Haruna Tamano

Insight into zinc signaling from dietary zinc deficiency.

Zinc is necessary for not only brain development but also brain function. Zinc homeostasis in the brain is tightly regulated by the brain barrier system and is not easily disrupted by dietary zinc deficiency. However, histochemically reactive zinc as revealed by Timms staining is susceptible to zinc deficiency, suggesting that the pool of Zn(2+) can be reduced by zinc deficiency. The hippocampus is also susceptible to zinc deficiency in the brain. On the other hand, zinc deficiency causes abnormal glucocorticoid secretion from the adrenal cortex, which is observed prior to the decrease in extracellular zinc concentration in the hippocampus. The hippocampus is enriched with glucocorticoid receptors and hippocampal functions are changed by abnormal glucocorticoid secretion. Zinc deficiency elicits neuropsychological symptoms and affects cognitive performance. It may also aggravate glutamate excitotoxicity in neurological diseases. Abnormal glucocorticoid secretion is associated with these symptoms in zinc deficiency. Furthermore, the decrease in Zn(2+) pool may cooperate with glucocorticoid action in zinc deficiency. Judging from susceptibility of Zn(2+) pool in the brain to zinc deficiency, it is possible that the decrease in Zn(2+) pool in the peripheral tissues triggers abnormal glucocorticoid secretion. To understand the importance of zinc as a signaling factor, this paper analyzes the relationship among the changes in hippocampal functions, abnormal behavior and pathophysiological changes in zinc deficiency, based on the data from experimental animals.

Susceptibility to kainate-induced seizures under dietary zinc deficiency

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 Timms stain was performed to estimate zinc concentrations in synaptic vesicles, Timms stain in the brain was attenuated in the zinc‐deficient 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 zinc‐deficient 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.

Decreased brain zinc availability reduces hippocampal neurogenesis in mice and rats

In the adult brain, neurogenesis occurs in the subgranular zone of the dentate gyrus (DG), where high levels of vesicular zinc are localized in the presynaptic terminals. To determine whether zinc has a role in modulating hippocampal neurogenesis under normal or pathologic conditions, we manipulated the level of vesicular zinc experimentally. To reduce hippocampal vesicular zinc, rats were either fed a zinc-deficient diet or treated with a zinc chelator, clioquinol (CQ). The number of progenitor cells and immature neurons was decreased significantly in the DG after 6 weeks of dietary zinc deprivation. Conversely, the number of progenitor cells and immature neurons was restored after a 2-week reversal to a normal zinc-containing diet. Similarly, a 1-week treatment with the zinc chelator, CQ, reduced the number of progenitor cells. The results of our previous study showed that hypoglycemia increased hippocampal neurogenesis. This study shows that zinc chelation reduced hypoglycemia-induced progenitor cell proliferation and neurogenesis. Finally, the role of vesicular zinc on neurogenesis was further assessed in zinc transporter 3 (ZnT3) gene deleted mice. Zinc transporter 3 knockout (KO) mice had significantly fewer proliferating progenitor cells and immature neurons after hypoglycemia. Our data provide converging evidence in support of the essential role zinc has in modulating hippocampal neurogenesis.

Anxiety-like behavior of young rats after 2-week zinc deprivation.

The relationship between neuronal function in the brain and neuropsychological behavior were analyzed in young rats fed a zinc-deficient diet for 1-2 weeks. Serum zinc concentration was less than 50% of that of the control. However, zinc concentration in the hippocampal perfusate measured by the in vivo microdialysis was not decreased after 2-week zinc deprivation. Timms stain, with which histochemically reactive zinc in the presynaptic vesicle is detected, was not also attenuated in the brain. On the other hand, serum corticosterone concentration, which was determined in the morning, was markedly increased after 2-week zinc deprivation and intracellular calcium signal, which was determined by fura-2 AM, was also increased in the hippocampus. In the hippocampus in zinc deficiency, intracellular free calcium concentration may be altered prior to the decrease in zinc concentration in the extracellular fluid. When rats were subjected to the open-field test, the frequency of line crossing and the time of grooming were decreased after 2-week zinc deprivation. In the plus-maze test, the time spent in the open arms was also decreased in zinc-deficient rats, suggesting that anxiety-like behavior is increased in zinc deficiency. The present study indicates that the increase in anxiety-like behavior in zinc deficiency may be linked to the increased concentration of basal free calcium in hippocampal cells, probably due to the increase in serum corticosterone concentration.

