Effects of zinc on markers of glutamate and aspartate neurotransmission in rat hippocampus

Brain Research, 334 (1985) 281-286 Elsevier

281

BRE 10742

Effects of Zinc on Markers of Glutamate and Aspartate Neurotransmission in Rat Hippocampus JOHN T. SLEVIN and EDWARD J. KASARSKIS Departments of Neurology and Toxicology, University of Kentucky, Veterans Administration Medical Center, and The Sanders-Brown Research Center on Aging, Lexington, KY 40536 (U.S.A.

(Accepted August 21st, 1984) Key words: zinc - - zinc deficiency - - hippocampus - - glutamate - - aspartate

Receptor binding and synaptosomal uptake of L-[3H]glutamate and L-[3H]aspartate were measured in hippocampus derived from rats maintained on zinc restricted diets from weaning. Despite near lethal zinc deficiency, these markers of excitatory amino acid neurotransmission were unaffected compared to zinc-supplemented controls. However, addition of zinc in vitro markedly inhibited binding of glutamate and aspartate to hippocampal membranes. These data suggest that zinc can modulate the receptor affinities for glutamate and aspartate and may function as a tonic inhibitor of excitatory synapses in vivo.

INTRODUCTION The hippocampus contains the highest concentration of zinc (Zn) in the central nervous systemtt, 30 where it is particularly enriched in the dentate gyrus and subfields CA3-CA414,15. Although the physiological functions for Zn in the hippocampus have yet to be defined, histoanalytical and chemical evidence suggests that a unique pool of chelatable Zn may exist in association with mossy fibers 9`ISAT. Mossy fiber Zn, revealed by dithizone histochemistry, accounts for 8% of total hippocampal Zn and may be localized within synaptic vesicles of the giant boutont4,1L This unusual compartmentalization of Zn has led to the suggestion that Zn may function, in some as yet undefined way, in the process of neurotransmission at the mossy fiber synapse 6-9. Several lines of evidence support this hypothesis and implicate a potential interaction of Zn with excitatory amino acid neurotransmitters 3t. Crawford and Connor have noted a positive correlation in CA3 among Zn concentration, glutamate levels and glutamate release following entorhinal stimulation 7. In another study, severe Zn deficiency was associated

with a decrement in the amplitude of evoked responses over mossy fiber pathways 16. This effect however was not observed with repetitive stimulation via the non-Zn-rich commissural tracts. Further support for a possible interaction between Zn and mossy fiber neurotransmission comes from studies of 65Zn uptake by hippocampal slices in vitro which indicate that a high-affinity Zn uptake system is active in C A 3 - C A 4 which is enhanced by electrical pulse stimulation of the granule cells 17. There is now considerable evidence to suggest that excitatory amino acids subserve neurotransmission in several hippocampal pathways 22,23.29.33. Efferent projections from CA3 pyramidal cells to either granule cells or to CA1 pyramidal cells are glutamatergic as are the afferent projections to granule cells from the entorhinal cortex via the perforant path. Although the neurotransmitter in the mossy fiber pathway is not known with certainty, most studies suggest that an excitatory amino acid subserves this role 7. Because alterations in Zn status have been proposed to interfere in hippocampal neurotransmission in mossy fibers in both experimental animals and in human pathological conditions such as Pick's dis-

Correspondence: E. J. Kasarskis, Present address: Department of Neurology, University of Kentucky, Lexington, KY 40536, U.S.A.

0006~8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

282 ease 5-~6, the following experiments were conducted to determine the effect of Zn deficiency on the integrity of pre- and postsynaptic markers of glutamate and aspartate neurotransmission in rat hippocampus. MATERIALS AND METHODS

Animal care and diet Weanling male Sprague-Dawley rats (40-50 g; Harlan Farms) were randomly assigned to 3 dietary groups and housed in a controlled 12 h light-dark, constant temperature-humidity environment. The first group (ZD) was fed a Zn-deficient diet containing < 1 ppm (mg/kg) Zn (Teklad Test diets; Madison, WI) ad fibitum. Consumption of this egg white-based diet, modified from the formulation of Luecke et al., produces severe clinical Zn deficiency over the 45-day experimental period 21. Food consumption by Z D rats was determined daily by weighing. Two control groups received the identical diet supplemented with 50 ppm (as ZnCO3) either ad libiturn (AL) or pair-fed (PF) based upon the measured food consumption of the Z D group. All groups were offered deionized water from glass bottles fitted with silicone rubber stoppers and stainless-steel sipper tubes. Rats were housed in pairs in stainless-steel cages, equipped with stainless-steel shields to prevent chewing of the rubber stoppers (a source of Zn contamination).

