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Circadian regulation of autonomic reflex pathways pairs physiological function with the daily light cycle. The brainstem nucleus of the solitary tract (NTS) is a key candidate for rhythmic control of the autonomic nervous system. Here we investigated circadian regulation of NTS neurotransmission and synaptic throughput using patch-clamp electrophysiology in brainstem slices from mice. We found that spontaneous quantal glutamate release on to NTS neurons showed strong circadian rhythmicity, with the highest rate of release during the light phase and the lowest in the dark, that were sufficient to drive day / night differences in constitutive postsynaptic action potential firing. In contrast, afferent-evoked action potential throughput was enhanced during the dark and diminished in the light. Afferent-driven synchronous release pathways showed a similar decrease in release probability that did not explain the enhanced synaptic throughput during the night. However, analysis of postsynaptic membrane properties revealed diurnal changes in conductance; which, when coupled with the circadian changes in glutamate release pathways, tuned synaptic throughput between the light and dark phases. These coordinated pre- / postsynaptic changes encode nuanced control over synaptic performance and pair NTS action potential firing and vagal throughput with time of day.

Vagal afferent fibers contact neurons in the nucleus of the solitary tract (NTS) and release glutamate via three distinct release pathways: synchronous, asynchronous, and spontaneous. The presence of TRPV1 in vagal afferents is predictive of activity-dependent asynchronous glutamate release along with temperature-sensitive spontaneous vesicle fusion. However, pharmacological blockade or genetic deletion of TRPV1 does not eliminate the asynchronous profile and only attenuates the temperature-dependent spontaneous release at high temperatures (>40°C), indicating additional temperature-sensitive calcium conductance(s) contributing to these release pathways. The transient receptor potential cation channel melastatin subtype 3 (TRPM3) is a calcium-selective channel that functions as a thermosensor (30–37°C) in somatic primary afferent neurons. We predict that TRPM3 is expressed in vagal afferent neurons and contributes to asynchronous and spontaneous glutamate release pathways. We investigated these hypotheses via measurements on cultured nodose neurons and in brainstem slice preparations containing vagal afferent to NTS synaptic contacts. We found histological and genetic evidence that TRPM3 is highly expressed in vagal afferent neurons. The TRPM3-selective agonist, pregnenolone sulfate, rapidly and reversibly activated the majority (∼70%) of nodose neurons; most of which also contained TRPV1. We confirmed the role of TRPM3 with pharmacological blockade and genetic deletion. In the brain, TRPM3 signaling strongly controlled both basal and temperature-driven spontaneous glutamate release. Surprisingly, genetic deletion of TRPM3 did not alter synchronous or asynchronous glutamate release. These results provide convergent evidence that vagal afferents express functional TRPM3 that serves as an additional temperature-sensitive calcium conductance involved in controlling spontaneous glutamate release onto neurons in the NTS. NEW & NOTEWORTHY 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.

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 that 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 nonpsychotropic 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, whereas TRPV1 knockout (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 in C-fiber afferents. To simulate adaptation, resulting from chronic cannabis use, we administered cannabis extract vapor daily for 3 wk. 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. NEW & NOTEWORTHY CBD usage has increased with its legalization. The clinical efficacy of CBD has been demonstrated for conditions including some forms of epilepsy, depression, and anxiety that are also treatable by vagus nerve stimulation. We found CBD exhibited direct excitatory effects on vagal afferent neurons that required TRPA1, were augmented by TRPV1, and attenuated following chronic cannabis vapor exposure. These effects may contribute to vagal mimetic effects of CBD and adaptation after chronic cannabis use.

Circulating blood glucocorticoid levels are dynamic and responsive to stimuli that impact autonomic function. In the brain stem, vagal afferent terminals release the excitatory neurotransmitter glutamate to 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 brain stem 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 signaling, which has been reported to transduce nongenomic GR effects. Pharmacological or genetic elimination of the cannabinoid type 1 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.