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

Abstract

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.

Thermally Active TRPV1 Tonically Drives Central Spontaneous Glutamate Release

“…Likely other glutamate terminals including from NTS interneurons contributed to our total activities but evidence suggests that their numbers are relatively low compared to ST contacts (McDougall et al , 2009). FM1-43 tracking of multiple individual terminals on NTS neurons shows that capsaicin and potassium depolarization activated the same terminals (Jin et al, 2010), a finding consistent with co-localization of the TRPV1-dependent pool with the depolarization-evoked vesicle pool.…”

“…To better understand the mechanisms of action of TRPV1 and reduce the possibility of interactions with adjacent neurons, we mechanically isolated medial NTS neurons using a microharvesting approach that retained functioning synaptic terminals (Jin et al, 2010). All neurons displayed glutamatergic and GABAergic events.…”

Localization of TRPV1 and P2X3 in unmyelinated and myelinated vagal afferents in the rat

“…This focus on synaptic function misses other potential functions that could be detected by other means. Likewise, our anatomical labeling studies focused on varicosities which are suspected as vagal afferent synaptic release sites (Andresen and Peters, 2008; Jin et al , 2010). The presence of substantial proportions of varicosities without staining for either TRPV1 or P2X3 may reflect incomplete detection, but raises the possibility of non-releasing varicosities or alternative functions that are unrelated to the myelinated/unmyelinated phenotypic differences.…”

Chronic intermittent hypoxia induces changes in expression of synaptic proteins in the nucleus of the solitary tract

“…Although the central mechanisms that may induce sympathetic long-term facilitation and baroreceptor reflex depression are not known, these mechanisms may be important in the development and maintenance of the hypertension observed in individuals suffering from chronic obstructive sleep apnea. Interestingly, the NTS region has been identified as one of the few neurogenic sites within the central nervous system (Bauer et al 2005;Chigr et al 2009), and the NTS has a high level of expression of factors associated with neuroplasticity including the neurite outgrowth protein brain-derived neurotrophic factor (BDNF), and its receptor tropomyosin receptor kinase B (TrkB), growthassociated protein-43 (GAP-43), and synaptophysin a synaptic vesicle glycoprotein (Jin et al, 2010;Korshunova and Mosevitsky, 2010;Moyse et al, 2006). Previous studies have suggested that BDNF, TrkB, GAP-43 and/or synaptophysin may be important in mediating alterations associated with activity-dependent functional changes in cardiovascular afferent fibers that terminate within the NTS (Martin et al, 2009).…”