Tissue preparation Rats were decapitated between 08.00 and 09.00 h and a sample of blood was collected in acid-washed polypropylene tubes for analysis of serum Zn content. Both hippocampi were rapidly removed by dissection on an ice-chilled glass plate. The fresh tissue was homogenized in 20 vols. of ice-cold 0.32 M sucrose with a Teflon-glass homogenizer (10 strokes). The homogenate was centrifuged at 1000 g. A known volume of the supernatant was centrifuged at 11,000 g for 15 rain, and the crude synaptosomal pellet was resuspended in isotonic sucrose-~k

Synaptosomal uptake of L-[3H]glutamate The synaptosomal uptake of L-[3H]glutamic acid (43.5 Ci/mmol; New England Nuclear) was performed using the method of Logan and Snyder 20. Briefly, homogenates of the crude synaptosomal pellet (P~

fraction) containing the eqmvalent of i m g of fresi~ hippocampal tissue (0.04 mg membrane protein) were incubated with L-[-~H]glutamate ~t uM finai substrate concentration) in 1.0 ml of Kret~s-Ringerphosphate buffer for 5 rain at 2(t ~'C. h~ order t~ measure non-specific glutamate uptake, a buffer m which choline chloride was substituted for sodium chloride was used. Incubations were terminated by addition of ice-cold, Na-free buffer followed immediately by centrifugation at 20,000 g for 15 rain at 4 X?. The supernatant was discarded and the pellets were superficially washed twice with ice-cold, Na-free buffer. The pellets were dissolved in Protosol (New England Nuclear) and assayed for 3H by liquid scintillation spectrometry.

Receptor bindings studies Glutamate. For measurement of specific L-[3H]glutamic acid binding, modification of the procedure of Foster and Roberts was used 13. Aliquots of the crude synaptosomal fraction were pelleted, resuspended in 100 vols. of ice-cold glass-distilled deionized water (GDW) and sonicated for 30 s with a cell disruptor (Ultrasonics; setting 3). This suspension was centrifuged at 50,000 g for 15 rain; the pellet was then resuspended in 70 vols. of 50 mM Tris-HCl, pH 7.4. This tissue suspension was incubated at 37 °C for 30 min to remove endogenous glutamate and then centrifuged at 50,000 g for 10 min. The membranes were resuspended in 50 mM Tris-HCl buffer, pH 7.4; a 500 ~1 aliquot was added to 1.5 ml Eppendoff polypropylene microcentrifuge tubes containing 350 pmol of L-[3H]glutamic acid (43.5 Ci/mmol; New England Nuclear) in 0.5 ml for a final ligand concentration of 350 nM. In order to study the effect of Ca enhancement of glutamate binding, some samples contained CaCI 2 (1 mM final concentration). For the displacement experiments, various concentrations of ZnC12 were added to some samples. Zinc concentrations were verified by flame atomic absorbtion spectrophotometry (AAS). To measure non-specific binding, 500 nmol of unlabeled L-glutamic acid were added to the radioligand (500pM final concentration). All samples were incubated at 37 °C for 20 rain and the reaction was terminated by centrifugation at 50,000 g at 4 °C. The supernatant was removed by aspiration, and the pellet was immediately and superficially washed with 1 ml of G D W at 4 °C. The pellets

283 were then dissolved in Protosol for 3H determination. A s p a r t a t e . Binding of L-[3H]aspartic acid to hippocampal membranes was measured by the method of DiLauro et al. 10. Aliquots of the crude synaptosomal preparation were lysed by sonic disruption and hypotonic shock, successively frozen, thawed and finally extensively washed with Tris-HC1 (5 mM, pH 7.4). The pellet was resuspended in 15 vols. of 50 mM Tris-HCl, pH 7.4. Two hundred microliters of the membrane suspension were added to 1.5 ml Eppendoff polypropylene microcentrifuge tubes containing 200 pmol of L-[3H]aspartic acid (17.3 Ci/mmol; New England Nuclear). Some samples contained MgCI 2 (1.0 mM final concentration) and some contained various concentrations of ZnCl 2. To measure nonspecific binding, 250 nmol of unlabeled L-aspartic acid were added to the radioligand. The tubes were preincubated for 10 min at 20 °C in a shaking water bath. The incubation was initiated by addition of the radioligand, continued for 30 min at 20 °C and stopped by addition of 4 ml of cold Tris-HCl buffer (50 mM, pH 7.4), followed by rapid filtration through Millipore glass fiber filters (Millipore, Bedford, MA). Filters were then rinsed twice with 4 ml of cold Tris-HC1 buffer. The retained radioactivity was measured by liquid scintillation spectrometry in 10 ml of Aquasol (New England Nuclear). Zn Determination

