consisted of continuous twin pulse stimulation at 2 Hz (pulses
40 ms apart) combined with a 10-s, single pulse, 20 Hz stimulus train delivered every minute for 24 h (1440 stimulus trains). As a result of sustained granule cell firing, the cells that are innervated by the mossy fiber pathway (CA3 pyramidal cells and hilar interneurons) are irreversibly damaged and this pattern of damage closely resembles the pattern of hippocampal damage caused by a variety of convulsant drugsl9, 20.22 or seen in the brains of chronic temporal lobe epileptics 18. A detailed description of this seizure-induced neuropathology has been published previously 25. Two separate control groups were used in this study. 'Unstimulated controls' were rats subjected to all experimental procedures including perforant path electrode placement but no electrical stimuli were delivered. 'Stimulated controls' were rats in which the electrode tips were placed in the cortex at the minimum distance above the perforant path at which stimuli did not evoke a hippocampal potential. These rats were stimulated with the same electrode for the same duration and at the same frequency and voltage (20V; 0.1 ms) as the experimental rats. These two groups therefore controlled for the effects of anesthesia, surgery and electrode placement, as well as for hydrolysis and deposition of metal ions at the electrode tips.
Correspondence: R. S. Sloviter, Neurology Center, Helen Hayes Hospital, West Haverstraw, NY 10993, U.S.A. 0006-8993/85/$03.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)
151 The sulphide/silver (Timm) histochemical stain for visualizing synaptic transitional metals8,12,24,28 was used to determine in vivo the effect sustained neurotransmission might have on the zinc contained in the mossy fiber terminals. The specific technique used to visualize zinc is a modified Timm stain that utilizes formaldehyde fixation and routine paraffin embedding, thus allowing histochemical and morphological analysis of the same sections. The details of this histochemical technique have been published previously24. After intracardiac perfusion with sulphide and formalin, brains were embedded in paraffin. Blocks containing control and experimental brains were then cut, processed for silver development and developed together. Each slide rack contained mounted sections from experimental and control groups to permit valid comparisons between treatment groups. Perforant path stimulation for 24 h evoked granule cell population spikes and epileptiform discharges throughout the stimulus period and produced, in all 5 experimental rats, the pattern of selective hippocampal damage described in detail previously25. No obvious hippocampal damage was produced in the hippocampi of the 'unstimulated' or 'stimulated' controls. Blind, qualitative evaluation of sulphide/silver stained sections of stimulated (n = 5) and control brains (n = 3 in each group) revealed a selective decrease and sometimes a virtually complete loss of Timm staining in both granule cell axon terminal fields on the stimulated side of the brain (Fig. 1). Since seizure activity evoked by perforant path stimulation is usually restricted to the ipsilateral hippocampus 25, the contralateral hippocampus usually exhibited little or no neuronal damage and negligible loss of mossy fiber Timm staining. In cases in which contralateral granule cell epileptiform discharges did occur, neuronal damage to contralateral hilar interneurons and CA3 pyramidal cells was evident as was a contralateral decrease in mossy fiber staining. When a decrease in both contralateral and ipsilateral mossy fiber staining was evident, the loss was greater on the ipsilateral side. The decrease or loss of mossy fiber staining was selective in that the other 3 laminas of the same hippocampus appeared relatively normally stained. These 3 regions include: (1) the Timmstained layer in the outer third of the dentate molecular layer that reflects synaptic metal contained in the