Release of glutamate and GABA in the hippocampus under zinc deficiency

Zinc homeostasis in the brain is affected by dietary zinc deficiency, and its alteration may cause brain dysfunctions. On the basis of the previous evidence that hippocampal zinc was responsive to 12‐week zinc deprivation, responsiveness of hippocampal zinc to dietary zinc deficiency was examined in rats fed a zinc‐deficient diet for 4 weeks. Zinc concentration in the hippocampus was not decreased by zinc deprivation for 4 weeks. However, Timms stain was extensively attenuated in the brain of the zinc‐deficient rats. In the brain of the zinc‐deficient rats, moreover, zinc concentration in the hippocampal extracellular fluid was approximately 30% of that of control rats. These results demonstrate that vesicular zinc is responsive to dietary zinc and may decrease easily under zinc deficiency. Zinc concentration in the hippocampal extracellular fluid during stimulation with high K+ was significantly increased even in zinc‐deficient rats, although the increased levels of zinc were lower than the basal levels of zinc in control rats. The basal glutamate concentration in the hippocampal extracellular fluid was not significantly different between the control and zinc‐deficient rats. However, glutamate concentration in the hippocampal extracellular fluid during stimulation with high K+ was more increased in the zinc‐deficient rats than in the control rats. Gamma aminobutyric acid (GABA) concentration in the hippocampal extracellular fluid during stimulation with high K+ was increased in the control rats, but not in the zinc‐deficient rats. The present study suggests that the excitability of hippocampal glutamatergic neurons is enhanced by dietary zinc deficiency.

Behavior in the forced swim test and neurochemical changes in the hippocampus in young rats after 2-week zinc deprivation.

Abnormal behavior in zinc deficiency and its cause are poorly understood. In the present paper, behavior in the forced swim test and neurochemical changes in the brain associated with its behavior were studied focused on abnormal corticosterone secretion in zinc deficiency. The effect of chronic corticosterone treatment was also studied. Immobility time in the forced swim test was increased in young rats fed a zinc-deficient diet for 2 weeks, as well as corticosterone (40mg/kg/dayx14 days)-treated control rats. The basal Ca(2+) levels in the hippocampus, which were determined by fluo-4FF, AM, were increased in both brain slices from zinc-deficient and corticosterone-treated rats. Serum glucose level was decreased in zinc deficiency and hippocampal glucose metabolism, which is determined by [(14)C]2-deoxyglucose uptake, was elevated. Hippocampal ATP level was not decreased, whereas, the concentrations of glutamate, GABA and glutamine in the hippocampus, unlike the whole brain, were decreased in zinc deficiency. However, the decrease in these amino acids was restored by adrenalectomy prior to zinc deficiency. These results suggest that glucose is insufficient for the synthesis of amino acids in the hippocampus of zinc-deficient rats. It is likely that the neurochemical and metabolic changes in the hippocampus, which may be associated with abnormal corticosterone secretion, is the base of abnormal behavior associated with neuropsychological symptoms in zinc deficiency.

Zinc homeostasis in the hippocampus of zinc-deficient young adult rats.

On the basis of the evidence of the transient learning impairment of young adult rats fed a zinc-deficient diet for 4 weeks, zinc concentration in the hippocampus was examined in the zinc-deficient rats to understand the mechanism of brain dysfunction in zinc deficiency. Zinc concentration in the hippocampus, as well as that in other brain regions, was not decreased by 4-week zinc deprivation. When Timms stain, with which histochemically reactive zinc in the presynaptic vesicles is detected, was compared between the control and zinc-deficient rats, the intensity of Timms stain in the hippocampus was almost the same between them. In the hippocampus, zinc concentration in the synaptosomal fraction was not also decreased by 4-week zinc deprivation, whereas that in the crude nuclear fraction was significantly increased. These results suggest that zinc concentration in the presynaptic vesicles is not decreased in young adults rats by 4-week zinc deprivation. It is likely that zinc-requiring systems in the nucleus are more responsive to zinc deficiency than vesicular zinc. This responsiveness appears to be involved in the transient learning impairment.