Hippocampi were placed in tared, acid-washed quartz crucibles and dried for 24 h at 100-105 °C. After cooling in a dessicator over silica gel, samples were weighed to determine dry matter and ashed 24 h at 450 °C in a muffle furnace. Ash was dissolved

in 0.5 ml HNO 3 (Ultrex Grade; J. T. Baker Chemicals) and diluted to a known volume with deionized water in volumetric flasks. Serum (0.25 ml) was diluted with 1.0 ml deionized water. Samples were aspirated directly into a Varian AA-475 flame atomic absorption spectrophotometer. Working standards (0, 0.1, 0.3, 0.5, 0.7 and 1.0 ¢tg/ml) were prepared in polypropylene tubes by dilution of certified reference Zn standards (1000 #g/ml; Fischer Scientific). Triplicate readings were made for all samples. RESULTS Rats fed Zn-restricted diets displayed typical signs of the Zn deficiency syndrome such as growth failure, anorexia, cyclic pattern of eating and dermatitis 21,30. ZD rats had significantly decreased serum Zn levels compared to either PF or AL controls (Table I) whereas the concentration of Zn in whole hippocampus was unaffected by dietary treatment. Neither chronic Zn deprivation nor caloric restriction (PF group) significantly affected the specific activity of high-affinity glutamate uptake into hippocampal synaptosomes (Table II). Similarly, neither dietary treatment significantly affected binding activity of either glutamic or aspartic acids. Although not at the level of statistical significance (1.3-fold), a tendency toward reduced L-[3H]glutamate uptake and increased binding was observed in the ZD group compared to either PF or AL controls, Glutamate binding to hippocampal membranes was enhanced (1.5-fold increase) by the presence of 1.0 mM CaC12, as reported previously3,1L Similarly,

TABLE I Effect o f dietary zinc restriction on tissue mass and zinc concentration

Treatment groups described in Materials and Methods section. Hippocampalweights derived from the average weight of both hippocampi for each rat. Significancelevels (Student's t-test): a p < 0.001 vs AL control; ~P < 0.001 vs PF control; c p < 0.02 vs PF control: d not significant. Numbers with parentheses represents the number of observations contributing to the mean _+S.E.M. Dietary treatment

Zinc deficient Pair-fed control Adlibitum control

Weight

Zinc level

Body (g)

Hippocampus (mg)

Serum (ng/ml)

Hippocampus (ng/mg wet wt. )

61 _+ 2.8~,b (15) 97 __ 5.3" (16) 240 + 3.9 (16)

41.1 + 1.1.,c (15) 44.8 _+ 0.8~ (16) 49.5 + 0.7 (16)

312 + 37a.b (10) 1460 +_ 61d (12) 1358 + 62 (12)

103 _+ 2.4d (10) 104 +_ 1.6a (12) 100 + 1.1 (12)

284 TABLE II z-/SH]Glutamic acid uptake by hippocampal [~ /?actions (prnoles uptake, mg protein t. min - i)

Fresh hippocampi from Zn-deficient and control rats were prepared as described in the Materials and Methods section. Assays were done in duplicate with duplicate blanks for each hippocampus. There was no significant difference among the groups. Mean _+S.E.M, Treatment

J

l

*I~ o

LL GLUTAMATE d080)~ G~-OU~OA~ATE(

~

~

t

o

e~

Uptake ,

Zinc deficient (n = 7) Pair-fed control (n = 8) Ad libitum control (n = 8)

ASPARTATI!

..

362 ± 21 398 ± 24 391 ± 29

~-

" ,, ,. •

i__ -6

1

-5

_ _ _ _ L

-4

o

o

t ............