projection from the lateral entorhinal cortex14; (2) the stained inner third of the molecular layer that refleets the metal-containing axon terminals of the hilar cells that form the ipsilateral associational/commissural projection to this regionl7; and (3) the metalcontaining axons of the CA3 pyramidal cells that innervate stratum oriens, stratum radiatum and stratum moleculare of regio inferior and regio superior 4. The staining of control and normal (naive) brains did not appear different but the possibility of subtle differences due to anesthesia, surgery, etc., cannot be discounted on the basis of this qualitative evaluation. Perforant path stimulation for 24 h did not cause obvious changes in sulphide/silver staining in the other heavily stained cortical regions, such as the amygdala and temporal neocortex. These results show that after 24 h of perforant path stimulation that evoked repetitive firing of the hippocampal granule cells, there is a near total loss of transitional metal staining in the presynaptic terminals of these neurons. There is no obvious explanation why the staining of the lateral perforant path, part of the fiber system being directly stimulated, was not similarly reduced. The decrease in mossy fiber Timm staining that accompanies granule cell firing could be secondary to granule cell damage. However, previous studies showed that granule cells discharge throughout the 24-h period of stimulation and that mossy fiber morphology after stimulation is norma121. The two most likely explanations for the decreased staining are: (1) a loss of presynaptic metal as a result of a hypothetical physiological release mechanism or, (2) that continuous neuronal firing results in a change in presynaptic zinc disposition that renders the zinc unavailable for reaction with the sulphide that is the basis of the sulphide/silver stain. In view of the recent reports that potassium, kainic acid or electrical stimulation releases zinc in vitro from hippocampal slices2,15, the present results are consistent with a zinc-release hypothesis. If zinc is released as a result of neuronal discharge, the possibility should be considered that its release could play a role in the neuropathology that results from seizure activity. A presynaptic role for zinc in neurotransmission distinct from a simple release mechanism is suggested by the observations that the zinc-containing mossy fiber pathway and other excitatory pathways of the
152
Fig. 1. Effect of 24 h perforant path stimulation on Timm stainability in rat hippocampus. A: hippocampus contralateral to stimulated side showing typical sulphide/silver staining pattern. B: same hippocampus as in A above at higher magnification to show distinct staining of the zinc-containing subgranular granule cell axon collateral terminals (H) that innervate interneurons in the hilus and the mossy fiber terminals (CA3) that innervate CA3 pyramidal cells. C: same hippocampus as in A and B above showing lateral CA3 pyramidal cells and their innervation by the mossy fibers (MF). D - F : Similar views of the hippocampus on the stimulated side of the same brain. Note decreased sulphide/silver staining in the regions innervated by the hilar axon collaterals and by the mossy fibers. Coronal, paraffin-embedded sections cut 10/~m thick, stained for 45 min in physical developer and then counterstained with cresyl violet. 26× in A and D; 104× in B, C, E and F.
h i p p o c a m p u s m a y use g l u t a m a t e as a transmitter6, 27 and that g l u t a m a t e and zinc i n t e r a c t c h e m i c a l l y to
that zinc is b o u n d n o r m a l l y to mossy fiber g l u t a m a t e o r a n o t h e r a m i n o acid suggests that zinc m a y play a
f o r m g l u t a m a t o z i n c (II) dihydrate3, 9. T h e possibility
role in regulating the a m o u n t of t r a n s m i t t e r available
153 for release. According to this hypothesis, altered transmitter status as a result of sustained seizure activity could result in altered zinc binding and there-
nisms 16.
