Zinc's role in seizures

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Interesting article. Especially given the fact that zinc levels are

off in many of our kids.

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Susceptibility to kainate-induced seizures under dietary zinc

deficiency

Atsushi Takeda, Maki Hirate, Haruna Tamano, Daisuke Nisibaba and

Naoto Oku

Department of Medical Biochemistry, School of Pharmaceutical

Sciences, University of Shizuoka, Shizuoka, Japan

Abstract

Zinc homeostasis in the brain is altered by dietary zinc deficiency,

and its alteration may be associated with the etiology

and manifestation of epileptic seizures. In the present study,

susceptibility to kainate-induced seizures was enhanced in

mice fed a zinc-deficient diet for 4 weeks. When Timm's stain

was performed to estimate zinc concentrations in synaptic

vesicles, Timm's stain in the brain was attenuated in the

zincdeficient

mice. In rats fed the zinc-deficient diet for 4 weeks,

susceptibility to kainate-induced seizures was also enhanced.

When the release of zinc and neurotransmitters in the hippocampal

extracellular fluid of the zinc-deficient rats was

studied using in vivo microdialysis, the zinc concentration in

the perfusate was less than 50% of that of the control rats and

the increased levels of zinc by treatment with kainate were

lower than the basal level in control rats, suggesting that vesicular

zinc is responsive to dietary zinc deficiency. The levels

of glutamate in the perfusate of the zinc-deficient rats were

more increased than in the control rats, whereas the levels of

GABA in the perfusate were not at all increased in the zincdeficient

rats, unlike in the control rats. The present results

demonstrate an enhanced release of glutamate associated

with a decrease in GABA concentrations as a possible

mechanism for the increased seizure susceptibility under zinc

deficiency.

Keywords: c-amino butyric acid, glutamate, hippocampus,

kainate, seizure, zinc deficiency.

J. Neurochem. (2003) 85, 1575–1580.

Zinc has been reported to act either as an anticonvulsant

(on and Spencer 1995) or a proconvulsant (Pei et al.

1983). Alteration of zinc homeostasis in the brain may be

associated with the etiology and manifestation of epileptic

seizures (Sterman et al. 1988; Buhl et al. 1996). Zinc

concentration in the hippocampal dentate area of seized EL

(epilepsy) mice is significantly lower than that of control

mice (Fukahori et al. 1988). Elimination of 65Zn from the

brain of EL mice is facilitated during induction of seizures

(Takeda et al. 1999). Seizure susceptibility of EL mice is

attenuated by dietary zinc loading, while it is enhanced by

dietary zinc deficiency (Fukahori and Itoh 1990). Susceptibility

to kindled seizures is also attenuated by dietary zinc

loading, while this susceptibility in cats is enhanced by zinc

deprivation (Sterman et al. 1986). These findings suggest

that susceptibility to epileptic seizures is enhanced by zinc

deficiency and that zinc homeostasis in the brain is important

for prevention of seizure development.

Zinc homeostasis in the brain may be affected by dietary

zinc deficiency; zinc concentration in the hippocampus is

significantly decreased by dietary zinc deficiency (Takeda

et al. 2001). It is currently a proven fact that the hippocampus

possesses zinc-containing glutamatergic neuron terminals

(Assaf and Chung 1984; Howell et al. 1984; Frederickson

1989; Frederickson and Danscher 1990). Zinc concentration in

the vesicles in the giant boutons of hippocampal mossy fibres is

estimated to be approximately 300 lM (Frederickson et al.

1983). There is the possibility that hippocampal vesicular

zinc is responsive to dietary zinc (Takeda 2001). The

attenuation of N-[6-methoxy-8-quinolyl]-P-toluenesulfonamide

(TSQ) stain, by which histochemically reactive zinc in

the presynaptic vesicles is detected, is observed in the

hippocampal mossy fibre of rats fed a zinc-deficient diet (Lu

et al. 2000). However, zinc is important for the function of

many enzymes and other proteins, including some unique to

the brain and important to neurotransmission (Prohaska

1987; Golub et al. 1995; Sandstead et al. 2000). There is

also the possibility that zinc-requiring proteins are responsive

Received January 2, 2003; revised manuscript received February 24,

2003; accepted March 14, 2003.

Address correspondence and reprint requests to Atsushi Takeda,

Department of Medical Biochemistry, School of Pharmaceutical

Sciences,

University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan.

E-mail: takedaa@...

Abbreviations used: ACSF, artificial cerebrospinal fluid; EL,

epilepsy;

TSQ, N-[6-methoxy-8-quinolyl]-P-toluenesulfonamide; Zn, zinc.

