In the brainstem nucleus of the solitary tract (NTS), primary vagal afferent neurons express the transient receptor potential vanilloid subfamily member 1 (TRPV1) at their central terminals where it contributes to quantal forms of glutamate release. The endogenous membrane lipid anandamide (AEA) is a putative TRPV1 agonist in the brain, yet the extent to which AEA activation of TRPV1 has a neurophysiological consequence is not well established. We investigated the ability of AEA to activate TRPV1 in vagal afferent neurons in comparison to capsaicin (CAP). Using ratiometric calcium imaging and whole-cell patch clamp recordings we confirmed that AEA excitatory activity requires TRPV1, binds competitively at the CAP binding site, and has low relative affinity. While AEA-induced increases in peak cytosolic calcium were similar to CAP, AEA-induced membrane currents were significantly smaller. Removal of bath calcium increased the AEA current with no change in peak CAP currents revealing a calcium sensitive difference in specific ligand activation of TRPV1. Both CAP- and AEA-activated TRPV1 currents maintained identical reversal potentials, arguing against a major difference in ion selectivity to resolve the AEA differences in signaling. In contrast with CAP, AEA did not alter spontaneous glutamate release at NTS synapses. We conclude: (1) AEA activation of TRPV1 is markedly different from CAP and produces different magnitudes of calcium influx from whole-cell current; and (2) exogenous AEA does not alter spontaneous glutamate release onto NTS neurons. As such, AEA may convey modulatory changes to calcium-dependent processes, but does not directly facilitate glutamate release.

Vagal afferent signaling coordinates autonomic reflex function and informs associated behaviors. Thermosensitive transient receptor potential (TRP) channels detect temperature and nociceptive stimuli in somatosensory afferent neurons, however their role in vagal signaling remains less well understood. We report that the TRPM3 ion channel provides a major thermosensitive point of control over vagal signaling and synaptic transmission. We conclude that TRPM3 translates physiological changes in temperature to neurophysiological outputs and can serve as a cellular integrator in vagal afferent signaling.

Primary vagal afferent neurons express a multitude of thermosensitive ion channels. Within this family of ion channels, the heat-sensitive capsaicin receptor (TRPV1) greatly influences vagal afferent signaling by determining the threshold for action-potential initiation at the peripheral endings, while controlling temperature-sensitive forms of glutamate release at central vagal terminals. Genetic deletion of TRPV1 does not completely eliminate these temperature-dependent effects, suggesting involvement of additional thermosensitive ion channels. The warm-sensitive, calcium-permeable, ion channel TRPV3 is commonly expressed with TRPV1; however, the extent to which TRPV3 is found in vagal afferent neurons is unknown. Here, we begin to characterize the genetic and functional expression of TRPV3 in vagal afferent neurons using molecular biology (RT-PCR and RT-quantitative PCR) in whole nodose and isolated neurons and fluorescent calcium imaging on primary cultures of nodose ganglia neurons. We confirmed low-level TRPV3 expression in vagal afferent neurons and observed direct activation with putative TRPV3 agonists eugenol, ethyl vanillin (EVA), and farnesyl pyrophosphate (FPP). Agonist activation stimulated neurons also containing TRPV1 and was blocked by ruthenium red. FPP sensitivity overlapped with EVA and eugenol but represented the smallest percentage of vagal afferent neurons, and it was the only agonist that did not stimulate neurons from TRPV3(-/-1) mice, suggesting FPP has the highest selectivity. Further, FPP was predictive of enhanced responses to capsaicin, EVA, and eugenol in rats. From our results, we conclude TRPV3 is expressed in a discrete subpopulation of vagal afferent neurons and may contribute to vagal afferent signaling either directly or in combination with TRPV1.

Vagal afferent neurons abundantly express excitatory transient receptor potential (TRP) channels which strongly influence afferent signaling. Cannabinoids have been identified as direct agonists of TRP channels, including TRPA1 and TRPV1, suggesting exogenous cannabinoids may influence vagal signaling via TRP channel activation. The diverse therapeutic effects of electrical vagus nerve stimulation also result from administration of the non-psychotropic cannabinoid cannabidiol (CBD); however, the direct effects of CBD on vagal afferent signaling remain unknown. We investigated actions of CBD on vagal afferent neurons using calcium imaging and electrophysiology. CBD produced strong excitatory effects in neurons expressing TRPA1. CBD responses were prevented by removal of bath calcium, ruthenium red, and the TRPA1 antagonist A967079; but not the TRPV1 antagonist SB366791; suggesting an essential role for TRPA1. These pharmacological experiments were confirmed using genetic knockouts where TRPA1 KO mice lacked CBD responses while TRPV1 KO mice exhibited CBD-induced activation. We also characterized CBD-provoked inward currents at resting potentials in vagal afferents expressing TRPA1 that were absent in TRPA1 KO mice, but persisted in TRPV1 KO mice. CBD also inhibited voltage-activated sodium conductances in A-fiber, but not C-fiber afferents. To simulate adaptation resulting from chronic cannabis use, we administered cannabis extract vapor daily for three weeks. Cannabis exposure reduced the magnitude of CBD responses likely due to a loss of TRPA1 signaling. Together these findings detail a novel excitatory action of CBD at vagal afferent neurons which requires TRPA1 and may contribute to the vagal mimetic effects of CBD and adaptation following chronic cannabis use.

Cholecystokinin (CCK) is a gut-derived peptide that potently promotes satiety and facilitates gastric function in part by activating G protein-coupled CCK1 receptors on primary vagal afferent neurons. CCK signaling is dynamic and rapidly desensitizes, due to decreases in either receptor function and the resulting signal cascade, ion channel effectors, or both. Here we report a decay-time analytical approach using fluorescent calcium imaging that relates peak and steady-state calcium responses in dissociated vagal afferent neurons, enabling discrimination between receptor and ion channel effector functions. We found desensitization of CCK-induced activation was predictable, consistent across cells, and strongly concentration dependent. The decay-time constant (tau) was inversely proportional to CCK concentration, apparently reflecting the extent of receptor activation. To test this possibility, we directly manipulated the ion channel effector(s) with either decreased bath calcium or the broad-spectrum pore blocker ruthenium red. Conductance inhibition diminished the magnitude of the CCK responses without altering decay kinetics, confirming changes in tau reflect changes in receptor function selectively. Next, we investigated the contributions of the PKC and PKA signaling cascades on the magnitude and decay-time constants of CCK calcium responses. While inhibition of either PKC or PKA increased CCK calcium response magnitude, only general PKC inhibition significantly decreased the decay-time constant. These findings suggest that PKC alters CCK receptor signaling dynamics, while PKA alters the ion channel effector of the CCK response. This analytical approach should prove useful in understanding receptor/effector changes underlying acute desensitization of G-protein coupled signaling and provide insight into CCK receptor dynamics.

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