16
Brain Researct,,. 529 I J!.~9o) 1t~- i!2 ~ .l~;cvic!
BRES 15885
Effects of dietary zinc status on seizure susceptibility and hippocampal zinc content in the El (epilepsy) mouse Motofumi Fukahori and Masatoshi Itoh Department of Neuropsychiatry, Faculty of Medicine, Kyushu University, Fukuoka (Japan) (Accepted 27 March 1990) Key words: Zinc; Hippocampus; Dentate gyrus; Epilepsy; El mouse; Zinc deficiency; Zinc supplementation
The effects of dietary zinc status on the development of convulsive seizures, and zinc concentrations in discrete hippocampal areas and other parts of the limbic system were studied in the El mouse model receiving zinc-adequate, zinc-deficient or zinc-loaded diets. Seizure susceptibility of the El mouse was increased by zinc deficiency, and decreased by zinc loading, while an adequate diet had no effect. Zinc loading was accompanied by a marked increase in hippocampal zinc content in the El mouse. Conversely, hippocampal zinc content declined in the El mouse fed a zinc-deficient diet. These results suggest that zinc may have a preventive effect on the development of seizures in the El mouse, and hippocampal zinc may play an important role in the pathophysiology of convulsive seizures of epilepsy.
INTRODUCTION The highest concentration of zinc, an essential trace element, is found in the hippocampus 1~'45, particularly within the mossy fiber axons of the dentate gyrus granule neurons 16'42. Zinc localized in the mossy fiber terminals may play a role in the neurotransmitter regulation on the hippocampus 8"19. Furthermore, activation of the dentate gyrus granule cells with electrical stimulation facilitates both the uptake and release of zinc from hippocampus, indicating that the metabolic turnover of zinc is sensitive to electrophysiologic activity 1'4'2a. Recent evidence suggests that the zinc pool associated with the mossy fiber terminals may be released by nerve stimulation. Sloviter demonstrated a selective loss of Timm's staining in the hippocampal mossy fiber terminals during seizures 36, while Frederickson's histofluorescence study indicated a loss of zinc staining from hippocampal mossy fibers during kainic acid induced seizures 15. The literature and recent findings indicate that zinc may be associated with the etiology and manifestation of epileptic seizures. Intracerebroventricular injection of zinc has been shown to cause epileptic seizures in the rat 24, and intracerebral injections of ZnC1 have increased audiogenic seizure susceptibility in mice 5. More recent evidence, however, suggests that zinc can minimize the development of seizures. Howell and colleagues 2° reported that audiogenic seizure activity decreased following subcutaneous zinc injections. Sterman and col-
leagues 37 demonstrated that dietary zinc loading decreased susceptibility to kindled seizures, while zinc deprivation increased susceptibility in cats. Thus, recent findings suggest that zinc may have a preventive effect on the development of seizures 3s. The El mouse is an animal model with genetically determined epilepsy 23. Convulsive seizures occur in response to postural stimulation, particularly paraboloid movements 4°. These seizures are characterized as complex partial seizures, with secondary generalization (i.e. temporal lobe epilepsy) 43. Seizures in this model appear to originate in the hippocampus and then spread to other brain regions 39m. Experiments in our laboratory revealed that the zinc content was significantly lower in the hippocampal dentate area of the El mouse compared with control ( d d Y and CBA strains) mice 17, suggesting possible involvement of hippocampal zinc in the pathophysiology of convulsive seizures in the epileptic mouse. The effect of dietary zinc status on the development of convulsive seizures of the El mouse has not been studied previously. This study was designed to investigate this question and corroborate the preventive effect of zinc on the development of seizures. In order to evaluate the possible relationship between the hippocampal zinc levels and seizure susceptibility, zinc concentrations were measured in discrete hippocampal areas (e.g. the dentate gyrus, subfields CA1, and CA3 areas in both the rostral and caudal hippocampus) and other limbic systems of the zinc-deficient, zinc-loaded and zinc-adequate El mouse.
Correspondence: M. Fukahori, Department of Neuropsychiatry; Faculty of Medicine, Kyushu University, Maidashi 3-1-1, Fukuoka, 812, Japan. 0006-8993/90/$03.50 (~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
17 M A T E R I A L S A N D METHODS
Experiment 1 For this study, 180, 4-week-old male El mice, weighing 7.5-11.5 g (mean = 10.5 g) at the beginning of the study served as subjects. El mice were reared in our institution in a specific pathogen-free, constant temperature-humidity environment, with a 12-h light-dark cycle. Care of the mice included cleansing their cages with 10% nitric acid and subsequent rinsing with distilled water. They were offered distilled, deionized water from glass bottles fitted with silicone stoppers and stainless steel spouts ad libitum to minimize trace metal contamination. The mice were randomly assigned to 3 dietary groups of 60 mice each and housed in groups of 5 in clean stainless steel cages. During a 1-week period of acclimatization, all mice received the identical standard laboratory diet (CLEA Japan, Inc.), which contained 38.9 mg Zn/kg by analysis. During a mean of 8 weeks (range 5-13 weeks) after acclimatization, one group was fed a zinc-deficient (1.1 mg Zn/kg) diet, animals of a second group were fed a zinc-loaded (248 mg Zn/kg) diet, and a third group (control) was fed an adequatezinc (38.9 mg Zn/kg) diet ad libitum. All groups were weighed weekly. Postural stimulation of the El mouse is most commonly achieved by tossing them to a height of approximately 20 cm, 30-40 times. In this study, this was done once each week starting at 4 weeks of age. Generalized seizures were induced at about 7 or 8 weeks of age. The procedure involved removing a mouse from its cage carefully, observing it for several minutes, and then tossing it into the air. As the El mice grew older, less postural stimulation was required to elicit seizures (i.e. the seizure threshold was lowered gradually through repetition of stimulation), and seizure activity persisted throughout life4°. With repetition of stimulation, the pattern of seizure activity changed as the animals developed 4°. Although susceptibility to seizure showed considerable individual variation, by 4-7 weeks of age seizures generally took an abortive form, most commonly squeaks and catatonic posture. Occasionally, simple muscular twitching, which was sometimes accompanied by wild running fits, was observed. By 