Dynamic action of neurometals at the synapse

There are synaptic vesicles that are labeled by Timms sulfide-silver staining method in the brain, suggesting that synaptic vesicles contain metals such as zinc and copper. Zinc is co-released with glutamate and the importance of zinc signaling in the intracellular compartment, in addition to extracellular compartment, is becoming recognized. Zinc can pass through calcium channels, while blocking them. Calcium signaling plays a critical role for synaptic activity and crosstalk between zinc signaling with calcium signaling through calcium channels may participate in synaptic neurotransmission including synaptic plasticity such as long-term potentiation. Copper released into the synaptic cleft during synaptic excitation may also participate in synaptic neurotransmission. Other metals including copper potentially serve as calcium channel blockers and also influence calcium signaling and zinc signaling via the interaction with metal-binding proteins such as metallothioneins. Homeostasis of metals needs to be controlled spatiotemporally for proper brain function, and their dyshomeostasis is associated with neurological diseases. However, the data on the dynamic action of metals at synapses is limited and their significance poorly understood. This paper summarizes the action of metals in synaptic neurotransmission focused on calcium signaling at glutamatergic synapses.

Susceptibility to stress in young rats after 2-week zinc deprivation

Dietary zinc deficiency elicits abnormal behavior in stressful environment. It is possible that abnormal corticosterone secretion in zinc deficiency is linked to abnormal behavior. To understand the increase in depression-like behavior in zinc deficiency, in the present study, serum corticosterone concentration was checked in young rats fed a zinc-deficient diet for 2 weeks after exposure to acute stress. Serum corticosterone concentration was higher in zinc-deficient rats after exposure to water-immersed and forced swim stress. Immobility time in the forced swim test was significantly increased in zinc-deficient rats, but not in pair-fed rats, suggesting that the increase in depression-like behavior is due to zinc deficiency rather than decreased food intake. The increase in immobility time in zinc deficiency was restored to the control level by feeding of the control diet. In dexamethasone suppression test, serum corticosterone concentration was markedly decreased in both the control and zinc-deficient rats. These results suggest that excessive corticosterone secretion after exposure to stress is linked to the increase in depression-like behavior in zinc deficiency. It has been reported that exposure to stress and glucocorticoids facilitates the increase in extracellular glutamate in the hippocampus. When the hippocampus was stimulated with 100mM KCl, the concentration of extracellular glutamate was more increased in zinc-deficient rats. In hippocampal slices from zinc-deficient rats, the decrease in FM4-64 fluorescence (exocytosis) was more facilitated. It is likely that zinc deficiency excessively excites glutamatergic neurons in the hippocampus after exposure to acute stress. This excessive excitation seems to contribute to susceptibility to stress after 2-week zinc deprivation and its related behavior such as the increase in depression-like behavior.

Suppressive effect of Yokukansan on excessive release of glutamate and aspartate in the hippocampus of zinc-deficient rats

Abstract Yokukansan (TJ-54), a herbal medicine, has been used as a cure for insomnia and irritability in children. Yokukansan also improves behavioral and psychological symptoms such as agitation, aggression and irritability in patients with dementia including Alzheimers disease, in which the glutamatergic neurotransmitter system is perturbed. However, the action of Yokukansan in synaptic neurotransmission is unknown. In the present study, the action of Yokukansan in the glutamatergic neurotransmitter system was examined in zinc-deficient rats, a neurological disease model, in which the glutamatergic neurotransmitter system is perturbed. Administration of Yokukansan significantly suppressed the increase in extracellular concentrations of glutamate and aspartate in the hippocampus after stimulation with 100 mM KCl, but not the increase in extracellular concentrations of glycine and taurine, suggesting that Yokukansan is involved in modulation of excitatory neurotransmitter systems. The present study demonstrates that Yokukansan is a possible medicine for prevention or cure of neurological diseases associated with excitotoxicity.