Optical tracking of phenotypically diverse individual synapses on solitary tract nucleus neurons

The solitary tract nucleus (NTS) is the termination site for cranial visceral afferents-peripheral primary afferent neurons which differ by phenotype (e.g. myelinated and unmyelinated). These afferents have very uniform glutamate release properties calculated by variance mean analysis. In the present study, we optical measured the inter-terminal release properties across individual boutons by assessing vesicle membrane turnover with the dye FM1-43. Single neurons were mechanically micro-harvested from medial NTS without enzyme treatment. The TRPV1 agonist capsaicin (CAP, 100 nM) was used to identify afferent, CAP-sensitive terminals arising from unmyelinated afferents. Isolated NTS neurons retained both glutamatergic and inhibitory terminals that generated EPSCs and IPSCs, respectively. Visible puncta on the neurons were stained positively with monoclonal antibody for synaptophysin, a presynaptic marker. Elevating extracellular K⁺ concentration to 10 mM increased synaptic release measured at individual terminals by FM1-43. Within single neurons, CAP destained some but not other individual terminals. FM1-43 positive terminals that were resistant to CAP could be destained with K⁺ solution. Individual terminals responded to depolarization with similar vesicle turnover kinetics. Thus, vesicular release was relatively homogenous across individual release sites. Surprisingly, conventionally high K⁺ concentrations (> 50 mM) produced erratic synaptic responses and at 90 mM K⁺ overt neuron swelling-results that suggest precautions about assuming consistent K⁺ responses in all neurons. The present work demonstrates remarkably uniform glutamate release between individual unmyelinated terminals and suggests that the homogeneous EPSC release properties of solitary tract afferents result from highly uniform release properties across multiple contacts on NTS neurons.