Questioning the Role of Zinc in Epileptogenesis

Lack of Effect of Mossy Fiber-Released Zinc on Granule Cell GABA_A Receptors in the Pilocarpine Model of Epilepsy

Molnar P

Nadler JV

J Neurophysiol 2001;85:1932–1940

The recurrent mossy fiber pathway of the dentate gyrus expands dramatically in the epileptic brain and serves as a mechanism for synchronization of granule cell epileptiform activity. It has been suggested that this pathway also promotes epileptiform activity by inhibiting GABA_A receptor function through release of zinc. Hippocampal slices from pilocarpine-treated rats were used to evaluate this hypothesis. The rats had developed status epilepticus after pilocarpine administration, followed by robust recurrent mossy fiber growth. The ability of exogenously applied zinc to depress GABA_A receptor function in dentate granule cells depended on removal of polyvalent anions from the superfusion medium. Under these conditions, 200 μM zinc reduced the amplitude of the current evoked by applying muscimol to the proximal portion of the granule cell dendrite (23%). It also reduced the mean amplitude (31%) and frequency (36%) of miniature inhibitory postsynaptic currents. Nevertheless, repetitive mossy fiber stimulation (10 Hz for 1 s, 100 Hz for 1 s, or 10 Hz for 5 min) at maximal intensity did not affect GABA_A receptor-mediated currents evoked by photorelease of GABA onto the proximal portion of the dendrite, where recurrent mossy fiber synapses were located. These results could not be explained by stimulation-induced depletion of zinc from the recurrent mossy fiber boutons. Negative results were obtained even during exposure to conditions that promoted transmitter release and synchronized granule cell activity (6 mM [K⁺]_o, nominally Mg²⁺-free medium, 33°C). These results suggest that zinc released from the recurrent mossy fiber pathway did not reach a concentration at postsynaptic GABA_A receptors sufficient to inhibit agonist-evoked activation.

Commentary

Zn²⁺ is a divalent cation that is prominently concentrated in synaptic terminals where it is intermixed with vesicles and may also be observed in synaptic clefts. In this study, the authors have investigated the possibility that release of the divalent cation Zn²⁺ from synaptic terminals may alter GABA_A receptor dependent inhibition in the hippocampus. Pharmacological studies have demonstrated that certain subsets of GABA_A receptors are sensitive to blockade by Zn²⁺. This is potentially an interesting question for epilepsy research, as there is also evidence in epilepsy models that Zn²⁺ may modify inhibition acutely in experimental status epilepticus, and in cultures of dissociated granule cells from the dentate gyrus of epileptic rats. Zn²⁺ sensitivity of GABA_A receptors is of particular interest in the hippocampus, where sprouted mossy fiber synaptic terminals containing relatively large amounts of releasable Zn²⁺ are observed in the supragranular layer of the dentate gyrus in both experimental models and resected human epileptic hippocampus. Release of Zn²⁺ from these sprouted mossy fiber terminals could reduce inhibition in hippocampal circuitry reorganized by preceding seizures, and therefore could play a role in epileptogenesis.

In this physiological study, responses evoked in granule cells of the dentate gyrus from pilocarpine treated rats by application of the GABA_A agonist muscimol were reduced by 200 μM Zn²⁺, which also reduced the amplitude and frequency of miniature IPSCs. The effects were noted only when polyvalent anions (PO₄^-3, SO₄^-2) were removed from the bathing media, presumably because these anions bind Zn²⁺ and render it inactive. In the same conditions, repetitive stimulation of mossy fibers in CA3 had no effect on GABA_A currents evoked by photorelease of caged GABA in the proximal dendritic region of granule cells, which is the site of sprouted mossy fiber terminals. The lack of evidence for Zn²⁺ blockade of GABA_A currents in physiologically relevant conditions suggests that the effects of Zn²⁺ may be less dramatic than previously suspected, or may be encountered only in unusual metabolic conditions, e.g., status epilepticus. The study provides another example of how a relatively clearly demonstrated in vitro phenomenon, such as the in vitro ability of Zn²⁺ to block inhibitory currents, may have very conditional or variable effects in a complex in vivo system of neural circuitry and epileptogenesis.