Zn is the second most abundant
Zn2+ is the second most abundant trace metal in many cells and neurons in the brain. It regulates cellular functions such as gene expression, epigenetic enzymatic activity, protein structural stability, and neuronal plasticity. Zn2+ is particularly abundant in the cortex and limbic system, including hippocampus and amygdala (Frederickson, 1989, Frederickson et al., 2000, Frederickson et al., 2005, Gower-Winter and Levenson, 2012, Szewczyk, 2013). Zn2+ homeostasis in the brain is tightly controlled and primarily regulated by Zn2+ transporters and binding proteins. High level of “chelatable” Zn2+ is sequestered in the synaptic vesicles of glutamatergic neurons through Zn2+ transporter protein ZnT3. Other transporters like ZIP proteins promote the efflux of vesicular Zn2+ and increase the concentration of cytoplasmic Zn2+ (Cole et al., 1999, Kambe et al., 2004, Lee et al., 2011). Zn2+ is synaptically co-released with glutamate from presynaptic vesicles of the hippocampal mossy fibers, the Cy5.5 NHS ester of DGGCs (Frederickson et al., 1983, Assaf and Chung, 1984, Molnar and Nadler, 2001, Paoletti et al., 2009). Zn2+ has been shown to modulate both excitatory and inhibitory transmission (Westbrook and Mayer, 1987, Harrison and Gibbons, 1994, Vogt et al., 2000). Zn2+ inhibits GABA-A receptors and has distinct sensitivities at different receptor subtypes partly due to the accessibility of Zn2+ binding sites (Smart et al., 1991, Barberis et al., 2000, Ruiz et al., 2004). The δ-containing extrasynaptic receptors exhibit greater sensitivity to Zn2+ blockade than γ-containing synaptic receptors (Hosie et al., 2003). Alterations in Zn2+ homeostasis have implications in several neurological disorders (Frederickson et al., 2005, Bitanihirwe and Cunningham, 2009, Qian et al., 2011, Szewczyk, 2013, Prakash et al., 2015). Aberrant Zn2+-rich mossy fiber sprouting is a classical feature in limbic epileptogenesis (Cavazos et al., 1991). Excessive release of Zn2+ drastically alters the excitability of hippocampal circuits and plays an important role in the pathophysiology of epilepsy (Takeda et al., 1999, Coulter, 2000, Foresti et al., 2008, Elsas et al., 2009). Allopregnanolone (3α-hydroxy-5α-pregnan-20-one, AP) and related neurosteroids are endogenous modulators of neuronal excitability and seizure susceptibility (Chuang and Reddy, 2018a, Chuang and Reddy, 2018b, Younus and Reddy, 2018). Neurosteroids modulate GABA-A receptors primarily via allosteric potentiation of GABA at nanomolar concentrations and through direct activation of the channel at micromolar concentrations (Hosie et al., 2007). AP has powerful antiseizure activity (Kokate et al., 1996, Kaminski et al., 2003, Kaminski et al., 2004, Carver et al., 2016, Reddy and Estes, 2016). Ganaxolone (3α-hydroxy-3β-methyl-5α-pregnan-20-one, GX), a synthetic analog of AP, is designed to treat epilepsy and related seizure conditions. GX produces robust positive allosteric modulation of GABA-A receptors, with preferential sensitivity at δ-subunit-containing extrasynaptic receptors (Chuang and Reddy, 2018b). GX has a more favorable biopharmaceutical profile than AP due to its higher bioavailability and less hormonal side effects (Carter et al., 1997; Reddy and Woodward, 2004; Carver and Reddy, 2016). GX produces powerful antiseizure activity in a wide range of experimental models, and is being evaluated in clinical trials for epilepsy (Kerrigan et al., 2000, Laxer et al., 2000, Nohria and Giller, 2007, Pieribone et al., 2007, Reddy and Rogawski, 2012, Bialer et al., 2015, Braat et al., 2015, Sperling et al., 2017). Recently, Zn2+ has been shown to selectively block neurosteroid-sensitive, extrasynaptic δGABA-A receptors in the hippocampus (Carver et al., 2016). Therefore, light reactions is likely that Zn2+ may exert antagonistic effects on GX modulation of tonic inhibition and seizure protection. In the present study, we investigated the pharmacodynamic interactions of Zn2+ and GX at extrasynaptic GABA-A receptors and its functional relevance on the anticonvulsant activity of GX in the kindling model. Our results demonstrate that Zn2+ diminishes the antiseizure effects of GX by selectively blocking the extrasynaptic δGABA-A receptors in the hippocampus.