3

L

-2

LOglo[Zinc]

Figl t. Inhibition of the specific binding of [3H}amino acid to the presence of 1.0 mM MgCI 2 resulted in a 25% increase in measurable [3H]aspartate bindingl0. The effect of CaC12 and MgCI 2 on glutamate and aspartate binding, respectively, was i n d e p e n d e n t of dietary Z n manipulation. In contrast, binding of these two amino acids to m e m b r a n e homogenates was inhibited by addition of ZnC12 in vitro to the preparation (Fig. 1). Aspartate binding appeared especially sensitive with 50% inhibition with as little as 50 ~ M Z n (verified by direct A A S analysis) in the m e d i u m , compared to L-[3H]glutamate binding (IC50 of 131 ktM).

hippocampal membranes. Washed Pz membranes prepared from adult rat hippocampus were incubated with either 350 nM L-pH]glutamate (0) or 200 nM t,-[3H]aspartate (O) in the presence of various concentrations of ZnC12. The results, the mean of at least 3 assays done in triplicate, presented a Schild plot with the slopes indicated in parentheses.

50% of either PF or AL, Z n - s u p p l e m e n t e d controls and exhibited characteristic clinical signs such as skin lesions and growth failure 21. Despite severe extracerebral Z n deficiency, all 3 groups maintained essentially identical levels of hippocampal Z n in agreement with the study of Wallwork et al. 30. U n d e r

DISCUSSION In the present study, rats fed diets restricted in Z n had serum Z n levels depressed to approximately TABLE II Effect of dietary zinc restriction on excitatory amino acid binding to hippocampus (pmoles bound.rag protein -I + S. E. M. )

Fresh hippocampi were prepared as described in the Materials and Methods section. Assays were done in duplicate with duplicate blanks for each hippocampus, 7-8 animals comprised each group. No significant difference was measured across animal groups. CaCI2 or MgCI2 enhanced glutamate or aspartate binding respectively: " P < 0.005; b p < 0.0l; c p < 0.002; a NS. Mean + S.E.M. L-I- H]glutamic acid binding

CaCIz, 0 mM CaCI2, 1.0mM

Zinc deficient

Pair-fed

A d libitum

29.2 _+ 3.7 42.9 _+ 5.4"

23.1 + 1.9 32.3 _+ 3.8h

26.5 _+ 2.3 39.5 _+ 4.9~'

c-[SH]aspartic acid binding Zinc deficient

MgCI2,0 mM 1.93 _+ 0.21 MgCI2, 1.0mM 2.79 + 0.19c

Pair-fed

Ad libitum

2.15 _+ 0.45 2.5(/± 0.43d

1.96 _+ 0.26 2.41 + (I.420

our experimental conditions (which were similar to those of Hessel6), dietary Z n status did not affect either the uptake of L-[3H]glutamate or L-[3H]aspar tate by hippocampal synaptosomes or binding of these ligands to isolated synaptosomal hippocampal membranes. However, there was a trend to increased glutamate binding in Z D rats compared to PF controls, suggesting that an inverse relationship may exist between Z n status and receptor binding of [3H]glutamate. Because Z n and calcium are mutually antagonistic in many biological systems, the effect of 1 m M CaC12 on glutamate binding to hippocampal m e m b r a n e s derived from Z n deficient rats was investigated2.4. Our data confirm the observations of Baudry and Lynch -~ and Fagg et al. 12, that in vitro Ca augments glutamate binding; however, they did not disclose a C a - Z n interaction on glutamate binding to hippocampal membranes of Z n deficient animals. Similarly, magnesium added in vitro increased measurable aspartate binding. However, the present study failed to identify any change in aspartate binding due to Z n nutriture either in the presence or absence of added Mg.

285 The results of these experiments d e m o n s t r a t e that Zn can antagonize the binding of g l u t a m a t e and aspartate to their respective receptors in the h i p p o c a m pus. The inhibitory effect of Zn a d d e d in vitro was striking and supports the hypothesis that only a m i n o r redistribution of Z n among its pools in vivo could p r o f o u n d l y affect central neurotransmission by m o d ulating the r e c e p t o r affinities for excitatory amino acid transmitterslL It is difficult to reconcile our data with the results of Hesse which d e m o n s t r a t e d a reduction in neural transmission through mossy fiber pathways, presumably due to depletion of Zn in this p a t h w a y 16. Conceivably, our animals were not sufficiently Zn deficient to affect either the binding or u p t a k e of glutamate and/or aspartate. Essentially nothing is known regarding the effect of Zn deficiency on Zn m e t a b o lism in mossy fibers. O u r data and those of others suggest that the concentration of Zn in the whole hipp o c a m p u s is not altered30; however, these data do not preclude a redistribution of available Z n within the h i p p o c a m p u s iS. Alternatively, Zn m a y have been d e p l a t e d in mossy fiber boutons, but g l u t a m a t e and/or aspartate may not function at that synapse 23. The animals in the present study were c o m p a r a b l y Zn deficient to those described by Hesse, and an alteration in m a r k e r s of glutamate and/or aspartate neurotransmission might be anticipated if the hypothesized interaction exists 16. H o w e v e r , our data and those of N a d l e r et al. 23, do not provide support for a Z n - g l u t a m a t e - a s p a r t a t e interaction in the hipp o c a m p u s under conditions of near-lethal Zn defi-

