br Funding This work was supported by Arena Pharmaceuticals

Funding This work was supported by Arena Pharmaceuticals, Inc, San Diego, CA, USA.
Introduction 5-hydroxytryptamine 1B (5-HT1B) receptors are widely distributed in the central nervous system (CNS); they have various modulatory functions in drug reinforcement, appetitive behaviors, stress, mood, and aggression (Clark and Neumaier, 2001; Sari, 2004). 5-HT1B receptors are inhibitory G protein-coupled receptors that are located predominantly on the nerve terminals of both serotonergic and non-serotonergic neurons, where they act as auto- and heteroreceptors, respectively (Boschert et al., 1994; Morikawa et al., 2000; Sari, 2004). This receptor subtype has recently been suggested as a putative target for the pharmacological treatment of depression based on the observation that overexpression of the 5-HT1B receptors or administration of 5-HT1B receptor agonists produces antidepressant-like effects (Tatarczyńska et al., 2005; Carr and Lucki, 2011; McDevitt et al., 2011; Murrough and Neumeister, 2011; Nautiyal and Hen, 2017). Furthermore, the antidepressant-like effects of selective serotonin reuptake inhibitors were absent in 5-HT1B knockout mice, and this effect was antagonized by the blockade of 5-HT1B receptors in wild-type mice (Redrobe and Bourin, 1999; O'Neill and Conway, 2011). These findings indicate that 5-HT1B receptor agonists are potential antidepressant candidates; however, the exact mechanism of action remains unclear. One hypothesis suggests that the antidepressant-like effect of 5-HT1B receptor agonists is mediated by the activation of 5-HT1B heteroreceptors (Chenu et al., 2008). These heteroreceptors are primarily located in the striatum and hippocampus (Bruinvels et al., 1994), and thay regulate the release of numerous neurotransmitters such as glutamate (Sari, 2004; Ruf and Bhagwagar, 2009). Data from in vitro electrophysiological experiments and in vivo microdialysis studies have shown that 5-HT1B receptor activation can inhibit glutamate release in several saha hdac regions (Gołembiowska and Dziubina, 2002; Lemos et al., 2006; Choi et al., 2012; Hwang and Chung, 2014); however, this effect has not been examined directly in nerve terminal preparations. The mechanism through which 5-HT1B receptor agonists act at the presynaptic level to inhibit glutamate release are particularly pertinent to depression because excessive glutamate release has been implicated in the pathogenesis of depression and in the mechanism of action of antidepressant drugs (Mitani et al., 2006; Hashimoto et al., 2007; Hashimoto, 2011; Lin et al., 2011a, Lin et al., 2011b; Musazzi et al., 2013). To elucidate the presynaptic effects of 5-HT1B receptors on glutamate release, we studied the effect of CGS12066, a selective 5-HT1B receptor agonist, on glutamate release in rat hippocampal nerve terminals (synaptosomes). Synaptosomes are capableof accumulating, storing, and releasing neurotransmitters and is devoid of functional glial and nerve cell body elements that might obscure the interpretation of findings because of modulatory loci at non-neuronal, postsynaptic, or network levels (Nicholls, 1993). Therefore, synaptosomes are extensively used to evaluate presynaptic effects on neurotransmitter release (Nicholls and Sihra, 1986). The present results demonstrated that CGS12066 acts on 5-HT1B receptors located on the hippocampal glutamate nerve terminals, thus inhibiting Ca2+ influx and glutamate release. Moreover, the inhibition is a consequence of reduced the activity in Gi/Go-protein-coupled adenylate cyclase (AC)/protein kinase A (PKA) cascade. This investigation is crucial for understanding 5-HT1B receptor function and for exploiting its potential for therapeutic interventions.
Materials and methods
Results
Discussion Electrophysiological studies have suggested that synaptically released 5-HT produces presynaptic inhibition of glutamate transmission by impinging on 5-HT1B heteroreceptors on the excitatory terminals (Lemos et al., 2006; Choi et al., 2012; Hwang and Chung, 2014; Liao and Lee, 2014). In these studies, the effects of presynaptic 5-HT1B receptors on glutamate release are monitored indirectly by observing changes in postsynaptic potentials. The use of isolated nerve terminals (synaptosomes) enables a relatively direct evaluation of presynaptic receptor modulation of transmitter release. In the present study, we used the synaptosomal model to explore the characteristics and mechanisms of action of 5-HT1B receptors involved in regulating glutamate release in the hippocampus. Our results show that rat hippocampal glutamatergic nerve terminals possess presynaptic 5-HT1B receptors because they are colocalized with the glutamatergic terminal marker protein VGLUT1. In our study, the activation of these receptors by CGS12066 reduced 4-AP-evoked glutamate release, and this phenomenon was completely blocked by a 5-HT1B receptor antagonist. The effect of CGS12066 was concentration dependent with half-maximal inhibition occurring between 3 and 5 μM, consistent with the values reported to activate 5-HT1B receptors in various systems (Sayer et al., 1999; Laurent et al., 2002). Furthermore, the electrophysiological experiments revealed that CGS12066 pretreatment reduced glutamatergic mEPSC frequency without affecting the current amplitude, and the postsynaptic response to glutamate in the presence of CGS12066 was not significantly altered. Taken together, our results provide evidence that functional 5-HT1B receptors are expressed on the hippocampal glutamatergic nerve terminals and that their activation by CGS12066 reduces the probability of glutamate release.