Journal of Neurochemistry, 2003, 85, 1575–1580 doi:10.1046/j.1471-

4159.2003.01803.x

2003 International Society for Neurochemistry, J.Neur ochem.

(2003) 85, 1575–1580 1575

to dietary zinc. Thus, alteration of the function of zincdependent

systems in the brain might be involved in the

pathophysiology of epileptic seizures under dietary zinc

deficiency.

The present study deals with the enhancement of susceptibility

to kainate-induced seizures under dietary zinc deficiency,

which may cause a decrease in the levels of synaptic

zinc. It is likely that an imbalance of inhibition–excitation,

which may be induced by zinc deficiency, is involved in the

enhanced susceptibility.

Materials and methods

Chemicals

Control (44 mg Zn/kg) and zinc-deficient (2.7 mg Zn/kg) diets were

purchased from Oriental Yeast Co. Ltd. (Yokohama, Japan).

Artificial cerebrospinal fluid (ACSF) used as a perfusate was

composed of 127 mM NaCl, 2.5 mM KCl, 1.3 mM CaCl2, 0.9 mM

MgCl2, 1.2 mM Na2HPO4, 21 mM NaHCO3 and 3.4 mM D-glucose

(pH 7.3). The zinc concentration in ACSF, which was measured

with a flameless atomic absorption spectrophotometer (Shimadzu

AA6800F, Kyoto, Japan), was 18.7 ± 3.8 nM.

Experimental animals

Male ddY mice and Wistar rats (both 3 weeks old) were

purchased from Japan SLC (Hamamatsu, Japan). They were

housed under the standard laboratory conditions (23 ± 1C,

55 ± 5% humidity) and had access to tap water and diet

ad libitum. Feeding the zinc-deficient diet was begun at 4 weeks of

age. The lights were automatically turned on at 08.00 and off at

20.00 h. All experiments were performed in accordance with the

Principles of Laboratory Animal Care of the National Institute of

Health and the University of Shizuoka.

Seizure induction under zinc deficiency

Seizures are induced in control animals and in animals experimentally

subjected to the zinc-deficient diet for 4 weeks. The control and

zinc-deficient mice were intraperitoneally injected with kainate

(12 mg/kg body weight) every 60 min three times (n ¼ 10). The

behaviour of mice was recorded with a video camera and seizure

scores were taken according to the procedure reported previously

(Racine 1972; Sperk et al. 1985).

In another experiment, the control and zinc-deficient rats were

intraperitoneally injected with kainate (5–10 mg/kg body weight)

(n ¼ 6–7). Twenty-six days after the start of feeding the

zincdeficient

diet, a guide tube was surgically implanted into the

hippocampal CA3 subregion (stereotaxic co-ordinates: AP ¼ )5.6 mm,

ML ¼ )4.6 mm, DV ¼ + 5.0 mm) of a chloral

hydrate-anaesthetized rat. Seventy-two hours after implantation

of the guide tube, the hippocampus was perfused with ACSF by

using a microdialysis probe (3-mm membrane CMA12 probe,

CMA Microdialysis, Solna, Sweden) at a flow rate of 5.0 lL/min

before and after the injection of kainate under the conscious

condition. The perfusate samples were collected at intervals of

every 20 min. The behaviour of the rats was recorded with a

video camera and seizure scores for every 20 min were taken.

Timm's sulphide-silver staining

Control and zinc-deficient mice were deeply anaesthetized with

chloral hydrate and then perfused transcardially with 0.1% Na2S

in phosphate buffer (pH 7.4) (n ¼ 6). Timm's staining was

performed according to the procedure described previously

(Danscher 1981).

HPLC analysis

The perfusate samples were analyzed for glutamate, GABA, and

glycine contents by HPLC [column, capillary column C18 (BAS,

Tokyo, Japan); mobile phase, 0.1 M KH2PO4, 0.1 M Na2HPO4, 10%

CH3CN, 0.5 mM EDTA-2Na, 3% tetrahydrofuran, pH 6.0] using the

pre-column derivatization technique with o-phthaldialdehyde and a

fluorescence detector (CMA/280, CMA Microdialysis) (Lindroth

and Mopper 1979). The concentration of neurotransmitters in the

perfusate samples was calculated from each peak area of the

neurotransmitter standard solution, which was analyzed before and

after the analysis of the samples.