fore less zinc being available for reaction with sulphide. Regardless of the mechanism underlying the
The author thanks D a w n Peters for photographic assistance and Laurie Sanchez for secretarial assistance. This work was presented at the Neurobiology
seizure-associated loss of histochemically stained zinc, the present results support the hypothesis that
of Zinc Symposium, Boston, M A , N o v e m b e r 1983. This investigation was supported by research grants
synaptic zinc plays a role in neurotransmissi0n2,7,13,15,26,29,30 and, possibly, seizure mecha-
from the Epilepsy F o u n d a t i o n of America and the
1 Andersen, P., Holmqvist, B. and Voorhoeve, P. E., Entorhinal activation of dentate granule cells, Acta. physiol. scand., 66 (1966) 448-460. 2 Assaf, S. Y. and Chung, S.-H., Release of endogenous Zn2+ from brain tissue during activity, Nature (Lond.), 308 (1984) 734-736. 3 Bere, A. and Helene, C., Formation of ternary complexes involving zinc or copper ions, polynucleotides and polypeptides containing glutamic acid and tyrosine residues, Biopolymers, 18 (1979) 2659-2672. 4 Blackstad, T. W., Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination, J. comp. Neurol., 105 (1956) 417-538. 5 Cotman, C. W. and Nadler, J. V., Glutamate and aspartate as hippocampal transmitters: biochemical and pharmacological evidence. In P. J. Roberts, J. Storm-Mathisen and G. A. R. Johnston (Eds.), Glutamate: Transmitter in the Central Nervous System, Wiley, New York, 1981, 117-154. 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 Crawford, I. L. and Connor, J. D., Zinc and hippocampal function, J. Orthomolec Psych., 4 (1975) 39-52. 8 Danscher, G. and Zimmer, J., An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues, Histochemistry, 55 (1978) 27-40. 9 Freeman, H., Crystal structure of metal-peptide complexes, Adv. Protein Chem., 22 (1967) 257-424. 10 Hassler, O. and Soremark, R., Accumulation of zinc in mouse brain. An autoradiographic study with 65Zn, Arch. Neurol., 19 (1968) 117-120. 11 Haug, F.-M. S., Electron microscopical localization of the zinc in hippocampal mossy fiber synapses by a modified sulphide silver procedure, Histochemie, 8 (1967) 355-368. 12 Haug, F.-M. S., Heavy metals in the brain, Adv. Anat. Emryol. Cell Biol., 47 (1973) 1-71. 13 Hesse, G. W., Chronic zinc deficiency alters neuronal function of hippocampal mossy fibers, Science, 205 (1979) 1005-1007. 14 Hjorth-Simonsen, A., Projection of the lateral part of the entorhinal area to the hippocampus and fascia dentata, J. comp. Neurol., 146 (1972) 219-232. 15 Howell, G. A., Welch, M. G. and Frederickson, C. J., Stimulation-induced uptake and release of zinc in hippocampal slices, Nature (Lond.), 308 (1984) 736-738. 16 Itoh, M. and Ebadi, M., The selective inhibition of hippo-
campal glutamic acid decarboxylase in zinc-induced epileptic seizures, Neurochem. Res., 7 (1982) 1287-1298. 17 Laurberg, S. and Sorensen, K. E., Associational and commissural collaterals of neurons in the hippocampal formation (hilus fasciae dentatae and subfield CA3), Brain Research, 212 (1981) 287-300. 18 Margerison, J. H. and Corsellis, J. A. N., Epilepsy and the temporal lobes, Brain, 89 (1966) 499-530. 19 Meldrum, B. S., Horton, R. W. and Brierley, J. B., Epileptic brain damage in adolescent baboons following seizures induced by allylglycine, Brain, 97 (1974) 407-418. 20 Nadler, J. V., Perry, B. W. and Cotman, C. W., Intraventricular kainic acid preferentially destroys hippocampal pyramidal cells, Nature, (Lond.) 271 (1978) 676-677. 21 Olney, J. W., deGubareff, T. and Sloviter, R. S., 'Epileptic' brain damage in rats induced by sustained electrical stimulation of the perforant path. II. Ultrastructural analysis of acute pathology, Brain Res. Bull., 10 (1983) 699-712. 22 Purpura, D. P. and Gonzalez-Monteagudo, O., Acute effects of methoxypyridoxine on hippocampal end-blade neurons: an experimental study of special pathodisis in the cerebral cortex, J. Neuropath. exp. Neurol., 19 (1960) 421-432. 23 Ramon y Cajal, S., Histologie du Systeme Nerveux de l'Homme et des Vertebres, Vol. 2, Maioine, Paris, 1911. 24 Sloviter, R. S., A simplified Timm stain procedure compatible with formaldehyde fixation and routine paraffin embedding of rat brain, Brain Res. Bull., 8 (1982) 771-774. 25 Sloviter, R. S., 'Epileptic' brain damage in rats induced by sustained electrical stimulation of the perforant path. I. Acute electrophysiological and light microscopic studies, Brain Res. Bull., 10 (1983) 675-697. 26 Smart, T. G. and Constanti, A., Pre- and postsynaptic effects of zinc on in vitro prepyriform neurones, Neurosci. Lett., 40 (1983) 205-211. 27 Storm-Mathisen, J., Leknes, A. K., Bore, A. T., Vaaland, J. L., Edminson, P., Haug, F.-M. S. and Ottersen, O. P., First visualization of glutamate and GABA in neurons by immunocytochemistry, Nature, (Lond.) 301 (1983) 517-520. 28 Timm, F., Sur Histochemie der Schwermetalle, das SulfidSilber-Ferfahren, Dr. Z. ges. gerichtl. Med., 46 (1958) 706-711. 29 Vallee, R. B., Biochemistry, physiology and pathology of zinc, Physiol. Rev., 39 (1959) 443-490. 30 Von Euler, C., On the significance of the high zinc content in the hippocampal formation. In P. Passouant (Ed.), Physiologie de l'Hippocampe, Editions du Centre National de la Recherche Scientifique, Paris, 1962, 135-145.
