Synaptic Integration of Subquantal Neurotransmission by Colocalized G Protein-Coupled Receptors in Presynaptic Terminals

Abstract

In presynaptic terminals, membrane-delimited G_i/o-mediated presynaptic inhibition is ubiquitous and acts via Gβγ to inhibit Ca²⁺ entry, or directly at SNARE complexes to inhibit Ca²⁺-dependent synaptotagmin-SNARE complex interactions. At CA1-subicular presynaptic terminals, 5-HT_1B and GABA_B receptors colocalize. GABA_B receptors inhibit Ca²⁺ entry, whereas 5-HT_1B receptors target SNARE complexes. We demonstrate in male and female rats that GABA_B receptors alter P_r, whereas 5-HT_1B receptors reduce evoked cleft glutamate concentrations, allowing differential inhibition of AMPAR and NMDAR EPSCs. This reduction in cleft glutamate concentration was confirmed by imaging glutamate release using a genetic sensor (iGluSnFR). Simulations of glutamate release and postsynaptic glutamate receptor currents were made. We tested effects of changes in vesicle numbers undergoing fusion at single synapses, relative placement of fusing vesicles and postsynaptic receptors, and the rate of release of glutamate from a fusion pore. Experimental effects of P_r changes, consistent with GABA_B receptor effects, were straightforwardly represented by changes in numbers of synapses. The effects of 5-HT_1B receptor-mediated inhibition are well fit by simulated modulation of the release rate of glutamate into the cleft. Colocalization of different actions of GPCRs provides synaptic integration within presynaptic terminals. Train-dependent presynaptic Ca²⁺ accumulation forces frequency-dependent recovery of neurotransmission during 5-HT_1B receptor activation. This is consistent with competition between Ca²⁺-synaptotagmin and Gβγ at SNARE complexes. Thus, stimulus trains in 5-HT_1B receptor agonist unveil dynamic synaptic modulation and a sophisticated hippocampal output filter that itself is modulated by colocalized GABA_B receptors, which alter presynaptic Ca²⁺ In combination, these pathways allow complex presynaptic integration.SIGNIFICANCE STATEMENT Two G protein-coupled receptors colocalize at presynaptic sites, to mediate presynaptic modulation by Gβγ, but one (a GABA_B receptor) inhibits Ca²⁺ entry whereas another (a 5-HT_1B receptor) competes with Ca²⁺-synaptotagmin binding to the synaptic vesicle machinery. We have investigated downstream effects of signaling and integrative properties of these receptors. Their effects are profoundly different. GABA_B receptors alter P_r leaving synaptic properties unchanged, whereas 5-HT_1B receptors fundamentally change properties of synaptic transmission, modifying AMPAR but sparing NMDAR responses. Coactivation of these receptors allows synaptic integration because of convergence of GABA_B receptor alteration on Ca²⁺ and the effect of this altered Ca²⁺ signal on 5-HT_1B receptor signaling. This presynaptic convergence provides a novel form of synaptic integration.

Keywords: fusion pore; membrane delimited; presynaptic G proteins; presynaptic calcium; synaptic integration; synaptic vesicle fusion.