SUMMARY TRPV1 receptors feature prominently in nociception of spinal primary afferents but are also expressed in unmyelinated cranial visceral primary afferents linked to homeostatic regulation. Cranial visceral afferents enter the brain at the solitary tract nucleus (NTS) to control the heart, lungs and other vital organs. Here we identify a novel role for central TRPV1 in the activity-dependent facilitation of glutamatergic transmission from solitary tract (ST) afferents. Fast, synchronous ST-NTS transmission from capsaicin sensitive (TRPV1+) and insensitive (TRPV1−) afferents was similar. However, afferent activation triggered long lasting asynchronous glutamate release only from TRPV1+ synapses. Asynchronous release was proportional to synchronous EPSC amplitude, activity, and calcium entry. TRPV1 antagonists and low temperature blocked asynchronous release but not evoked EPSCs. At physiological afferent frequencies, asynchronous release strongly potentiated the duration of postsynaptic spiking. This activity dependent TPRV1-mediated facilitation is a novel form of synaptic plasticity that brings a unique central integrative feature to the CNS and autonomic regulation.

Cranial visceral afferents travel via the solitary tract (ST) to contact neurons within the ST nucleus (NTS)and activate homeostatic reflexes. Hypothalamic projections from the paraventricular nucleus (PVN) release oxytocin (OT) to modulate visceral afferent communication with NTS neurons. However, the cellular mechanisms through which OT acts are poorly understood. Here, we electrophysiologically identified second-order NTS neurons in horizontal brainstem slices by their low-jitter, ST-evoked glutamatergic EPSCs. OT increased the frequency of miniature EPSCs in half of the NTS second-order neurons (13/24) but did not alter event kinetics or amplitudes. These actions were blocked by a selective OT receptor antagonist. OT increased the amplitude of ST-evoked EPSCs with no effect on event kinetics. Variance-mean analysis of ST-evoked EPSCs indicated OT selectively increased the release probability of glutamate from the ST afferent terminals. In OT-sensitive neurons, OT evoked an inward holding current and increased input resistance. The OT-sensitive current reversed at the K ϩ equilibrium potential. In in vivo studies, NTS neurons excited by vagal cardiopulmonary afferents were juxtacellularly labeled with Neurobiotin and sections were stained to show filled neurons and OT-immunoreactive axons. Half of these physiologically characterized neurons (5/10) showed close appositions by OT fibers consistent with synaptic contacts. Electron microscopy of medial NTS found immunoreactive OT within synaptic boutons. Together, these findings suggest that OT released from PVN axons acts on a subset of second-order neurons within medial NTS to enhance visceral afferent transmission via presynaptic and postsynaptic mechanisms.

Central synapses spontaneously release neurotransmitter at low rates. In the brainstem, cranial visceral afferent terminals in caudal solitary tract nucleus (NTS) display pronounced, activity-dependent, asynchronous release of glutamate and this extra release depends on TRPV1 receptors (TRPV1ϩ). Asynchronous release is absent for afferents lacking TRPV1 (TRPV1Ϫ) and resting EPSC frequency was greater in TRPV1ϩ. Here, we studied this basal activity difference by assessing thermal sensitivity of spontaneous and miniature synaptic events in TRPV1ϩ and TRPV1Ϫ second-order NTS neurons. The spontaneous EPSC rate decreased when temperature was decreased, increased steeply between 30 and 42°C only in TRPV1ϩ neurons, and was calcium-dependent. TRPV1-specific antagonist SB366791, but not TTX, strongly attenuated thermal responses. Temperature changes failed to alter EPSC frequency in TRPV1Ϫ neurons. EPSC amplitudes and decay kinetics changed little with temperature. IPSCs in these second-order NTS neurons were unaltered by temperature. Such results suggest that activated, presynaptic TRPV1ϩ receptors trigger continuous resting release of glutamate vesicles at physiological temperatures only in capsaicin-responsive terminals. In mechanically isolated individual neurons harvested from medial NTS, increases in temperature increased the rate of glutamate release only in TRPV1ϩ neurons, whereas IPSC rates were unaffected. Cadmium failed to block thermal increases in glutamate release, suggesting that calcium entry through TRPV1 channels may trigger glutamate release independently of voltage-activated calcium channels. Together, our findings indicate a new form of afferent signaling in which TRPV1 channels within central terminals of peripheral afferents tonically generate glutamate release in NTS at 37°C in the absence of afferent action potentials.

Obesity is associated with consumption of energy-dense diets and development of systemic inflammation. Gut microbiota play a role in energy harvest and inflammation and can influence the change from lean to obese phenotypes. The nucleus of the solitary tract (NTS) is a brain target for gastrointestinal signals modulating satiety and alterations in gut-brain vagal pathway may promote overeating and obesity. Therefore, we tested the hypothesis that high-fat diet-induced changes in gut microbiota alter vagal gut-brain communication associated with increased body fat accumulation. Sprague-Dawley rats consumed a low energy-dense rodent diet (LFD; 3.1 kcal/g) or high energy-dense diet (HFD, 5.24 kcal/g). Minocycline was used to manipulate gut microbiota composition. 16S Sequencing was used to determine microbiota composition. Immunofluorescence against IB4 and Iba1 was used to determine NTS reorganization and microglia activation. Nodose ganglia from LFD rats were isolated and co-cultured with different bacteria strains to determine neurotoxicity. HFD altered gut microbiota with increases in Firmicutes/Bacteriodetes ratio and in pro-inflammatory Proteobacteria proliferation. HFD triggered reorganization of vagal afferents and microglia activation in the NTS, associated with weight gain. Minocycline-treated HFD rats exhibited microbiota profile comparable to LFD animals. Minocycline suppressed HFD-induced reorganization of vagal afferents and microglia activation in the NTS, and reduced body fat accumulation. Proteobacteria isolated from cecum of HFD rats were toxic to vagal afferent neurons in culture. Our findings show that diet-induced shift in gut microbiome may disrupt vagal gut-brain communication resulting in microglia activation and increased body fat accumulation.

To test the hypothesis that leptin can directly activate vagal afferent neurons, we used fluorescence imaging to detect acute changes in cytosolic calcium after leptin application to primary cultures of vagal afferent neurons dissociated from adult rat nodose ganglia. We found that approximately 40% of vagal afferent neurons exposed to leptin (40 ng/ml) responded with rapid and reversible increases in cytosolic calcium. These responses were dependent upon extracellular calcium. As previously reported, about 35% of vagal afferents increase cytosolic calcium in response to the gut-peptide cholecystokinin (CCK). A majority (74%) of neurons that responded to CCK also exhibited increases in cytosolic calcium in response to leptin. In addition, synergistic increases in cytosolic calcium were observed when leptin and CCK were applied in combination. These results demonstrate that leptin acts directly on vagal afferent neurons to trigger acute influxes of extracellular calcium. Our results also suggest cooperation between leptin and CCK in the activation of some vagal afferent neurons. Acute activation of vagal afferents by leptin alone and in combination with CCK may contribute to modulation of visceral reflexes and control of food intake.