7-8 weeks of age, typical generalized tonic-clonic convulsions began in most mice. No method is currently available for objective, numerical quantification of the level of susceptibility of individual El mice to the induction of convulsions. Therefore, we devised a system whereby the convulsions in each mouse could be categorized into 3 main types: complete tonic-clonic convulsions ( + + ) , abortive seizure activity (+), and muscular twitching or wild running fits only (+). Scores were then adjusted for age in weeks so that the level of susceptibility of individual El mice to the induction of convulsions could be expressed as the total of the scores from 4 to 13 weeks of age. For example, if a mouse was 4 weeks of age and the pattern of seizure was ' + ' , then the mouse was assigned a score of 10 (see Table I). If the same (or different) mouse was 13 weeks old with a ' + + ' pattern of seizure activity, a score of 21 was assigned. Thus, the result of each stimulation was expressed numerically with that score reflecting the age of the mouse, the pattern of seizure activity, and seizure susceptibility. The series of scores were then totaled. Mice with high total scores were categorized as having high susceptibility; those with low scores were categorized as having low susceptibility. Finally, the total scores were compared among the 3 diet groups using the Wilcoxon t-test. Experiment 2 At 13 weeks of age, mice were selected from each of the 3 dietary groups based on their seizure susceptibility scores. This included 10 mice with low scores in the zinc-loaded group, 10 mice with high scores in the zinc-deficient group, and 10 mice with average scores among those treated with adequate amounts of zinc. Animals were decapitated between 13.00 and 13.30 h at 7 days after the last seizure. The brain was removed, frozen quickly in liquid nitrogen,
(A)
(B)
""i"
(C)
(D)
Fig. 1. Schematic drawing of microdissections of the hippocampus and the other areas that were dissected for assay. A: the rostral hippocampus. B: the caudal hippocampus. The regions assayed were the dentate gyrus (stipple), the subfield CA1 (vertical lines), and the subfield CA3 (oblique lines). C: the amygdaloid areas (vertical lines). D: the caudatus putamen (vertical lines), and septum (oblique lines).
and then carefully sliced with a razor blade on ice. A series of frontal sections were made at a thickness of 100/~m in a cryostat at-15 °C. The sections were freeze-dried overnight at -30 °C and 10 -3 m m Hg and stored in evacuated tubes at -20 °C until used. The fresh, discrete hippocampal areas and the peripheral areas were carefully dissected under a stereomicroscope according to the method of Uchimura and colleagues~. Dissected areas included: the dentate gyrus, subfields CA1 and CA3 areas in both the rostral and caudal hippocampus, amygdala, caudatus putamen and septum. Schematic drawings of the dissected areas are shown in Fig. 1. Each sample was weighed using an electronic microbalance (Type 4152, Sartorius Co.) with a digital voltmeter (Type Eo-12, Eto Co.). The sensitivity of the microbalance was 0.1 /~g. The weight of each sample was 20-70 pg. The tissue was pooled from individual cryostat sections.
TABLE I
Quantitative analysis of seizure susceptibility of El mice Seizures were categorized into 3 types: complete tonic-clonic convulsions ( + + ) ; incomplete abortive forms (+); and muscular twitching or wild running fits (+). The degree of sensitivity to induction of convulsions in each mouse was expressed through the sum of each weekly score from 4 to 13 weeks of treatment.
Weeksofage
++ + ±
4
5
6
7
8
9
10
11
12
13
30 20 10
29 19 9
28 18 8
27 17 7
26 16 6
25 15 5
24 14 4
23 13 3
22 12 2
21 11 1
18
20
25-
c~
~'
-g
0 td, U)
20-
10
Z
..c
W
"o O rn
15-
0 5
I 5
I 6
I 7 WEEKS
I 8
I 9 ON
I 10
I 11
I 12
I 13
DIET
Fig. 2. The effect of dietary zinc manipulations on the growth of El mice; zinc-adequate mice (rn), zinc-deficient mice (A), zinc-loaded mice (©).
All glassware was soaked in 10% nitric acid and washed 5 times with deionized distilled water. All chemicals used during the study were the purest grade available. The concentrations of zinc were determined by a timeless atomic absorption spectrophotometer (Hitachi, Type 180-70 Polarized Zeeman) using a graphite furnace atomizer. Measurement of the zinc concentration was done by the method previously reported 17. We used the method of standard addition, which is theoretically considered the most precise method, because it minimizes the interference effects of other trace metals 27. Statistical analyses of the data were done using the two-tailed Wilcoxon t-test, with 0.05 as the level of probability required for significance in all tests.
6
7
8
9
10
11
12
13
ON DIET
WEEKS
Fig. 3. Weekly alteration of mean scores of the 3 dietary groups; zinc-adequate mice (v1), zinc-deficient mice (A), zinc-loaded mice
(0). deficient El mice was significantly higher (P < 0.01) than in the zinc-adequate group and the zinc-loaded group (Fig. 4). The mean (+ S.E.M.) score in the zinc-loaded group was significantly lower than in the zinc-deficient group (P < 0.01) and the zinc-adequate group (P < 0.05). These findings indicate that the seizure susceptibility of the El mouse was increased by zinc deficiency, while zinc loading decreased seizure susceptibility.
Experiment 2 Zinc content was highest in the dentate gyrus, with values approximately twice those of the remaining dis-
RESULTS
Experiment 1 Mice fed zinc-deficient diets showed markedly depressed body weights compared with mice receiving the zinc-loaded or zinc-adequate diets (Fig. 2). In addition, zinc-deficient mice displayed typical symptoms of the zinc deficiency during the experimental period 29'45. These included growth retardation, anorexia, blepharitis, alopecia, depigmentation of hair, and decreased behavioral activity. On the other hand, El mice fed zinc-loaded diets showed no behavioral or clinical changes. The weekly alteration of mean scores of the 3 dietary groups of mice is shown in Fig. 3. The mean score increased weekly, reaching a plateau at approximately 10 weeks of age in the zinc-adequate group of mice. These findings support the use of the numerical quantification process described above as a reliable tool to evaluate seizure susceptibility of El mice, since generalized seizures are induced at about 7 or 8 weeks of age and then persist throughout life. The mean (+ S.E.M.) of the sum of the individual scores used to quantitate seizure susceptibility in zinc-
r
140
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(52) T
120 (I)
100
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:! !: :!:!: :I(?I:T ::::::::::::::::::::::: .......-....... -.......-....... -._ -............
