The cellular mechanism underlying the CGS mediated inhibitio
The cellular mechanism underlying the CGS12066-mediated inhibition of glutamate release from hippocampal nerve terminals through presynaptic 5-HT1B receptors remains to be elucidated. 5-HT1B receptors are coupled to PTX-sensitive G proteins (Gi or Go) that have been shown to inhibit AC activity as well as promote the opening of potassium channels and closing of VDCCs (Barnes and Sharp, 1999; Brown and Sihra, 2008). Each of these pathways may be crucial in the CGS12066-mediated inhibition of glutamate release observed in this study. In the present study, preincubation of nerve terminals with PTX prevented the inhibitory effect of CGS12066 on glutamate release. This PTX sensitivity of the CGS12066 inhibitory pathway in nerve terminals has also been observed in the inhibitory effect of 5-HT1B receptor agonists on glutamate and GABA release in aqz slices and neuron cultures (Matsuoka et al., 2004; Xiao et al., 2008; Choi et al., 2012). Therefore, the inhibitory effect of CGS12066 on glutamate release appears to be mediated by PTX-sensitive G proteins, as for many other presynaptic inhibitory receptors, including histamine H3, 5-hydroxytryptamine 6, and metabotropic glutamate receptors (Millan et al., 2002; Lu et al., 2017; Wang et al., 2016). In addition, signaling through 5-HT1B receptor-mediated presynaptic inhibition involves reduced levels of cAMP and PKA (Mathur et al., 2011; Hwang and Chung, 2014). Moreover, the AC inhibitor MDL12330A and the PKA inhibitor H89 blocked the CGS12066-mediated inhibition of glutamate release, suggesting the involvement of a cAMP-dependent pathway. The cAMP/PKA pathway is known to be present at the presynaptic level, where it facilitates glutamate release through multiple mechanisms, including the enhancement of VDCC activity, the inhibition of K+ channels, and the upregulation of release machinery (Herrero and Sánchez-Prieto, 1996; Catterall, 2000; Schrader et al., 2002). Our data suggest the involvement of a cAMP/PKA-mediated mechanism in the inhibition of glutamate release by presynaptic 5-HT1B receptor activation with CGS12066. This finding raises the question of which target substrate of PKA underlies the observed effect. In this study, we found that that CGS12066 significantly reduced the evoked increase in intrasynaptosomal Ca2+ levels and KCl-evoked glutamate release, a process that involves Ca2+ influx primarily through VDCC opening (Barrie et al., 1991). Furthermore, the extent of CGS12066-mediated inhibition of glutamate release was significantly reduced by ω-CgTX MVIIC (an N- and P/Q-type Ca2+ channel blocker) but not by dantrolene and CGP37157 (intracellular Ca2+ release blockers). These results suggest that the CGS12066-mediated inhibition of glutamate release is associated with a reduction in Ca2+ influx through N- and P/Q-type Ca2+ channel activity in the hippocampal nerve terminals. Our finding is consistent with previous electrophysiological data, which showed that the activation of presynaptic 5-HT1B receptors inhibits VDCCs to reduce glutamate release at central synapses (Mizutani et al., 2006; Choi et al., 2012). However, the observation that the mechanism underlying 5-HT1B receptor-induced presynaptic inhibition was independent of VDCC modulation has also been reported by other workers (Mizutani et al., 2006; McCamphill et al., 2008; Nishijo and Momiyama, 2016). We are uncertain whether the discrepancies are attributable to the use of adult rats, synaptosomes, different 5-HT1B receptor agonists, or other methodological differences. On the other hand, 5-HT1B receptor-mediated presynaptic inhibition of glutamate release was reported to result from the activation of K+ channels (Hwang and Chung, 2014). The activation of K+ channels can cause the hyperpolarization of nerve terminals, which would reduce action potentials. This reduction would decrease the presynaptic Ca2+ influx, which in turn, would affect neurotransmitter release (Nicoll, 1988; Rehm and Tempel, 1991). However, this mechanism was excluded owing to our finding that CGS12066 did not affect the synaptosomal plasma membrane potential either in the resting state or after depolarization with 4-AP. Hence, the activation of presynaptic 5-HT1B receptors by CGS12066 observed here may inhibit the cAMP/PKA pathway, thus reducing VDCC activity and glutamate release.