National Institutes of Health (NS 18201).
Brain Research, 330 (1985) 150-153 Elsevier
BRE 20668
A selective loss of hippocampal mossy fiber Timm stain accompanies granule cell seizure activity induced by perforant path stimulation ROBERT S. SLOVITER Neurology Center, Helen Hayes Hospital, West Haverstraw, NY 10993 and Departments of Pharrnacology and Neurology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (U.S.A.) (Accepted October 23rd, 1984) Key words: hippocampus - - zinc - - epilepsy - - Timm stain
Perforant path stimulation for 24 h evoked hippocampal granule cell spikes and epileptiform discharges throughout the stimulation period. After stimulation, hippocampal neurons that receive granule cell output were irreversibly damaged and the stimulated hippocampus of each rat revealed a selective loss of Timm stain in the mossy fiber pathway. These results provide evidence in vivo for a role for synaptic zinc in neurotransmission and/or seizure mechanisms.
Although the function of synaptic metals present in the normal brain is unclear, considerable evidence suggests a role for metal ions in neurotransmission2,7,t3,15,26,29,30. We have used a recently developed focal seizure model in rats 25 to address the possible role of zinc in hippocampal function or dysfunction and report that the electrical stimulation that evokes hippocampal granule cell discharges also causes a selective, nearly total loss of histochemically stainable transitional metal in the zinc-containing a0,H hippocampal mossy fiber pathway. Eleven male S p r a g u e - D a w l e y descendant rats (300-450 g) were allowed free access to food and water and were housed on a 12-h light/dark cycle. Rats were anesthetized with urethane (Sigma Chemicals; 1.25 g/kg i.p.) and placed in a stereotaxic instrument. Rectal temperature was monitored and maintained at 37 + 1 °C for the 24 h duration of the experiment. Hippocampal granule cell firing was evoked in vivo by electrical stimulation of the perforant path 1, the main excitatory afferent to the hippocampus 23. Evoked granule cell firing was maintained and recorded for 24 h as described in detail previously25. The stimulation paradigm used to evoke granule cell spikes and epileptiform discharges consisted of continuous twin pulse stimulation at 2 Hz (pulses
40 ms apart) combined with a 10-s, single pulse, 20 Hz stimulus train delivered every minute for 24 h (1440 stimulus trains). As a result of sustained granule cell firing, the cells that are innervated by the mossy fiber pathway (CA3 pyramidal cells and hilar interneurons) are irreversibly damaged and this pattern of damage closely resembles the pattern of hippocampal damage caused by a variety of convulsant drugsl9, 20.22 or seen in the brains of chronic temporal lobe epileptics 18. A detailed description of this seizure-induced neuropathology has been published previously 25. Two separate control groups were used in this study. 'Unstimulated controls' were rats subjected to all experimental procedures including perforant path electrode placement but no electrical stimuli were delivered. 'Stimulated controls' were rats in which the electrode tips were placed in the cortex at the minimum distance above the perforant path at which stimuli did not evoke a hippocampal potential. These rats were stimulated with the same electrode for the same duration and at the same frequency and voltage (20V; 0.1 ms) as the experimental rats. These two groups therefore controlled for the effects of anesthesia, surgery and electrode placement, as well as for hydrolysis and deposition of metal ions at the electrode tips.