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::::::::::::::::::::: :i i:ii i :i::::i/ :i !i !i~i ::
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7inc
adequate group
I
Zinc deficient group
Zinc loaded group
Fig. 4. Seizure susceptibility of the 3 dietary groups is expressed as mean (+ S.E.M.) of the sum of the scores for each week (i.e. weeks 4-13, see Table I); (*P < 0.01, **P < 0.05; the two-tailed Wilcoxon t-test). The numbers of animals are given above each column.
19
v-q I-]
I--7vq
[--] v7
f--qv-q
v-q v-7
The rostral dent. gyrus
CA1
CA3
The caudal dent. gyrus
CA1
v7
200
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150
100
== \ C~ O.
50
CA3
Fig. 5. The zinc content in discrete hippoeampal regions of the 3 dietary groups of mice, 10 mice per group: control (shaded), zinc-loaded (black), zinc-deficient group (white column). The zinc contents are expressed in pg//~gdry weight (mean + S.E.M.); (*P < 0.01, **P < 0.05; two-tailed Wilcoxon t-test).
crete areas measured in this study (Fig. 5). The distribution of zinc in the hippocampus of the zinc-adequate group of El mice is in close agreement with our previous study 17. In the dentate gyrus, the subfields CA1 and the subfields CA3 areas in the rostral hippocampus, the zinc level in the zinc-loaded group of mice was significantly higher ( P < 0.05 to P < 0.01) than in the corresponding areas of both the zinc-deficient and zinc-adequate groups of mice. A tendency toward reduced zinc level (P < 0.1) was observed in the zinc-deficient group of mice compared with either the zinc-adequate or zinc-loaded groups of mice in all discrete areas in the hippocampus. There were no significant differences in the zinc content in the amygdala, caudatus putamen, or septum among all 3 dietary groups (Table II).
TABLE II Zinc content in the amygdala, caudatus putamen and septum of 3 dietary groups of mice Zinc content is expressed in pg/~g dry weight and values are the means + S.E.M. of 10 mice per group. Statistical differences were determined by the two-tailed Wilcoxon t-test. There were no significant differences in the zinc content in the amygdala, caudatus putamen and septum among the 3 dietary groups of mice. Groups Zinc-adequate Zinc-deficient Zinc-loaded Amygdala Caudatusputamen Septum
120.94 + 2.48 125.09+ 1.87 120 + 0.91 76.95+ 0.72 74.73+ 1.53 71.78+ 1.29 108.66 + 1.54 104.49+ 1.96 99.69+0.95
DISCUSSION Preliminary evidence from our laboratory indicated that zinc content was significantly lower in the hippocampal dentate area of the E! mouse compared with the control ( d d Y and CBA strains) mice 17. However,it was unclear whether the low zinc level in the hippocampal dentate area of E1 mouse was due to the repetition of convulsions or was the primary factor in the seizure mechanism of the El mouse. In this study, therefore, the effect of dietary zinc status on seizure susceptibility and hippocampal zinc content of El mouse was investigated to determine whether the low zinc level in the hippocampus could be a primary factor in the seizure mechanism of the El mouse. The results indicate that seizure susceptibility of the El mouse is increased by zinc deficiency, while zinc loading decreases seizure susceptibility. Furthermore, the results suggest that seizure susceptibility of the El mouse is subject to regulation. Seizures are stimulated in the zinc-deficient state and inhibited following zinc supplementation. These findings confirm earlier studies by Sterman et al. 37 in cats where dietary zinc deprivation increased susceptibility to kindled seizures, while dietary zinc loading decreased susceptibility. In the dentate gyrus, and in the subfields CA1 and CA3 in the rostral hippocampus, the zinc level in the zinc-loaded mice was significantly higher than in the zinc-deficient and zinc-adequate groups (Fig. 5). A tendency toward reduced zinc level (P < 0.1) was observed in the zinc-deficient group of mice compared with the zinc-adequate group of mice in all discrete areas in the hippocampus; and, as mentioned, these changes
20 were associated with significant and opposite alterations in the susceptibility of the El mouse with regard to the development of generalized seizures. There were no significant differences among the 3 dietary groups in zinc content in the amygdala, caudatus putamen, or septum (Table II). This suggests that most of the changes of zinc content in the limbic structures of El mice following dietary zinc manipulations are confined to the hippocampus. Furthermore, physiological data suggest that the seizures in El mice originate in the hippocampus and then spread to the other brain regions 39,41" Hippocampal zinc may play an important role in the pathophysiologic mechanisms that lead to convulsive seizures in the El mouse, and may be critically involved in the development of the seizure mechanism of the El mouse. Moreover, it is reasonable to assume that the seizure susceptibility of the El mice may be related to a genetically mediated lower zinc concentration in the hippocampus. A study to address this question by measuring the zinc content in developing El mice and other strains of mice from 4 to 13 weeks or older is underway in our laboratory. Although not at the level of statistical significance (P < 0.1), zinc concentrations were not depressed significantly in the zinc-deficient group of mice compared with the zinc-adequate group of mice in all discrete areas in the hippocampus. The predominant view in the literature concerning zinc deficiency, is that no reduction in regional zinc content has been observed in the hippocampus, even under conditions of severe zinc deficiency 12'45. Wensink et al. 46, however, recently demonstrated that dietary zinc deficiency for 90 days caused a significant reduction in the mossy fiber zinc content, while dietary zinc deficiency for 28 days did not. In this study, the period of dietary zinc deficiency was 56 days. It is possible, therefore, to speculate that our animals were not sufficiently zinc deficient to approach statistical significance, because of a relatively short period of dietary zinc deficiency. A potential interaction of zinc with excitatory amino acid neurotransmitters has been reported 7'8'35'47 and observed. Recently, it was demonstrated that zinc inhibited the binding of [3H]glutamate to N-methyl-Daspartate (NMDA) receptors, one of the subtypes of excitatory amino acid receptors 3°. Forsythe et al. 14 also suggested that the major action of physiological concentrations of zinc on excitatory synaptic transmission in the hippocampus was as a NMDA receptor antagonist, and that zinc may be effective as a channel blocker of NMDA receptor, as demonstrated by Reynolds and Miller 33. In addition, zinc is located within certain synaptic vesicles in excitatory synaptic boutons 22'31, and a parallel relation-