ciency induced by dietary restriction. The results of this study add aspartate and glutamate to the list of putative neurotransmitters whose receptor affinities can be modified by Zn. The 7-aminobutyric acid, muscarinic cholinergic, benzodiazepine and opiate receptors all can be m o d u l a t e d by Zn in vitro, although this effect is shared by a variety of other divalent cations as well IA8,19.24-28. It is tempting to speculate that the behavioral changes described in Zn-deficient animals may be related to these alterations in r e c e p t o r affinities which could theoretically occur in the absence of large changes in total Zn content. The present findings also suggest that total hippocampal Zn content is effectively regulated and most p r o b a b l y rigidly c o m p a r t m e n t a l i z e d . It is conceivable that low concentrations of extracellular free Zn may act as a tonic inhibitor of excitatory synapses in hippocampus in the resting state and transiently be sequestered during depolarization and transmitter release 17. Such a mechanism of postsynaptic signal amplification, due to 'disinhibition' related to presynaptic t r a n s m e m b r a n e Z n influx, remains to be tested.

REFERENCES

brain: hippocampal concentration and localization, J. Neurochem., 19 (1972) 1451-1458. 7 Crawford, I. L. and Connor, J. D., Localization and release of glutamic acid in relation to the hippocampal mossy fibre pathway, Nature (Lond.), 244 (1973) 442-443. 8 Crawford, I. L. and Connor, J. D., Zinc and hippocampal function, J. Orthomolec. Psychiat., 4 (1975) 34-52. 9 Crawford, I. L., Zinc and the hippocampus: histology, neurochemistry, pharmacology, and putative functional relevance. In I. E. Dreosti and R. M. Smith (Eds.), Neurobiology o f the Trace Element, Vol. 1, Humana, Clifton, NJ, 1983, pp. 163-211. 10 Dilauro, A., Meek, J. L. and Costa, E., Specific high-affinity binding of l,-[3H]aspartate to rat brain membranes, J. Neurochem., 38 (1982) 1261-1267. 11 Donaldson, J., St. Pierre, T., Minnich, J. L. and Barbeau, A., Determination of Na +, K +, Mg2÷, Ca 2+, Zn 2+, and Mn 2÷ in rat brain regions, Canad. J. Biochem., 51 (1973) 87-92.

1 Baraldi, M., Zinc and GABA binding, Soc. Neurosci. Abstr., 8 (1982) 575. 2 Baudier, J., Haglid, K., Haiech, J. and Gerard, D., Zinc ion binding to human brain calcium binding proteins, calmodulin and S100B protein, Biochem. Biophys. Res. Commun., 114 (1983) 1138-1146. 3 Baudry, M. and Lynch, G., Regulation of glutamate receptors by cations, Nature (Lond.), 282 (1979) 748-750. 4 Brewer, G. J., Aster, J. C., Knutsen, C. A. and Kruckeberg, W. C., Zinc inhibition of calmodulin: a proposed moleclar mechanism of zinc action on cellular functions, Amer. J. Hematol., 7 (1979) 53-60. 5 Constantinidis, J. and Tissot, R., Role of glutamate and zinc in the hippocampal lesions of Pick's disease. In G. Di Chiara and G. L. Gessa (Eds.), Glutamate as a Neurotransmitter, Raven Press, New York, 1981, pp. 413-422. 6 Crawford, I. L. and Connor, J. D., Zinc in maturing rat

ACKNOWLEDGEMENTS The authors thank D a n Walls, Joyce Fain and Lindley F e r r a r a for their expert technical assistance and Bonnie Edens for typing the manuscript. This research was s u p p o r t e d by the Research Service, Veterans A d m i n i s t r a t i o n , and Grants 507-RR05374 ( B . R . S . G . ) , NS00732 (J.T.S.) and NS0068 (E.J.K.).

