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Agonists of the mu-opioid receptor (MOR) can modulate the activity of visceral premotor neurons, including cardiac premotor neurons. Neurons in brainstem regions containing these premotor neurons also contain dense concentrations of the MOR1. This study examined the distribution of MOR1 within two populations of visceral premotor neurons: one located in the dorsal motor nucleus of the vagus and the other in the nucleus ambiguus. Visceral premotor neurons contained the retrograde tracer Fluoro-Gold following injections of the tracer into the pericardiac region of the thoracic cavity. MOR1 was localized using immunogold detection of an anti-peptide antibody. Visceral premotor neurons in both regions contained MOR1 at somatic and dendritic sites, although smaller dendrites were less likely to contain the receptor than larger dendrites, suggesting there may be selective trafficking of MOR1 within these neurons. MOR1 labeling in nucleus ambiguus neurons was more likely to be localized to plasma membrane sites, suggesting that ambiguus neurons may be more responsive to opioid ligands than neurons in the dorsal motor nucleus of the vagus. In addition, many of the dendrites of visceral premotor neurons were in direct apposition to other dendrites. MOR1 was often detected at these dendro-dendritic appositions that may be gap junctions. Together these findings indicate that the activity of individual visceral premotor neurons, as well as the coupling between neurons, may be regulated by ligands of the MOR.

Ligands of the mu-opiate receptor (MOR) are known to influence many functions that involve vagal afferent input to the nucleus tractus solitarius (NTS), including cardiopulmonary responses, gastrointestinal activity, and cortical arousal. The current study sought to determine whether a cellular substrate exists for direct modulation of vagal afferents and/or their neuronal targets in the NTS by ligands of the MOR. Anterograde tracing of vagal afferents arising from the nodose ganglion was achieved with biotinylated dextran amine (BDA), and the MOR was detected by using antipeptide MOR antiserum. The medial subdivision of the intermediate NTS was examined by electron microscopy for the presence of peroxidase-labeled, BDA-containing vagal afferents and immunogold MOR labeling. MOR was present in both presynaptic axon terminals and at postsynaptic sites, primarily dendrites. In dendrites, MOR immunogold particles usually were located along extrasynaptic portions of the plasma membrane. Of 173 observed BDA-labeled vagal afferent axon terminals, 33% contained immunogold labeling for MOR within the axon terminal. Many of these BDA-labeled terminals formed asymmetric, excitatory-type synapses with dendrites, some of which contained MOR immunogold labeling. MORs were present in 19% of the dendrites contacted by BDA-labeled terminals but were present rarely in both the vagal afferent and its dendritic target. Together, these results suggest that MOR ligands modulate either the presynaptic release from or the postsynaptic responses to largely separate populations of vagal afferents in the intermediate NTS. These results provide a cellular substrate for direct actions of MOR ligands on primary visceral afferents and their second-order neuronal targets in NTS., (Copyright 2000 Wiley-Liss, Inc.)

N-Methyl-D-aspartate receptors are present in the nodose ganglion, which contains the cell bodies of vagal afferents, and in the nucleus tractus solitarius, where these afferent fibers terminate. This suggests that N-methyl-D-aspartate receptors are located presynaptically on visceral vagal afferents and/or their target neurons in the nucleus tractus solitarius. To test this hypothesis, we combined anterograde transport of biotinylated dextran amine, following injections into the left nodose ganglion, with electron microscopic immunogold labeling of antipeptide antiserum against the R1 subunit of the N-methyl-D-aspartate receptor in the nucleus tractus solitarius of rat brain. Within the medial nucleus tractus solitarius, the N-methyl-D-aspartate receptor R1 immunoreactivity was seen in dendrites (39% of 639 profiles), axons and axon terminals (41%), and a few neuronal perikarya and glia. Many vagal afferent axons and terminals (40% of 468 profiles) contained N-methyl-D-aspartate receptor R1 immunogold labeling. In addition, 42% of the dendrites contacted by vagal afferent terminals (n = 206) contained N-methyl-D-aspartate receptor R1 immunoreactivity. In axons and dendrites, the gold particles were occasionally seen within asymmetric postsynaptic junctions or at non-synaptic sites on the plasma membrane. More commonly, however, N-methyl-D-aspartate receptor R1 labeling was seen on membranes of vesicular cytoplasmic organelles, suggesting that there is abundant N-methyl-D-aspartate receptor protein available for activity-dependent mobilization to the plasmalemma. Since many vagal afferents are glutamatergic, our results implicate N-methyl-D-aspartate receptors in autoregulation of the presynaptic release and postsynaptic responses to glutamate at the level of the first central synapse in the nucleus tractus solitarius.

Expt. 1 showed that electrical stimulation of either the main dorsal or ventral branch of the subdiaphragmatic vagus could produce inhibition of the nociceptive tail-flick reflex in lightly anesthetized rats. The antinociception produced by electrical stimulation of the dorsal subdiaphragmatic vagus was eliminated by resection of the right cervical vagus, but relatively unaffected by resection of the left cervical vagus. The opposite effects for cervical vagal resection were obtained with electrical stimulation of the ventral branch of the subdiaphragmatic vagus. These results indicate that the antinociceptive effects of subdiaphragmatic vagal stimulation are mediated via uncrossed afferents traveling in the cervical vagus to activate an inhibitory spinopetal system. These findings are consistent with the established anatomy of vagal afferents. Expt. 2 showed that degeneration of the dorsal subdiaphragmatic vagus did not alter the threshold intensity of right cervical vagal stimulation necessary to produce inhibition of the tail-flick reflex. These results demonstrate that the antinociceptive effects of cervical vagal stimulation are primarily due to activation of the cardiopulmonary component of the nerve, rather than the subdiaphragmatic component. The second experiment also demonstrated that the subdiaphragmatic branch of the vagus can be selectively degenerated with ricin while leaving the cervical branch intact, even though the cell bodies of both sets of afferents are located within the nodose ganglion. These data are discussed in terms of vagal afferents and their role in the modulation of nociceptive transmission.

Electrical stimulation of afferents of the right cervical vagus inhibited the tail-flick reflex elicited by noxious heat in barbiturate-anesthetized rats. This inhibitory effect was eliminated in rats receiving local anesthetic blockade of either the nucleus tractus solitarii (NTS), the lateral reticular nuclei, the nucleus raphe magnus-medullary reticular formation, or nucleus raphe obscurus regions of the medulla. Similarly, the vasodepressor and bradycardic effects of vagal stimulation were either attenuated or eliminated by local anesthetic blockade of these regions. Microinjection of the non-specific glutamate antagonist gamma-D-glutamylglycine (DGG) into the NTS region also eliminated vagally evoked inhibition of the tail-flick reflex, hypotension, and bradycardia. Conversely, microinjection of glutamate into the NTS region resulted in inhibition of the tail-flick reflex, hypotension, and bradycardia. These findings with DGG and glutamate are consistent with the view that glutamate serves as a neurotransmitter of the primary vagal afferents mediating these antinociceptive and cardiovascular responses. These results are discussed in terms of vagal afferent influences on somatosensory, somatomotor, and cardiovascular function.