Correspondence: R. S. Sloviter, Neurology Center, Helen Hayes Hospital, West Haverstraw, NY 10993, U.S.A. 0006-8993/85/$03.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)
151 The sulphide/silver (Timm) histochemical stain for visualizing synaptic transitional metals8,12,24,28 was used to determine in vivo the effect sustained neurotransmission might have on the zinc contained in the mossy fiber terminals. The specific technique used to visualize zinc is a modified Timm stain that utilizes formaldehyde fixation and routine paraffin embedding, thus allowing histochemical and morphological analysis of the same sections. The details of this histochemical technique have been published previously24. After intracardiac perfusion with sulphide and formalin, brains were embedded in paraffin. Blocks containing control and experimental brains were then cut, processed for silver development and developed together. Each slide rack contained mounted sections from experimental and control groups to permit valid comparisons between treatment groups. Perforant path stimulation for 24 h evoked granule cell population spikes and epileptiform discharges throughout the stimulus period and produced, in all 5 experimental rats, the pattern of selective hippocampal damage described in detail previously25. No obvious hippocampal damage was produced in the hippocampi of the 'unstimulated' or 'stimulated' controls. Blind, qualitative evaluation of sulphide/silver stained sections of stimulated (n = 5) and control brains (n = 3 in each group) revealed a selective decrease and sometimes a virtually complete loss of Timm staining in both granule cell axon terminal fields on the stimulated side of the brain (Fig. 1). Since seizure activity evoked by perforant path stimulation is usually restricted to the ipsilateral hippocampus 25, the contralateral hippocampus usually exhibited little or no neuronal damage and negligible loss of mossy fiber Timm staining. In cases in which contralateral granule cell epileptiform discharges did occur, neuronal damage to contralateral hilar interneurons and CA3 pyramidal cells was evident as was a contralateral decrease in mossy fiber staining. When a decrease in both contralateral and ipsilateral mossy fiber staining was evident, the loss was greater on the ipsilateral side. The decrease or loss of mossy fiber staining was selective in that the other 3 laminas of the same hippocampus appeared relatively normally stained. These 3 regions include: (1) the Timmstained layer in the outer third of the dentate molecular layer that reflects synaptic metal contained in the
projection from the lateral entorhinal cortex14; (2) the stained inner third of the molecular layer that refleets the metal-containing axon terminals of the hilar cells that form the ipsilateral associational/commissural projection to this regionl7; and (3) the metalcontaining axons of the CA3 pyramidal cells that innervate stratum oriens, stratum radiatum and stratum moleculare of regio inferior and regio superior 4. The staining of control and normal (naive) brains did not appear different but the possibility of subtle differences due to anesthesia, surgery, etc., cannot be discounted on the basis of this qualitative evaluation. Perforant path stimulation for 24 h did not cause obvious changes in sulphide/silver staining in the other heavily stained cortical regions, such as the amygdala and temporal neocortex. These results show that after 24 h of perforant path stimulation that evoked repetitive firing of the hippocampal granule cells, there is a near total loss of transitional metal staining in the presynaptic terminals of these neurons. There is no obvious explanation why the staining of the lateral perforant path, part of the fiber system being directly stimulated, was not similarly reduced. The decrease in mossy fiber Timm staining that accompanies granule cell firing could be secondary to granule cell damage. However, previous studies showed that granule cells