Statistical analysis

Student's t-test was used for comparison of the means of unpaired

data. For multiple comparison ANOVA followed by PLSD (Fisher's

Protected Least Significant Difference) was performed.

Results

Seizure susceptibility under zinc deficiency

The mean body weight (20.1 g) of mice fed the zinc-deficient

diet for 4 weeks was 50% of that of the control mice

(41.9 g). When kainate was injected into the control and

zinc-deficient mice, maximum seizure scores of the zincdeficient

mice were significantly higher than those of the

control mice (Fig. 1). All the zinc-deficient mice exhibited

status epilepticus, while only 30% of the control mice

exhibited it. Death occurred in 60 and 20% of the zincdeficient

and control mice, respectively, within 3 days. The

latency in myoclonic jerks of the zinc-deficient mice was

significantly shorter than in the control mice, while the

latency in clonic and tonic convulsions and in status

epilepticus was not different between the two groups.

Vesicular zinc under zinc deficiency

Timm's stain, by which histochemically reactive zinc in the

synaptic vesicles is detected, was performed to estimate zinc

concentrations in the synaptic vesicles. The attenuation of

Timm's stain was extensively observed in the brain of the

zinc-deficient mice (Fig. 2). In the hippocampus of the zincdeficient

mice, Timm's stain was attenuated in the mossy

fibre, stratum radiatum and stratum oriens.

Release of zinc and neurotransmitters

in the hippocampus under zinc deficiency

To study alteration of synaptic neurotransmission induced

with kainate under zinc deficiency, an in vivo microdialysis

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(2003) 85, 1575–1580

experiment was performed using zinc-deficient rats (the

mean body weight, 102 g) injected intraperitoneally with

kainate at a dose of 10 mg/kg body weight (Fig. 3a). In the

control rats (the mean body weight, 201 g), clonic

convulsion was observed approximately 2 h after the injection,

while all the rats fed the zinc-deficient diet for 4 weeks

exhibited status epilepticus and then died (data not shown).

In the case of injection with kainate at a dose of 5 mg/kg

body weight, wet-dog shakes, e.g. head bobbing and

twitching, were observed in the control rats and the

convulsion became severer with time (Fig. 3b). In the rats

fed the zinc-deficient diet for 4 weeks, however, clonic

convulsion was observed immediately after injection of

kainate.

When the hippocampus was perfused with ACSF, the

basal zinc concentration in the perfusate (before injection of

kainate) of the zinc-deficient rats was less than 50% of that of

the control rats (Fig. 3d). In contrast, the basal concentrations

of glutamate, GABA and glycine in the perfusate of the zincdeficient

rats were not appreciably different from those of the

control rats (Figs 3c and 4).

Zinc concentration in the perfusate was increased even in the

zinc-deficient rats after treatment with kainate (Fig. 3d).

However, the increased levels of zinc in the zinc-deficient rats

were lower than the basal levels of the control rats. Glutamate

concentration in the perfusate of the control rats was only

negligibly increased after treatment with kainate, whereas that

of the zinc-deficient rats was markedly increased after the

treatment (Fig. 3c). The increment of glutamate concentration

in the zinc-deficient rats was significantly more than in the

control rats (Fig. 4). GABA concentration in the perfusate was

significantly increased in the control rats after treatment with

kainate, whereas it showed no increase at all in the zincdeficient

rats. Glycine concentration in the perfusate was

significantly increased in both the control and zinc-deficient

groups after treatment with kainate.

Fig. 2 Timm's stain under zinc deficiency.

Timm's staining was performed to estimate

zinc concentrations in synaptic vesicles

under zinc deficiency. Coronal slices (30 lm

thickness) for zinc staining were prepared

from mice fed the control or zinc-deficient

diet for 4 weeks (n ¼ 6). The attenuation of

Timm's stain was observed in the brain of six

zinc-deficient mice. The top and the bottom

photographs were taken using a camera and

a phase microscope, respectively.

Fig. 1 Susceptibility to kainate-induced seizures. Mice were fed a

control or zinc-deficient diet for 4 weeks and then intraperitoneally

injected with 12 mg/kg kainate every 60 min three times (n ¼ 10). The

incidence represents the rate of seized mice to the total mice.

