Mammalian hippocampal circuitry contains copious vesicular Zn2+ and glutamate in the mossy fiber axon terminals which originate from the granule cells of the dentate gyrus. Temporal lobe epilepsy in humans and rodent epilepsy models reveal a peculiar feature in the brain, branches referred to as “recurrent mossy fibers” which sprouts off from mossy fibers. The recurrent mossy fiber terminals of pilocarpine induced epileptic rats were examined for Zn2+ and confirmed with Timm's staining and intracellular fluorescent zinc indicators. These zinc-rich terminals were investigated for release of Zn2+ into the extracellular space detected by a low affinity fluorescent indicator for Zn2+, namely Newport Green. This study provides evidence to support that high frequency electrical stimulation of the mossy fiber axons causes Zn2+ release from not only mossy fibers but also the Zn2+ rich recurrent mossy fiber terminals. This release is frequency dependent; the fluorescent response is attenuated in the presence of Zn2+ specific chelators, low calcium medium and a vesicular uptake inhibitor. Also, the concentration of Zn2+ released from the terminals was estimated to be in the low micromolar range, with significantly higher release observed in epileptic animals when compared to sham treated ones.
Evidence for a Zn2+ sensitive voltage gated sodium channel, Nav1.5/ SCN5A expression in the dentate gyrus was obtained by western blotting and real time quantitative PCR and using immunostaining they were seen localized to regions around the soma of granule cells and CA3 pyramidal cells in the dentate gyrus.
Interestingly, when epileptic rats were analyzed it was seen that animals that were 16 weeks epileptic had the maximal amount of releasable zinc as well as Nav1.5 protein expression. This was followed by a gradual decline with age. Since these channels could be blocked by Zn2+ and their localization was found near where synaptic Zn2+ is released, it could be postulated that the presence of increased zinc could block Nav1.5 channels and consequently decrease the excitatory activity in the dentate gyrus of epileptic rats. However, a causal effect between the increase in zinc and Nav1.5 channels could not be determined due to the presence of these channels in the age-matched controls. Still, the mere presence of this TTX resistant Zn2+ sensitive channel in the hippocampal pathway makes it an interesting candidate to be studied for the interactions of zinc.
To conclude, this study demonstrates the presence of functional Zn2+ releasing synapses on the anomalous sprouts that are seen in epilepsy, releasing Zn2+ in the low micromolar range. In the epileptic rat hippocampus, the fluorescent response to the released Zn2+ was seen not only in the molecular layer and CA3-hilar region but also observed in the granule cell layer albeit in lower amounts compared to the other areas. This Zn2+ may serve as a contributing factor to several postsynaptic and presynaptic interactions depending on the constituency of the targets in the particular region. Moreover, a potential target for Zn2+, a Zn2+ sensitive channel, SCN5A/Nav1.5 in the hippocampus was identified for the first time.