discharge throughout the 24-h period of stimulation and that mossy fiber morphology after stimulation is norma121. The two most likely explanations for the decreased staining are: (1) a loss of presynaptic metal as a result of a hypothetical physiological release mechanism or, (2) that continuous neuronal firing results in a change in presynaptic zinc disposition that renders the zinc unavailable for reaction with the sulphide that is the basis of the sulphide/silver stain. In view of the recent reports that potassium, kainic acid or electrical stimulation releases zinc in vitro from hippocampal slices2,15, the present results are consistent with a zinc-release hypothesis. If zinc is released as a result of neuronal discharge, the possibility should be considered that its release could play a role in the neuropathology that results from seizure activity. A presynaptic role for zinc in neurotransmission distinct from a simple release mechanism is suggested by the observations that the zinc-containing mossy fiber pathway and other excitatory pathways of the
152
Fig. 1. Effect of 24 h perforant path stimulation on Timm stainability in rat hippocampus. A: hippocampus contralateral to stimulated side showing typical sulphide/silver staining pattern. B: same hippocampus as in A above at higher magnification to show distinct staining of the zinc-containing subgranular granule cell axon collateral terminals (H) that innervate interneurons in the hilus and the mossy fiber terminals (CA3) that innervate CA3 pyramidal cells. C: same hippocampus as in A and B above showing lateral CA3 pyramidal cells and their innervation by the mossy fibers (MF). D - F : Similar views of the hippocampus on the stimulated side of the same brain. Note decreased sulphide/silver staining in the regions innervated by the hilar axon collaterals and by the mossy fibers. Coronal, paraffin-embedded sections cut 10/~m thick, stained for 45 min in physical developer and then counterstained with cresyl violet. 26× in A and D; 104× in B, C, E and F.
h i p p o c a m p u s m a y use g l u t a m a t e as a transmitter6, 27 and that g l u t a m a t e and zinc i n t e r a c t c h e m i c a l l y to
that zinc is b o u n d n o r m a l l y to mossy fiber g l u t a m a t e o r a n o t h e r a m i n o acid suggests that zinc m a y play a
f o r m g l u t a m a t o z i n c (II) dihydrate3, 9. T h e possibility
role in regulating the a m o u n t of t r a n s m i t t e r available
153 for release. According to this hypothesis, altered transmitter status as a result of sustained seizure activity could result in altered zinc binding and there-
nisms 16.
fore less zinc being available for reaction with sulphide. Regardless of the mechanism underlying the
The author thanks D a w n Peters for photographic assistance and Laurie Sanchez for secretarial assistance. This work was presented at the Neurobiology
seizure-associated loss of histochemically stained zinc, the present results support the hypothesis that
of Zinc Symposium, Boston, M A , N o v e m b e r 1983. This investigation was supported by research grants
synaptic zinc plays a role in neurotransmissi0n2,7,13,15,26,29,30 and, possibly, seizure mecha-
from the Epilepsy F o u n d a t i o n of America and the
1 Andersen, P., Holmqvist, B. and Voorhoeve, P. E., Entorhinal activation of dentate granule cells, Acta. physiol. scand., 66 (1966) 448-460. 2 Assaf, S. Y. and Chung, S.-H., Release of endogenous Zn2+ from brain tissue during activity, Nature (Lond.), 308 (1984) 734-736. 3 Bere, A. and Helene, C., Formation of ternary complexes involving zinc or copper ions, polynucleotides and polypeptides containing glutamic acid and tyrosine residues, Biopolymers, 18 (1979) 2659-2672. 4 Blackstad, T. W., Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination, J. comp. Neurol., 105 (1956) 417-538. 5 Cotman, C. W. and Nadler, J. V., Glutamate and aspartate as hippocampal transmitters: biochemical and pharmacological evidence. In P. J. Roberts, J. Storm-Mathisen and G. A. R. Johnston (Eds.), Glutamate: Transmitter in the Central Nervous System, Wiley, New York, 1981, 117-154. 