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.

Circulating blood glucocorticoid levels are dynamic and responsive to stimuli that impact autonomic function. In the brainstem, vagal afferent terminals release the excitatory neurotransmitter glutamate onto neurons in the nucleus of the solitary tract (NTS). Vagal afferents integrate direct visceral signals and circulating hormones with ongoing NTS activity to control autonomic function and behavior. Here we investigated the effects of corticosterone (CORT) on glutamate signaling in the NTS using patch-clamp electrophysiology on brainstem slices containing the NTS and central afferent terminals from male C57BL/6 mice. We found that CORT rapidly decreased both action-potential evoked and spontaneous glutamate signaling. The effects of CORT were phenocopied by dexamethasone and blocked by mifepristone, consistent with glucocorticoid receptor (GR) mediated signaling. While mRNA for GR was present in both the NTS and vagal afferent neurons, selective intracellular quenching of G-protein signaling in postsynaptic NTS neurons eliminated the effects of CORT. We then investigated the contribution of retrograde endocannabinoid (eCB) signaling, which has been reported to transduce non-genomic GR effects. Pharmacological or genetic elimination of the cannabinoid type 1 (CB1) receptor signaling blocked CORT suppression of glutamate release. Together our results detail a mechanism whereby the NTS integrates endocrine CORT signals with fast neurotransmission to control autonomic reflex pathways.