5-HT₃R–sourced calcium enhances glutamate release from a distinct vesicle pool

The serotonin 3 receptor (5-HT₃R) is a calcium-permeant channel heterogeneously expressed in solitary tract (ST) afferents. ST afferents synapse in the nucleus of the solitary tract (NTS) and rely on a mix of voltage-dependent calcium channels (CaVs) to control synchronous glutamate release (ST-EPSCs). CaV activation triggers additional, delayed release of glutamate (asynchronous EPSCs) that trails after the ST-EPSCs but only from afferents expressing the calcium-permeable, transient receptor potential vanilloid type 1 receptor (TRPV1). Most afferents express TRPV1 and have high rates of spontaneous glutamate release (sEPSCs) that is independent of CaVs. Here, we tested whether 5-HT₃R-sourced calcium contributes to these different forms of glutamate release in horizontal NTS slices from rats. The 5-HT₃R selective agonist, m-chlorophenyl biguanide hydrochloride (PBG), enhanced sEPSCs and/or delayed the arrival times of ST-EPSCs (i.e. increased latency). The specific 5-HT₃R antagonist, ondansetron, attenuated these effects consistent with direct activation of 5-HT₃Rs. PBG did not alter ST-EPSC amplitude or asynchronous EPSCs. These independent actions suggest two distinct 5-HT₃R locations; axonal expression that impedes conduction and terminal expression that mobilizes a spontaneous vesicle pool. Calcium chelation with EGTA-AM attenuated the frequency of 5-HT₃R-activated sEPSCs by half. The mixture of chelation-sensitive and resistant sEPSCs suggests that 5-HT₃R-activated vesicles span calcium diffusion distances that are both distal (micro-) and proximal (nanodomains) to the channel. Our results demonstrate that the calcium domains of 5-HT₃Rs do not overlap other calcium sources or their respective vesicle pools. 5-HT₃Rs add a unique calcium source on ST afferents as part of multiple independent synaptic signaling mechanisms.