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 Crawford, I. L. and Connor, J. D., Zinc and hippocampal function, J. Orthomolec Psych., 4 (1975) 39-52. 8 Danscher, G. and Zimmer, J., An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues, Histochemistry, 55 (1978) 27-40. 9 Freeman, H., Crystal structure of metal-peptide complexes, Adv. Protein Chem., 22 (1967) 257-424. 10 Hassler, O. and Soremark, R., Accumulation of zinc in mouse brain. An autoradiographic study with 65Zn, Arch. Neurol., 19 (1968) 117-120. 11 Haug, F.-M. S., Electron microscopical localization of the zinc in hippocampal mossy fiber synapses by a modified sulphide silver procedure, Histochemie, 8 (1967) 355-368. 12 Haug, F.-M. S., Heavy metals in the brain, Adv. Anat. Emryol. Cell Biol., 47 (1973) 1-71. 13 Hesse, G. W., Chronic zinc deficiency alters neuronal function of hippocampal mossy fibers, Science, 205 (1979) 1005-1007. 14 Hjorth-Simonsen, A., Projection of the lateral part of the entorhinal area to the hippocampus and fascia dentata, J. comp. Neurol., 146 (1972) 219-232. 15 Howell, G. A., Welch, M. G. and Frederickson, C. J., Stimulation-induced uptake and release of zinc in hippocampal slices, Nature (Lond.), 308 (1984) 736-738. 16 Itoh, M. and Ebadi, M., The selective inhibition of hippo-
campal glutamic acid decarboxylase in zinc-induced epileptic seizures, Neurochem. Res., 7 (1982) 1287-1298. 17 Laurberg, S. and Sorensen, K. E., Associational and commissural collaterals of neurons in the hippocampal formation (hilus fasciae dentatae and subfield CA3), Brain Research, 212 (1981) 287-300. 18 Margerison, J. H. and Corsellis, J. A. N., Epilepsy and the temporal lobes, Brain, 89 (1966) 499-530. 19 Meldrum, B. S., Horton, R. W. and Brierley, J. B., Epileptic brain damage in adolescent baboons following seizures induced by allylglycine, Brain, 97 (1974) 407-418. 20 Nadler, J. V., Perry, B. W. and Cotman, C. W., Intraventricular kainic acid preferentially destroys hippocampal pyramidal cells, Nature, (Lond.) 271 (1978) 676-677. 21 Olney, J. W., deGubareff, T. and Sloviter, R. S., 'Epileptic' brain damage in rats induced by sustained electrical stimulation of the perforant path. II. Ultrastructural analysis of acute pathology, Brain Res. Bull., 10 (1983) 699-712. 22 Purpura, D. P. and Gonzalez-Monteagudo, O., Acute effects of methoxypyridoxine on hippocampal end-blade neurons: an experimental study of special pathodisis in the cerebral cortex, J. Neuropath. exp. Neurol., 19 (1960) 421-432. 23 Ramon y Cajal, S., Histologie du Systeme Nerveux de l'Homme et des Vertebres, Vol. 2, Maioine, Paris, 1911. 24 Sloviter, R. S., A simplified Timm stain procedure compatible with formaldehyde fixation and routine paraffin embedding of rat brain, Brain Res. Bull., 8 (1982) 771-774. 25 Sloviter, R. S., 'Epileptic' brain damage in rats induced by sustained electrical stimulation of the perforant path. I. Acute electrophysiological and light microscopic studies, Brain Res. Bull., 10 (1983) 675-697. 26 Smart, T. G. and Constanti, A., Pre- and postsynaptic effects of zinc on in vitro prepyriform neurones, Neurosci. Lett., 40 (1983) 205-211. 27 Storm-Mathisen, J., Leknes, A. K., Bore, A. T., Vaaland, J. L., Edminson, P., Haug, F.-M. S. and Ottersen, O. P., First visualization of glutamate and GABA in neurons by immunocytochemistry, Nature, (Lond.) 301 (1983) 517-520. 28 Timm, F., Sur Histochemie der Schwermetalle, das SulfidSilber-Ferfahren, Dr. Z. ges. gerichtl. Med., 46 (1958) 706-711. 29 Vallee, R. B., Biochemistry, physiology and pathology of zinc, Physiol. Rev., 39 (1959) 443-490. 30 Von Euler, C., On the significance of the high zinc content in the hippocampal formation. In P. Passouant (Ed.), Physiologie de l'Hippocampe, Editions du Centre National de la Recherche Scientifique, Paris, 1962, 135-145.
National Institutes of Health (NS 18201).