ship exists between the localization of glutamate-containing synaptic boutons and the localization of zinc, especially in the mossy fiber area 6. Recent studies also suggest that zinc may be co-released during excitatory synaptic transmission at glutamatergic synapses, and may act at postsynaptic receptors to decrease actions mediated by NMDA receptors 28. In addition, the specific antagonist of NMDA receptors reportedly block convulsive seizures in some animal models of chronic epilepsy 1°:~2. In the present experiment, seizure susceptibility of the El mouse was decreased following dietary zinc loading. Together with these observations, it is suggested that excess zinc, delivered via dietary supplementation, might decrease the activation of NMDA receptors and would accordingly protect against further development of seizures in the El mouse. It is generally agreed that zinc is a necessary element in the biosynthesis and metabolism of y-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the central nervous system, which is synthesized by glutamic acid decarboxylase (GAD) 34. At physiological concentrations, zinc stimulates the activity of pyridoxal kinase and enhances the formation of pyridoxal phosphate, which in turn enhances the activity of GAD ~3. In addition, a decrease in brain zinc in pathological conditions is paralleled by a decrease in GABA receptors 2, and zinc deficiency causes decreased GABA levels in the hippocampus 3. Hesse TM demonstrated that depletion of zinc in the mossy fiber pathway produced abnormal neurotransmission in this system, possibly due to diminished glutamic acid release. Moreover, a reduction in the hippocampal zinc content by the chelating agent dithizone resulted in an altered electrophysiological response of the mossy fibers to stimuli 9. It is generally accepted that impairment of inhibitory synaptic transmission, mediated by GABA, can lead to convulsive disorders 25. Perhaps a reduction in zinc metabolism in the hippocampus could produce a disturbance in binding or storage of GABA, with the resulting low GABA concentrations leading to increased levels of neuronal excitability to normal stimuli 3. Thus, the increase of seizure susceptibility of El mice following dietary zinc deprivation may result from diminishing the GABA-induced neuronal inhibition, either caused by a reduction in mossy fiber zinc content or by effects secondary to the zinc deficiency in the hippocampus. Consequently, based on the present experimental results, we propose that zinc could antagonize further development of seizures by regulating both excitatory and inhibitory synaptic transmission in the hippocampus. Our findings reveal that zinc may be an important natural modulator of seizure susceptibility during seizure onset. Several lines of research suggest that increased
21 zinc will facilitate t h e seizure process 5'24. H o w e v e r , r e c e n t e v i d e n c e indicates t h a t physiological a m o u n t s of zinc a d d e d to t h e p e r f u s a t e o f rat h i p p o c a m p a l slices r e d u c e d s y n a p t i c r e s p o n s e 26. S t e r m a n et al. 38 r e p o r t e d t h a t t h e e a r l i e r h y p o t h e s i s (i.e. zinc i n d u c e d activation of seizures) m a y reflect a lack of a p p r e c i a t i o n for the n o r m a l p h y s i o l o g i c a l r e g u l a t i o n of this ion. T h e y indicated that the availability of excess zinc, delivered via dietary supplem e n t a t i o n protects against the d e v e l o p m e n t of seizures in animal m o d e l s o f epilepsy38. T h e present results in the El
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mouse corroborate and extend Sterman's assertion. T h e El mouse is an excellent animal m o d e l for studying complex partial seizures or t e m p o r a l lobe epilepsy 43. T h e results of our w o r k with this m o d e l indicate that the protective effect of zinc on convulsive seizures m a y have i m p o r t a n t clinical implications in humans. Acknowledgements. The authors are indebted to Professor N. Tashiro, Dr. H. Uchimura, Dr. M. Hirano, Dr. A. Nohtomi, Dr. T. Kuroki, Dr. H. Kawasaki and Dr. H. Takeshita for their guidance and to Miss T. Tanaka for technical assistance.
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22 relations in studies of the y-aminobutyric acid system, Brain
Research, 8 (1968) 1. 35 Slevin, J.T. and Kasarskis, E.J., Effects of zinc on markers of glutamate and aspartate neurotransmission in rat hippoeampus, Brain Research, 4 (1985) 281-286. 36 Sloviter, R.S., A selective loss of hippocampal mossy fiber Timm stain accompanies granule cell seizure activity induced by perforant path stimulation, Brain Research, 330 (1985) 150-153. 37 Sterman, M.B., Shouse, M.N., Fairchild, M.D. and Belsito, O., Kindled seizure induction alters and is altered by zinc absorption, Brain Research, 383 (1986) 382-386. 38 Sterman, M.B., Shouse, M.N. and Fairchild, M.D., Zinc and seizure mechanisms. In J.E. Morley, M.B. Sterman and J.H. Walsh (Eds.), Nutritional Modulation of Neural Function, Academic, San Diego, 1988, pp. 307-319. 39 Suzuki, J., Paroxysmal discharges in the electroencephalogram of the El mouse, Experientia, 32 (1976) 336-337. 40 Suzuki, J. and Nakamoto, Y., El mouse: a model of sensory precipitating epilepsy, Excerpta Medica, 427 (1977) 81-82. 41 Suzuki, J., Nakamoto, Y. and Shinkawa, Y., Local cerebral glucose utilization in epileptic seizures of the mutant El mouse,
Brain Research, 266 (1983) 359-363. 42 Timm, F., Zur histochemie des ammonshorn gebictes, Z. Zillforsch., 48 (1958) 548-555. 43 Seyfried, T.N. and Glaser, G.H., A review of mouse mutants as genetic models of epilepsy, Epilepsia, 26 (1985) 143-150. 44 Uchimura, H., Saito, M. and Hirano, M., Regional distribution of choline acetyltransferase in hypothalamus of the rat. Brain Research, 91 (1975) 161-164. 45 Wallwork, J.C., Milne, D.B. and Sims, R.L., 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) 1895-1905. 46 Wensink, J., Lenglet, W.J.M., Vis, R.D. and Van den Hamer, C.J.A., The effect of dietary zinc deficiency on the mossy fiber zinc content of the rat hippocampus, Histochemistry, 87 (1987) 65-69. 47 White, W.E, Nadler, J.V., Hamberger, H., Colman, C.W. and Cummins, J.T., Glutamate as transmitter of hippocampal perforant path, Nature, 270 (1977) 356-357.