Seizure

severity represents the maximum seizure score, which was observed

for 5 h after the treatment with kainate. Each bar and line

represent the

mean ± SEM. Asterisks represent significant differences (*p < 0.01;

**p < 0.001) from the control.

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Discussion

An inadequate dietary zinc supply causes changes in

behaviour such as reduced activity and responsiveness

(Golub et al. 1995). Learning behaviour in adult rats is

impaired by dietary zinc deprivation (Takeda et al. 2000). Lu

et al. (2000) suggested that the long-term potentiation in

hippocampal mossy fibre synapses is impaired by the

decrease of mossy fibre zinc. Zinc homeostasis in zinccontaining

glutamatergic synapses appears to be important

for excitatory neurotransmission (Takeda 2000). Zinc transporter-

3 (ZnT-3)-null mice, which lack histochemically

reactive zinc in synaptic vesicles, are more sensitive than

the control mice to seizures induced with kainate (Cole et al.

2000). However, spatial learning, memory and sensorimotor

functions were not impaired in ZnT-3-null mice (Cole et al.

2001). Thus, it may be important to examine the possibility

that zinc levels in the synaptic vesicles influence the degree

and balance of inhibition-excitation in zinc-containing

glutamatergic synapses (Xie and Smart 1991; Takeda 2001).

In the present study, susceptibility to kainate-induced

seizures was examined in mice fed the zinc-deficient diet for

4 weeks. Kainate-induced seizures were enhanced by dietary

zinc deficiency. The enhanced susceptibility to epileptic

seizures has been observed in zinc-deficient EL mice

(Fukahori and Itoh 1990) and kindled cats (Sterman et al.

1986). However, the mechanism of the enhanced susceptibility

is unknown. When Timm's stain was performed to

estimate zinc concentrations in the synaptic vesicles, Timm's

stain in the brain was attenuated in the mice fed the zincdeficient

diet for 4 weeks. Zinc concentration in the hippocampal

extracellular fluid was also studied by in vivo

microdialysis. In rats fed the zinc-deficient diet for 4 weeks,

Timm's stain was also extensively attenuated in the brain

(unpublished data) and zinc concentration in the perfusate

was less than 50% of that of the control rats. Zinc release into

the hippocampal extracellular fluid was increased even in the

zinc-deficient rats after treatment with kainate. However, the

increased levels of zinc in the zinc-deficient rats were lower

than the basal levels of the control rats. These results suggest

that vesicular zinc is responsive to dietary zinc deficiency,

and that zinc homeostasis in the synaptic vesicles is affected

by the zinc deficiency.

Alternatively, there is the possibility that other zincdependent

systems in the brain are affected by the zinc

deficiency. The concentrations of neurotransmitters, in addition

to zinc, in the hippocampal extracellular fluid of the

zinc-deficient rats were examined by the in vivo microdialysis.

The basal concentrations of glutamate, GABA and

glycine were not appreciably different between the

zinc-deficient and control rats. The alteration of synaptic

neurotransmission induced with kainate under zinc defici-

Fig. 3 Concentrations of glutamate and zinc in the hippocampal

extracellular fluid during kainate-induced seizures. (a) Position of

the

microdialysis probe in the hippocampus. Rats were fed a control or

zinc-deficient diet for 4 weeks and then intraperitoneally injected

with

5 mg/kg kainate (KA). The arrows represent the time of injection.

Open squares, control; closed circles, zinc-deficient. Seizure scores

( and the concentrations of glutamate © and zinc (d) in the

perfusate

were measured every 20 min. Each point and line represent the

mean ± SEM. (n ¼ 6–7).

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(2003) 85, 1575–1580

ency was then compared with the control rats. Seizure

severity was only gradually increased in the control rats after

treatment with kainate, while it was instantaneously

increased in the zinc-deficient rats. Glutamate release into

the hippocampal extracellular fluid of the zinc-deficient rats

was significantly more than in the control rats, whereas an

increase in GABA release was not observed at all in the zincdeficient

rats. Although the development process of seizures

is complicated, imbalance of inhibition-excitation, if it is

involved there, appears to proceed easily in the zinc-deficient

rats. Zinc is necessary for the physiological functions of

many enzymes and cellular processes, which may be

severely compromised under zinc deficiency (Prasad 1988;

Vallee and Falchuk 1993). The functions of zinc-requiring

proteins in the brain may be impaired by zinc deprivation,

resulting in severe changes in metabolic functions of neurons

and glial cells, which may be associated with the excessive

release of glutamate and the lack of enhanced release of

GABA in the zinc-deficient rats. However, glycine release

into the hippocampal extracellular fluid does not appear to be

affected by the zinc deficiency.

In conclusion, the present results demonstrate an enhanced

release of glutamate associated with a decrease in GABA

concentrations as a possible mechanism for the increased

seizure susceptibility under zinc deficiency.

Acknowledgement

This work was supported by a grant from COE21.

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