br Material and methods br Results br Discussion Neurosteroi

Material and methods
Results
Discussion Neurosteroids capable of blocking the NMDAR channel attract a lot of attention as possible therapeutic agents for the treatment of neurodegenerative diseases. A large number of new MAPK Inhibitor Library with different structures have been synthesized in order to create an effective antagonist of NMDAR with minimal side effects (Adla et al., 2017; Holubova et al., 2014; Kleteckova et al., 2014; Kudova et al., 2015; Rambousek et al., 2011; Vyklicky et al., 2016). As the neurosteroids can exhibit multi-target action (Korinek et al., 2011; O'Dell et al., 2005), the study of their ability to interact with other receptors, including inhibitory GABAAR and GlyR, is an important task. The effect of steroids on IGABA has been studied by many authors (for a review, see: Belelli and Lambert, 2005; King, 2013; Korinek et al., 2011; Majewska et al., 1988; Wu et al., 1990; Zorumski et al., 2013). It has been shown that progesterone, pregnanolone, and allopregnanolone enhance IGABA while pregnanolone sulfate and pregnenolone sulfate inhibit it. Due to the ability to enhance GABA responses, steroids exhibit anxiolytic, analgesic, anticonvulsant, sedative, hypnotic and anaesthetic properties (for a review see Belelli and Lambert, 2005). In our experiments, the effects of two groups of steroids in the concentration range 0.1–50 μM were studied on the IGABA of rat pyramidal hippocampal neurons: pregnanolone glutamate (PA-Glu) and its derivatives, and androstane glutamate (AND-Glu) and its derivatives. There is an important structural difference between these groups, namely, the presence of a C-17 acetyl moiety. It was found that only analogues of pregnanolone (PA-Glu, PA-hSuc and PA-hPim) and not analogues of androstane could modulate IGABA. These results suggest that a C-17 acetyl moiety is necessary to affect the GABAAR of hippocampal pyramidal neurons. Our data agree with the results of Harrison et al. (1987), who considered that a ketone moiety at C-20 is a structural requirement for the interaction of a steroid with GABAAR. We found that PA-Glu increased IGABA peak amplitude with a maximum potentiation of up to 422%, and an EC50 of 7 μM. Thus, PA-Glu appeared to be a weaker IGABA enhancer than pregnanolone, which effectively potentiated this current at nanomolar concentrations (Belelli and Lambert, 2005; Weir et al., 2004; Wu et al., 1990). In contrast to PA-Glu, its derivates PA-hSuc and PA-hPim decreased IGABA with IC50 values close to 10 μM. These values are close to or lower than those obtained in the study of the effects of pregnenolone sulfate on IGABA: IC50 = 60 μM (Majewska et al., 1988) and 7 μM (Wu et al., 1991). Our study is the first one describing acceleration of desensitization of the IGABA in the presence of steroids. The effect of PA-hSuc on the IGABA desensitization was stronger than on the IGABA peak, and this suggest that tonic inhibition in hippocampus can be suppressed stronger than phasic one with this steroid. In contrast, the effects PA-hPim on the peak and decay of the IGABA were similar, and this means that tonic and fast synaptic inhibition are suppressed in a similar way. Next, we studied the effects of the compounds PA-Glu, PA-hSuc, and PA-hPim on the IGly of rat hippocampal neurons. As in the experiments with IGABA, IGly was affected by steroids with a pregnane skeleton, but not those with an androstane skeleton. At the same time, androstane steroids were shown to be able to affect GlyRs with a substitution at C-17 (17-oxo or 20-oxo) (Maksay et al., 2001). In our experiments, all the pregnane steroids tested (PA-Glu, PA-hPim and PA-hSuc) inhibited IGly, causing a decrease in its peak amplitude and an acceleration of desensitization. The effect of steroids on IGly decay was stronger than on the IGly peak, and this means that steroids are more potent at inhibiting the steady-state than the peak responses mediated by GlyRs (Fig. 3D). These results suggest that the steroids tested are effective antagonists of tonically activated GlyRs with a smaller effect on fast synaptic transmission.