Brain Researct,,. 529 I J!.~9o) 1t~- i!2 ~ .l~;cvic!
BRES 15885
Effects of dietary zinc status on seizure susceptibility and hippocampal zinc content in the El (epilepsy) mouse Motofumi Fukahori and Masatoshi Itoh Department of Neuropsychiatry, Faculty of Medicine, Kyushu University, Fukuoka (Japan) (Accepted 27 March 1990) Key words: Zinc; Hippocampus; Dentate gyrus; Epilepsy; El mouse; Zinc deficiency; Zinc supplementation
The effects of dietary zinc status on the development of convulsive seizures, and zinc concentrations in discrete hippocampal areas and other parts of the limbic system were studied in the El mouse model receiving zinc-adequate, zinc-deficient or zinc-loaded diets. Seizure susceptibility of the El mouse was increased by zinc deficiency, and decreased by zinc loading, while an adequate diet had no effect. Zinc loading was accompanied by a marked increase in hippocampal zinc content in the El mouse. Conversely, hippocampal zinc content declined in the El mouse fed a zinc-deficient diet. These results suggest that zinc may have a preventive effect on the development of seizures in the El mouse, and hippocampal zinc may play an important role in the pathophysiology of convulsive seizures of epilepsy.
INTRODUCTION The highest concentration of zinc, an essential trace element, is found in the hippocampus 1~'45, particularly within the mossy fiber axons of the dentate gyrus granule neurons 16'42. Zinc localized in the mossy fiber terminals may play a role in the neurotransmitter regulation on the hippocampus 8"19. Furthermore, activation of the dentate gyrus granule cells with electrical stimulation facilitates both the uptake and release of zinc from hippocampus, indicating that the metabolic turnover of zinc is sensitive to electrophysiologic activity 1'4'2a. Recent evidence suggests that the zinc pool associated with the mossy fiber terminals may be released by nerve stimulation. Sloviter demonstrated a selective loss of Timm's staining in the hippocampal mossy fiber terminals during seizures 36, while Frederickson's histofluorescence study indicated a loss of zinc staining from hippocampal mossy fibers during kainic acid induced seizures 15. The literature and recent findings indicate that zinc may be associated with the etiology and manifestation of epileptic seizures. Intracerebroventricular injection of zinc has been shown to cause epileptic seizures in the rat 24, and intracerebral injections of ZnC1 have increased audiogenic seizure susceptibility in mice 5. More recent evidence, however, suggests that zinc can minimize the development of seizures. Howell and colleagues 2° reported that audiogenic seizure activity decreased following subcutaneous zinc injections. Sterman and col-
leagues 37 demonstrated that dietary zinc loading decreased susceptibility to kindled seizures, while zinc deprivation increased susceptibility in cats. Thus, recent findings suggest that zinc may have a preventive effect on the development of seizures 3s. The El mouse is an animal model with genetically determined epilepsy 23. Convulsive seizures occur in response to postural stimulation, particularly paraboloid movements 4°. These seizures are characterized as complex partial seizures, with secondary generalization (i.e. temporal lobe epilepsy) 43. Seizures in this model appear to originate in the hippocampus and then spread to other brain regions 39m. Experiments in our laboratory revealed that the zinc content was significantly lower in the hippocampal dentate area of the El mouse compared with control ( d d Y and CBA strains) mice 17, suggesting possible involvement of hippocampal zinc in the pathophysiology of convulsive seizures in the epileptic mouse. The effect of dietary zinc status on the development of convulsive seizures of the El mouse has not been studied previously. This study was designed to investigate this question and corroborate the preventive effect of zinc on the development of seizures. In order to evaluate the possible relationship between the hippocampal zinc levels and seizure susceptibility, zinc concentrations were measured in discrete hippocampal areas (e.g. the dentate gyrus, subfields CA1, and CA3 areas in both the rostral and caudal hippocampus) and other limbic systems of the zinc-deficient, zinc-loaded and zinc-adequate El mouse.
