In vitro and in vivo physiology of low nanomolar concentrations of Zn2+ in artificial cerebrospinal fluid.

Artificial cerebrospinal fluid (ACSF), i.e., brain extracellular medium, which includes Ca2+ and Mg2+, but not other divalent cations such as Zn2+, has been used for in vitro and in vivo experiments. The present study deals with the physiological significance of extracellular Zn2+ in ACSF. Spontaneous presynaptic activity is suppressed in the stratum lucidum of brain slices from young rats bathed in ACSF containing 10 nM ZnCl2, indicating that extracellular Zn2+ modifies hippocampal presynaptic activity. To examine the in vivo action of 10 nM ZnCl2 on long-term potentiation (LTP), the recording region was perfused using a recording electrode attached to a microdialysis probe. The magnitude of LTP was not modified in young rats by perfusion with ACSF containing 10 nM ZnCl2, compared to perfusion with ACSF without Zn2+, but attenuated by perfusion with ACSF containing 100 nM ZnCl2. Interestingly, the magnitude of LTP was not modified in aged rats even by perfusion with ACSF containing 100 nM ZnCl2, but enhanced by perfusion with ACSF containing 10 mM CaEDTA, an extracellular Zn2+ chelator. The present study indicates that the basal levels of extracellular Zn2+, which are in the range of low nanomolar concentrations, are critical for synaptic activity and perhaps increased age-dependently.

Significance of synaptic Zn2+ signaling in zincergic and non-zincergic synapses in the hippocampus in cognition.

A portion of zinc concentrates in the synaptic vesicles in the brain and is released from glutamatergic (zincergic) neuron terminals. It serves as a signaling factor (in a form of free Zn2+). Both extracellular Zn2+ signaling, which predominantly originates in Zn2+ release from zincergic neuron terminals, and intracellular Zn2+ signaling, which is often linked to extracellular Zn2+ signaling, are involved in hippocampus-dependent memory. At mossy fiber-CA3 pyramidal cell synapses and Schaffer collateral-CA1 pyramidal cell synapses, which are zincergic, extracellular Zn2+ signaling leads to intracellular Zn2+ signaling and is involved in learning and memory. At medial perforant pathway-dentate granule cell synapses, which are non-zincergic, intracellular Zn2+ signaling, which originates in the internal stores containing Zn2+, is involved in learning and memory. The blockade of Zn2+ signaling with Zn2+ chelators induces memory deficit, while the optimal amount range of Zn2+ signaling is unknown. It is possible that the degree and frequency of Zn2+ signaling, which determine the increased Zn2+ levels, modulates learning and memory as well as intracellular Ca2+ signaling. To understand the precise role of synaptic Zn2+ signaling in the hippocampus, the present paper summarizes the current knowledge on Zn2+ signaling at zincergic and non-zincergic synapses in the hippocampus in cognition and involvement of zinc transporters and zinc-binding proteins in synaptic Zn2+ signaling.

Preventive Effect of 3,5-dihydroxy-4-methoxybenzyl Alcohol (DHMBA) and Zinc, Components of the Pacific Oyster Crassostrea gigas, on Glutamatergic Neuron Activity in the Hippocampus.

The effects of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), and zinc--both components of the Pacific oyster Crassostrea gigas--were examined by glutamatergic neuron activity in rats in an in vivo microdialysis experiment and an in vitro brain slice experiment. The basal concentration of extracellular glutamate in the hippocampus was decreased under hippocampal perfusion with DHMBA (1 mmol l(-1)) or ZnCl2 (µmol l(-1)), indicating that DHMBA and Zn(2+) suppress glutamatergic neuron activity under basal (static) conditions. To assess the preventive effect of DHMBA and Zn(2+) on glutamate release from neuron terminals, brain slices were pretreated with DHMBA (1 mmol l(-1)) or ZnCl2 (100 nmol l(-1)) for 1 h, then stimulated with high K(+). A high, K(+)-induced increase in extracellular Zn(2+) level, an index of glutamate release, was suppressed with pretreatment with DHMBA or zinc. A high, K(+)-induced increase in intracellular Ca(2+) level was also suppressed with pretreatment with DHMBA or Zn(2+). These results suggest that DHMBA and Zn(2+), previously taken up in the hippocampal cells, suppress high, K(+)-induced glutamate release in the hippocampus, probably via presynaptic suppression of intracellular Ca(2+) signaling. It is likely that Zn(2+) and DHMBA play a preventive role in suppressing excess glutamatergic neuron activity in rats and mice.

Modification of hippocampal excitability in brain slices pretreated with a low nanomolar concentration of Zn2+.

Synaptic Zn2+ homeostasis may be changed during brain slice preparation. However, much less attention has been paid to Zn2+ in artificial cerebrospinal fluid (ACSF) used for slice experiments than has been paid to Ca2+ . The present study assesses addition of Zn2+ to ACSF, focused on hippocampal excitability after acute brain slice preparation. When the static levels of intracellular Zn2+ and Ca2+ were compared between brain slices prepared with conventional ACSF without Zn2+ and those pretreated with ACSF containing 20 nM ZnCl2 for 1 hr, both levels were almost the same. On the other hand, intracellular Ca2+ levels were significantly increased in the stratum lucidum of the control brain slices after stimulation with high K+, although the increase was significantly suppressed by the pretreatment with ACSF containing Zn2+, suggesting that neuronal excitation is enhanced in brain slices prepared with ACSF without Zn2+. The increase in extracellular Zn2+ level, an index of glutamate release, after stimulation with high K+ was also significantly suppressed by pretreatment with ACSF containing Zn2+. When mossy fiber excitation was assessed in brain slices with FM4-64, an indicator of presynaptic activity, attenuation of FM 4-64 fluorescence based on presynaptic activity was suppressed in the stratum lucidum of brain slices pretreated with ACSF containing Zn2+. The present study indicates that hippocampal excitability is enhanced in brain slices prepared with ACSF without Zn2+. It is likely that a low nanomolar concentration of Zn2+ is necessary for ACSF.