Differential content of vesicular glutamate transporters in subsets of vagal afferents projecting to the nucleus tractus solitarii in the rat

Abstract

The vagus nerve contains primary visceral afferents that convey sensory information from cardiovascular, pulmonary, and gastrointestinal tissues to the nucleus tractus solitarii (NTS). The heterogeneity of vagal afferents and their central terminals within the NTS is a common obstacle for evaluating functional groups of afferents. To determine if different anterograde tracers can be used to identify distinct subpopulations of vagal afferents within NTS, we injected cholera toxin B subunit (CTb) and isolectin B4 (IB4) into the vagus nerve. Confocal analyses of medial NTS following injections of both CTb and IB4 into the same vagus nerve resulted in labeling of two exclusive populations of fibers. The ultrastructural patterns were also distinct. CTb was found in both myelinated and unmyelinated vagal axons and terminals in medial NTS, while IB4 was found only in unmyelinated afferents. Both tracers were observed in terminals with asymmetric synapses, suggesting excitatory transmission. Since glutamate is thought to be the neurotransmitter at this first primary afferent synapse in NTS, we determined if vesicular glutamate transporters (VGLUTs) were differentially distributed among the two distinct populations of vagal afferents. Anterograde tracing from the vagus with CTb or IB4 was combined with immunohistochemistry for VGLUT1 or VGLUT2 in medial NTS and evaluated with confocal microscopy. CTb-labeled afferents contained primarily VGLUT2 (83%), while IB4-labeled afferents had low levels of vesicular transporters, VGLUT1 (5%) or VGLUT2 (21%). These findings suggest the possibility that glutamate release from unmyelinated vagal afferents may be regulated by a distinct, non-VGLUT, mechanism.

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

“…1A). As we have shown previously (Hermes et al , 2014), CTb-ir vagal afferents were scattered throughout the subnuclei of subpostremal NTS, with the most dense localization in more lateral subregions (Fig.1B). The overall distribution of CTb labeling was distinct from that of IB4 labeling within NTS with an overlapping distribution observed in the dorsomedial and medial subnucleus.…”

“…5). Previously we found that CTb was mostly associated with myelinated afferents, but the tracer was also detected in some unmyelinated afferents that were distinct from IB4 stained afferents (Hermes et al , 2014). The present findings suggest that the P2X3 distribution within vagal afferents is not strictly related to fiber type.…”

“…We previously showed by electron microscopy that IB4 is only detected in unmyelinated axons following vagal nerve applications (Hermes et al , 2014), thus this tract tracer is effective at detecting C-fiber afferents. TRPV1 in peripheral nociceptors requires high (42°C) temperatures to reach threshold for activation and elicit perceptions of pain (Caterina et al , 1999).…”

“…The antibodies directed against the tract tracers IB4 (Vector, Cat: AS-2104) and CTb (List labs, Cat: 703), have been previously utilized by our lab, as well as others (Aicher et al , 2013; Aicher and Reis, 1997; Borges and Sidman, 1982; Hermes et al , 2014; Llewellyn-Smith et al , 1990; Stucky and Lewin, 1999). The antibodies directed against the channels P2X3 (Neuromics, Cat: RA10109) and TRPV1 (Chemicon AB5566) have also been utilized in a number of prior studies (Amadesi et al , 2009; Guo et al , 1999; Hegarty et al , 2014; Saeed and Ribeiro-da-Silva, 2012).…”

“…Our previous studies indicate the tract tracer isolectin B4 (IB4) is only associated with unmyelinated vagal afferents (Hermes et al , 2014), allowing us to specifically mark distinct groups of fibers based upon their degree of myelination. Cholera Toxin B subunit (CTb) preferentially labels fibers with myelinated axons, but also detects a small population of unmyelinated afferents (Corbett et al , 2005).…”