Correspondence: M. Fukahori, Department of Neuropsychiatry; Faculty of Medicine, Kyushu University, Maidashi 3-1-1, Fukuoka, 812, Japan. 0006-8993/90/$03.50 (~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
17 M A T E R I A L S A N D METHODS
Experiment 1 For this study, 180, 4-week-old male El mice, weighing 7.5-11.5 g (mean = 10.5 g) at the beginning of the study served as subjects. El mice were reared in our institution in a specific pathogen-free, constant temperature-humidity environment, with a 12-h light-dark cycle. Care of the mice included cleansing their cages with 10% nitric acid and subsequent rinsing with distilled water. They were offered distilled, deionized water from glass bottles fitted with silicone stoppers and stainless steel spouts ad libitum to minimize trace metal contamination. The mice were randomly assigned to 3 dietary groups of 60 mice each and housed in groups of 5 in clean stainless steel cages. During a 1-week period of acclimatization, all mice received the identical standard laboratory diet (CLEA Japan, Inc.), which contained 38.9 mg Zn/kg by analysis. During a mean of 8 weeks (range 5-13 weeks) after acclimatization, one group was fed a zinc-deficient (1.1 mg Zn/kg) diet, animals of a second group were fed a zinc-loaded (248 mg Zn/kg) diet, and a third group (control) was fed an adequatezinc (38.9 mg Zn/kg) diet ad libitum. All groups were weighed weekly. Postural stimulation of the El mouse is most commonly achieved by tossing them to a height of approximately 20 cm, 30-40 times. In this study, this was done once each week starting at 4 weeks of age. Generalized seizures were induced at about 7 or 8 weeks of age. The procedure involved removing a mouse from its cage carefully, observing it for several minutes, and then tossing it into the air. As the El mice grew older, less postural stimulation was required to elicit seizures (i.e. the seizure threshold was lowered gradually through repetition of stimulation), and seizure activity persisted throughout life4°. With repetition of stimulation, the pattern of seizure activity changed as the animals developed 4°. Although susceptibility to seizure showed considerable individual variation, by 4-7 weeks of age seizures generally took an abortive form, most commonly squeaks and catatonic posture. Occasionally, simple muscular twitching, which was sometimes accompanied by wild running fits, was observed. By 7-8 weeks of age, typical generalized tonic-clonic convulsions began in most mice. No method is currently available for objective, numerical quantification of the level of susceptibility of individual El mice to the induction of convulsions. Therefore, we devised a system whereby the convulsions in each mouse could be categorized into 3 main types: complete tonic-clonic convulsions ( + + ) , abortive seizure activity (+), and muscular twitching or wild running fits only (+). Scores were then adjusted for age in weeks so that the level of susceptibility of individual El mice to the induction of convulsions could be expressed as the total of the scores from 4 to 13 weeks of age. For example, if a mouse was 4 weeks of age and the pattern of seizure was ' + ' , then the mouse was assigned a score of 10 (see Table I). If the same (or different) mouse was 13 weeks old with a ' + + ' pattern of seizure activity, a score of 21 was assigned. Thus, the result of each stimulation was expressed numerically with that score reflecting the age of the mouse, the pattern of seizure activity, and seizure susceptibility. The series of scores were then totaled. Mice with high total scores were categorized as having high susceptibility; those with low scores were categorized as having low susceptibility. Finally, the total scores were compared among the 3 diet groups using the Wilcoxon t-test. Experiment 2 At 13 weeks of age, mice were selected from each of the 3 dietary groups based on their seizure susceptibility scores. This included 10 mice with low scores in the zinc-loaded group, 10 mice with high scores in the zinc-deficient group, and 10 mice with average scores among those treated with adequate amounts of zinc. Animals were decapitated between 13.00 and 13.30 h at 7 days after the last seizure. The brain was removed, frozen quickly in liquid nitrogen,
(A)
(B)
""i"
(C)
(D)
Fig. 1. Schematic drawing of microdissections of the hippocampus and the other areas that were dissected for assay. A: the rostral hippocampus. B: the caudal hippocampus. The regions assayed were the dentate gyrus (stipple), the subfield CA1 (vertical lines), and the subfield CA3 (oblique lines). C: the amygdaloid areas (vertical lines). D: the caudatus putamen (vertical lines), and septum (oblique lines).
and then carefully sliced with a razor blade on ice. A series of frontal sections were made at a thickness of 100/~m in a cryostat at-15 °C. The sections were freeze-dried overnight at -30 °C and 10 -3 m m Hg and stored in evacuated tubes at -20 °C until used. The fresh, discrete hippocampal areas and the peripheral areas were carefully dissected under a stereomicroscope according to the method of Uchimura and colleagues~. Dissected areas included: the dentate gyrus, subfields CA1 and CA3 areas in both the rostral and caudal hippocampus, amygdala, caudatus putamen and septum. Schematic drawings of the dissected areas are shown in Fig. 1. Each sample was weighed using an electronic microbalance (Type 4152, Sartorius Co.) with a digital voltmeter (Type Eo-12, Eto Co.). The sensitivity of the microbalance was 0.1 /~g. The weight of each sample was 20-70 pg. The tissue was pooled from individual cryostat sections.
TABLE I
Quantitative analysis of seizure susceptibility of El mice Seizures were categorized into 3 types: complete tonic-clonic convulsions ( + + ) ; incomplete abortive forms (+); and muscular twitching or wild running fits (+). The degree of sensitivity to induction of convulsions in each mouse was expressed through the sum of each weekly score from 4 to 13 weeks of treatment.
Weeksofage
++ + ±
4
5
6
7
8
9
10
11
12
13
30 20 10
29 19 9
28 18 8
27 17 7
26 16 6
25 15 5
24 14 4
23 13 3
22 12 2
21 11 1
18
20
25-
c~
~'
-g
0 td, U)
20-
10
Z
..c
W
"o O rn
15-
0 5
I 5
I 6
I 7 WEEKS
I 8
I 9 ON
I 10
I 11
I 12
I 13
DIET
Fig. 2. The effect of dietary zinc manipulations on the growth of El mice; zinc-adequate mice (rn), zinc-deficient mice (A), zinc-loaded mice (©).
All glassware was soaked in 10% nitric acid and washed 5 times with deionized distilled water. All chemicals used during the study were the purest grade available. The concentrations of zinc were determined by a timeless atomic absorption spectrophotometer (Hitachi, Type 180-70 Polarized Zeeman) using a graphite furnace atomizer. Measurement of the zinc concentration was done by the method previously reported 17. We used the method of standard addition, which is theoretically considered the most precise method, because it minimizes the interference effects of other trace metals 27. Statistical analyses of the data were done using the two-tailed Wilcoxon t-test, with 0.05 as the level of probability required for significance in all tests.
6
7
8
9
10
11
12
13
ON DIET
WEEKS
Fig. 3. Weekly alteration of mean scores of the 3 dietary groups; zinc-adequate mice (v1), zinc-deficient mice (A), zinc-loaded mice
(0). deficient El mice was significantly higher (P < 0.01) than in the zinc-adequate group and the zinc-loaded group (Fig. 4). The mean (+ S.E.M.) score in the zinc-loaded group was significantly lower than in the zinc-deficient group (P < 0.01) and the zinc-adequate group (P < 0.05). These findings indicate that the seizure susceptibility of the El mouse was increased by zinc deficiency, while zinc loading decreased seizure susceptibility.