286 12 Fagg, G. E., Foster, A. C., Mena, E. E. and Cotmam C. W., Chloride and calcium ions separate L-glutamate receptor populations in synaptic membranes, Europ J. Pharmacol., 88 (1983) 105-110. 13 Foster, G. A. and Roberts, P. J., Pharmacology of excitatoI ry amino acid receptors mediating the stimulation of rat cerebellar cyclic GMP levels in vitro, Life Sci., 27 (1980) 215-221. 14 Frederickson, C. J., Howell, G. A. and Frederickson, M. H., Zinc dithizonate staining in the cat hippocampus: relationship to the mossy-fibre neuropil and postnatal development, Exp. Neurol., 73 (1981) 812-823. 15 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 Research, 273 (1983) 335-339. 16 Hesse, G. W., Chronic zinc deficiency alters neuronal function of hippocampal mossy fibers, Science, 205 (1979) 1005-1007. 17 Howell, G. A. and Frederickson, C. J., Selective uptake of zinc in sub-regions of hippocampal slices in vitro, Soc. Neurosci. Abstr., 8 (1982) 991. 18 Hulme, E. C., Berrie, C. P., Birdsall, N. J. M., Jameson, M. and Stockton, J, M., Regulation of muscarinic agonist binding by cations and guanine nucleotides, Europ. J. Pharmacol., 94 (1983) 59-72. 19 Lo, M. M. S. and Synder, S. H., Two distinct solubilized benzodiazepine receptors: differential modulation by ions, J. Neurosci., 3 (1983) 2270-2279. 20 Logan, W. J. and Snyder, S. H., High affinity uptake systems for glycine, glutamic and aspartic acids in synaptosomes of rat central nervous tissue, Brain Research, 42 (1972) 413-431. 21 Luecke, R. W., Olman, M. E. and Baltzer, V. B., Zinc deficiency in the rat: effect on serum and intestinal alkaline phosphatase activities, J. Nutr., 94 (1968) 344-35(I. 22 Nadler, J. V., Vaca, K. W., White, W. F., Lynch, G. S. and Cotman, C. W., Aspartate and glutamate as possible transmitters of excitatory hippocampal afferents, Nature (Lond.), 260 (1976) 538-540.

23 Nadler, J. V., White, W. F., Vaca, K. W., Perry, B. W and Cotman, C. W., Biochemical correlates ~! transmissiot~ mediated by glutamate and aspartate. J ~ e u r o c h e m ~i (1978) 147-155, 24 Sader, W., Pheiffer, A. and Herz, A., Opiate receptor: multiple effects of metal ions, J. Neurochem., 39 (1982) 659-600. 25 Smith, C. P. and Huger, F. P., Effect of zinc on [3HIONB displacement by cholinergic agonists and antagonists, Biochem. Pharmacol., 32 (1983) 377-380. 26 Smith, C. P. and Huger, F. P., Effect of zinc on 3H-oxotremorine displacement by muscarinic agonists and antagonists, Soc. Neurosci. Abstr., 9 (1983) 581. 27 Stengaard-Pederson, K., Fredens, K. and Larsson, L. I., Enkephalin and zinc in the mossy fiber system, Brain Research, 212 (1981) 230-233. 28 Stengaard-Pederson, K., Fredens, K. and Larsson, L. 1., Inhibition of opiate receptor binding by zinc ions: possible physiological importance in the hippocampus, Peptides, 2, Suppl. 1 (1981) 27-35. 29 Storm-Mathisen, J., Glutamate in hippocampal pathways. In G. Di Chiara and G. L. Gessa (Eds.), Glutamate as a Neurotransmitter, Raven Press, New York. 1981, pp. 43-55. 30 Wallwork, J. C., Milne, D. B., Sims, R. L. and Sandstead, H. H., Severe zinc deficiency: effects of the distribution of nine elements (potassium, phosphorus, sodium, magnesium, calcium, iron, zinc, copper, and manganese) in regions of the rat brain, J. Nutr., 113 (1983) 1985-1905. 31 White, W. F., Nadler, J. V., Hamberger, H., Cotman, C. W. and Cummins, J. T., Glutamate as transmitter of hippocampal perforant path, ,Nature (Lond. i, 270 (t977) 356-357. 32 Whittaker, V. P., The application of suhcel!ular fractionation techniques to the study of brain function, Prog. Biophys. Molec. Biol., 15 (1965) 39-96. 33 Wolf, G, and Schmidt, W., Zinc and glutamate dehydrogenase in putative glutaminergic brain structures, Acta histochem., 72 (1983) 15-23.