Experiment 2 Zinc content was highest in the dentate gyrus, with values approximately twice those of the remaining dis-
RESULTS
Experiment 1 Mice fed zinc-deficient diets showed markedly depressed body weights compared with mice receiving the zinc-loaded or zinc-adequate diets (Fig. 2). In addition, zinc-deficient mice displayed typical symptoms of the zinc deficiency during the experimental period 29'45. These included growth retardation, anorexia, blepharitis, alopecia, depigmentation of hair, and decreased behavioral activity. On the other hand, El mice fed zinc-loaded diets showed no behavioral or clinical changes. The weekly alteration of mean scores of the 3 dietary groups of mice is shown in Fig. 3. The mean score increased weekly, reaching a plateau at approximately 10 weeks of age in the zinc-adequate group of mice. These findings support the use of the numerical quantification process described above as a reliable tool to evaluate seizure susceptibility of El mice, since generalized seizures are induced at about 7 or 8 weeks of age and then persist throughout life. The mean (+ S.E.M.) of the sum of the individual scores used to quantitate seizure susceptibility in zinc-
r
140
*
;,k lr
(52) T
120 (I)
100
(57) -
0 0 ,r-
E
-
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40 20 0
i m
:! !: :!:!: :I(?I:T ::::::::::::::::::::::: .......-....... -.......-....... -._ -............
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(6O
::::::::::::::::::::: :i i:ii i :i::::i/ :i !i !i~i ::
-
- ........... ................. : .. :+:+:.. : :.: :. :::::::::::::::::::::: •....... :! i:i:i:i i:i:i:i: :!: :i
i~~:!~i:i:i~ii:i i:iii:i ii i:iiiii ~ii ;i: .! !
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::::::::::::::::::::::::::::::::::::: :::::::::::::::::::::
7inc
adequate group
I
Zinc deficient group
Zinc loaded group
Fig. 4. Seizure susceptibility of the 3 dietary groups is expressed as mean (+ S.E.M.) of the sum of the scores for each week (i.e. weeks 4-13, see Table I); (*P < 0.01, **P < 0.05; the two-tailed Wilcoxon t-test). The numbers of animals are given above each column.
19
v-q I-]
I--7vq
[--] v7
f--qv-q
v-q v-7
The rostral dent. gyrus
CA1
CA3
The caudal dent. gyrus
CA1
v7
200
.O)
"O
150
100
== \ C~ O.
50
CA3
Fig. 5. The zinc content in discrete hippoeampal regions of the 3 dietary groups of mice, 10 mice per group: control (shaded), zinc-loaded (black), zinc-deficient group (white column). The zinc contents are expressed in pg//~gdry weight (mean + S.E.M.); (*P < 0.01, **P < 0.05; two-tailed Wilcoxon t-test).
crete areas measured in this study (Fig. 5). The distribution of zinc in the hippocampus of the zinc-adequate group of El mice is in close agreement with our previous study 17. In the dentate gyrus, the subfields CA1 and the subfields CA3 areas in the rostral hippocampus, the zinc level in the zinc-loaded group of mice was significantly higher ( P < 0.05 to P < 0.01) than in the corresponding areas of both the zinc-deficient and zinc-adequate groups of mice. A tendency toward reduced zinc level (P < 0.1) was observed in the zinc-deficient group of mice compared with either the zinc-adequate or zinc-loaded groups of mice in all discrete areas in the hippocampus. There were no significant differences in the zinc content in the amygdala, caudatus putamen, or septum among all 3 dietary groups (Table II).
TABLE II Zinc content in the amygdala, caudatus putamen and septum of 3 dietary groups of mice Zinc content is expressed in pg/~g dry weight and values are the means + S.E.M. of 10 mice per group. Statistical differences were determined by the two-tailed Wilcoxon t-test. There were no significant differences in the zinc content in the amygdala, caudatus putamen and septum among the 3 dietary groups of mice. Groups Zinc-adequate Zinc-deficient Zinc-loaded Amygdala Caudatusputamen Septum
120.94 + 2.48 125.09+ 1.87 120 + 0.91 76.95+ 0.72 74.73+ 1.53 71.78+ 1.29 108.66 + 1.54 104.49+ 1.96 99.69+0.95
DISCUSSION Preliminary evidence from our laboratory indicated that zinc content was significantly lower in the hippocampal dentate area of the E! mouse compared with the control ( d d Y and CBA strains) mice 17. However,it was unclear whether the low zinc level in the hippocampal dentate area of E1 mouse was due to the repetition of convulsions or was the primary factor in the seizure mechanism of the El mouse. In this study, therefore, the effect of dietary zinc status on seizure susceptibility and hippocampal zinc content of El mouse was investigated to determine whether the low zinc level in the hippocampus could be a primary factor in the seizure mechanism of the El mouse. The results indicate that seizure susceptibility of the El mouse is increased by zinc deficiency, while zinc loading decreases seizure susceptibility. Furthermore, the results suggest that seizure susceptibility of the El mouse is subject to regulation. Seizures are stimulated in the zinc-deficient state and inhibited following zinc supplementation. These findings confirm earlier studies by Sterman et al. 37 in cats where dietary zinc deprivation increased susceptibility to kindled seizures, while dietary zinc loading decreased susceptibility. In the dentate gyrus, and in the subfields CA1 and CA3 in the rostral hippocampus, the zinc level in the zinc-loaded mice was significantly higher than in the zinc-deficient and zinc-adequate groups (Fig. 5). A tendency toward reduced zinc level (P < 0.1) was observed in the zinc-deficient group of mice compared with the zinc-adequate group of mice in all discrete areas in the hippocampus; and, as mentioned, these changes
20 were associated with significant and opposite alterations in the susceptibility of the El mouse with regard to the development of generalized seizures. There were no significant differences among the 3 dietary groups in zinc content in the amygdala, caudatus putamen, or septum (Table II). This suggests that most of the changes of zinc content in the limbic structures of El mice following dietary zinc manipulations are confined to the hippocampus. Furthermore, physiological data suggest that the seizures in El mice originate in the hippocampus and then spread to the other brain regions 39,41" Hippocampal zinc may play an important role in the pathophysiologic mechanisms that lead to convulsive seizures in the El mouse, and may be critically involved in the development of the seizure mechanism of the El mouse. Moreover, it is reasonable to assume that the seizure susceptibility of the El mice may be related to a genetically mediated lower zinc concentration in the hippocampus. A study to address this question by measuring the zinc content in developing El mice and other strains of mice from 4 to 13 weeks or older is underway in our laboratory. Although not at the level of statistical significance (P < 0.1), zinc concentrations were not depressed significantly in the zinc-deficient group of mice compared with the zinc-adequate group of mice in all discrete areas in the hippocampus. The predominant view in the literature concerning zinc deficiency, is that no reduction in regional zinc content has been observed in the hippocampus, even under conditions of severe zinc deficiency 12'45. Wensink et al. 46, however, recently demonstrated that dietary zinc deficiency for 90 days caused a significant reduction in the mossy fiber zinc content, while dietary zinc deficiency for 28 days did not. In this study, the period of dietary zinc deficiency was 56 days. It is possible, therefore, to speculate that our animals were not sufficiently zinc deficient to approach statistical significance, because of a relatively short period of dietary zinc deficiency. A potential interaction of zinc with excitatory amino acid neurotransmitters has been reported 7'8'35'47 and observed. Recently, it was demonstrated that zinc inhibited the binding of [3H]glutamate to N-methyl-Daspartate (NMDA) receptors, one of the subtypes of excitatory amino acid receptors 3°. Forsythe et al. 14 also suggested that the major action of physiological concentrations of zinc on excitatory synaptic transmission in the hippocampus was as a NMDA receptor antagonist, and that zinc may be effective as a channel blocker of NMDA receptor, as demonstrated by Reynolds and Miller 33. In addition, zinc is located within certain synaptic vesicles in excitatory synaptic boutons 22'31, and a parallel relation-
ship exists between the localization of glutamate-containing synaptic boutons and the localization of zinc, especially in the mossy fiber area 6. Recent studies also suggest that zinc may be co-released during excitatory synaptic transmission at glutamatergic synapses, and may act at postsynaptic receptors to decrease actions mediated by NMDA receptors 28. In addition, the specific antagonist of NMDA receptors reportedly block convulsive seizures in some animal models of chronic epilepsy 1°:~2. In the present experiment, seizure susceptibility of the El mouse was decreased following dietary zinc loading. Together with these observations, it is suggested that excess zinc, delivered via dietary supplementation, might decrease the activation of NMDA receptors and would accordingly protect against further development of seizures in the El mouse. It is generally agreed that zinc is a necessary element in the biosynthesis and metabolism of y-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the central nervous system, which is synthesized by glutamic acid decarboxylase (GAD) 34. At physiological concentrations, zinc stimulates the activity of pyridoxal kinase and enhances the formation of pyridoxal phosphate, which in turn enhances the activity of GAD ~3. In addition, a decrease in brain zinc in pathological conditions is paralleled by a decrease in GABA receptors 2, and zinc deficiency causes decreased GABA levels in the hippocampus 3. Hesse TM demonstrated that depletion of zinc in the mossy fiber pathway produced abnormal neurotransmission in this system, possibly due to diminished glutamic acid release. Moreover, a reduction in the hippocampal zinc content by the chelating agent dithizone resulted in an altered electrophysiological response of the mossy fibers to stimuli 9. It is generally accepted that impairment of inhibitory synaptic transmission, mediated by GABA, can lead to convulsive disorders 25. Perhaps a reduction in zinc metabolism in the hippocampus could produce a disturbance in binding or storage of GABA, with the resulting low GABA concentrations leading to increased levels of neuronal excitability to normal stimuli 3. Thus, the increase of seizure susceptibility of El mice following dietary zinc deprivation may result from diminishing the GABA-induced neuronal inhibition, either caused by a reduction in mossy fiber zinc content or by effects secondary to the zinc deficiency in the hippocampus. Consequently, based on the present experimental results, we propose that zinc could antagonize further development of seizures by regulating both excitatory and inhibitory synaptic transmission in the hippocampus. Our findings reveal that zinc may be an important natural modulator of seizure susceptibility during seizure onset. Several lines of research suggest that increased
21 zinc will facilitate t h e seizure process 5'24. H o w e v e r , r e c e n t e v i d e n c e indicates t h a t physiological a m o u n t s of zinc a d d e d to t h e p e r f u s a t e o f rat h i p p o c a m p a l slices r e d u c e d s y n a p t i c r e s p o n s e 26. S t e r m a n et al. 38 r e p o r t e d t h a t t h e e a r l i e r h y p o t h e s i s (i.e. zinc i n d u c e d activation of seizures) m a y reflect a lack of a p p r e c i a t i o n for the n o r m a l p h y s i o l o g i c a l r e g u l a t i o n of this ion. T h e y indicated that the availability of excess zinc, delivered via dietary supplem e n t a t i o n protects against the d e v e l o p m e n t of seizures in animal m o d e l s o f epilepsy38. T h e present results in the El
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mouse corroborate and extend Sterman's assertion. T h e El mouse is an excellent animal m o d e l for studying complex partial seizures or t e m p o r a l lobe epilepsy 43. T h e results of our w o r k with this m o d e l indicate that the protective effect of zinc on convulsive seizures m a y have i m p o r t a n t clinical implications in humans. Acknowledgements. The authors are indebted to Professor N. Tashiro, Dr. H. Uchimura, Dr. M. Hirano, Dr. A. Nohtomi, Dr. T. Kuroki, Dr. H. Kawasaki and Dr. H. Takeshita for their guidance and to Miss T. Tanaka for technical assistance.
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