Chemical receptors of the arytenoid: A comparison of human and mouse.

OBJECTIVES/HYPOTHESIS: The larynx is a highly responsive organ exposed to mechanical, thermal, and chemical stimuli. Chemicals elicit responses both in intraepithelial nerve fibers and in specialized chemosensory cells, including scattered solitary cells as well as taste cells organized into taste buds. Activation of both chemosensory cells and taste buds in the larynx elicit cough, swallow, or apnea with exposure to sour or bitter substances, and even by water or sweet-tasting chemicals. In an effort to begin understanding their function, we sought to compare the distribution, density, and types of chemosensory cells and chemoresponsive nerve fibers in laryngeal epithelium of humans and mice. STUDY DESIGN: Animal and human laboratory analysis. METHODS: Using immunohistochemistry, we identified taste cells and polymodal nociceptive nerve fibers in the arytenoid area of the laryngeal epithelium of the following: 1) infants undergoing supraglottoplasty for laryngomalacia, and 2) a cadaveric specimen procured from a 34-year-old donor. We then compared these findings to both preweanling and mature mouse tissue. RESULTS: Arytenoid tissue from both human and mouse contained many taste buds containing type II taste cells-bitter, sweet, or umami sensing-which were innervated by nerve fibers expressing P2X3 type adenosine triphosphate receptors. Type III cells (acid responsive) were also present, but they were fewer in human tissue than in equivalent tissue from mice. In both species, the epithelium was densely innervated by free nerve endings. CONCLUSIONS: Our findings suggest that from a standpoint of chemosensation, human and mouse larynges are biologically similar. This suggests that a murine model can be used effectively in laryngeal chemosensory research. LEVEL OF EVIDENCE: NA Laryngoscope, 130:423-430, 2020.

Hydrogen sulfide activates the carotid body chemoreceptors in cat, rabbit and rat ex vivo preparations.

We and others previously reported experimental evidence suggesting an important role for hydrogen sulfide (H2S) in oxygen sensing in murine carotid body chemoreceptors. More recent data implicated abnormal H2S-mediated chemoreceptor signaling in pathological conditions such as chronic heart failure and hypertension. However, the idea of H2S as a mediator of oxygen-sensing in chemoreceptors has been challenged. In particular, it was shown that exogenous H2S inhibited the release of neurotransmitters (ACh and ATP) from the cat carotid body, raising the possibility that there exists significant species difference in H2S-mediated signaling in chemoreceptors. This study was designed specifically to determine the effect of H2S on chemoreceptors in different species. We conducted multiunit extracellular recordings of the sinus nerve in the ex vivo carotid body preparation taken from the rat, the cat and the rabbit. As observed in the mouse carotid body, H2S donors (NaHS or Na2S) evoked qualitatively similar excitatory responses of the afferent sinus nerves of the species studied here. The excitatory effects of the H2S donors were concentration-dependent and reversible. The sinus nerve responses to H2S donors were prevented by blockade of the transmission between type I cells and the afferent terminals, as was the response to hypoxia. These results demonstrate that exogenous H2S exerts qualitatively similar excitatory effects on chemoreceptor afferents of different species. The role of endogenous H2S-mediated signaling in carotid body function in different species awaits further investigation.

Trpc2-expressing sensory neurons in the main olfactory epithelium of the mouse.

The mouse olfactory system contains two distinct chemosensory epithelia, the main olfactory epithelium (MOE) and the vomeronasal epithelium (VNE). Their sensory neurons express odorant receptor genes and vomeronasal receptor genes, respectively, and differ fundamentally in their signal transduction pathways. Genes required for chemosensory transduction are the cyclic nucleotide-gated channel subunit Cnga2 and the transient receptor potential cation channel Trpc2, respectively. Here, we document two previously unrecognized types of Trpc2+ neurons in the MOE of mice of various ages, including adults. These cell types express Cnga2 and can be distinguished by expression of adenylate cyclase Adcy3 (positive: type A; negative: type B). A third of MOE neurons that express the odorant receptor genes Olfr68/Olfr69 coexpress Trpc2 and are type A cells. In Trpc2-IRES-taulacZ gene-targeted mice, some labeled axons coalesce into glomeruli in the main olfactory bulb. Our findings have implications for the conventional VNE-centric interpretation of the behavioral phenotypes of Trpc2 knockout mice.

Distribution and innervation of putative arterial chemoreceptors in the bullfrog (Rana catesbeiana).

Peripheral arterial chemoreceptors have been located previously in the carotid labyrinth, the aortic arch, and the pulmocutaneous artery of frogs. In the present study we used cholera toxin B neuronal tract tracing and immunohistochemical markers for cholinergic cells (vesicular acetylcholine transporter [VAChT]), tyrosine hydroxylase (TH), and serotonin (5HT) to identify putative O2-sensing cells in Rana catesbeiana. We found potential O2-sensing cells in all three vascular areas innervated by branches of the vagus nerve, whereas only cells in the carotid labyrinth were innervated by the glossopharyngeal nerve. Cells containing either 5HT or TH were found in all three sites, whereas cells containing both neurotransmitters were found only in the carotid labyrinth. Cell bodies containing VAChT were not found at any site. The morphology and innervation of putative O2-sensing cells were similar to those of glomus cells found in other vertebrates. The presence of 5HT- and TH-immunoreactive cells in the aorta, pulmocutaneous artery, and carotid labyrinth appears to reflect a phylogenetic transition between the major neurotransmitter seen in the putative O2-sensing cells of fish (5HT) and those found in the glomus cells of mammals (acetylcholine, adenosine, and catecholamines).

Synaptic communication and signal processing among sensory cells in taste buds.

Taste buds (sensory structures embedded in oral epithelium) show a remarkable diversity of transmitters synthesized and secreted locally. The known transmitters accumulate in a cell type selective manner, with 5-HT and noradrenaline being limited to presynaptic cells, GABA being synthesized in both presynaptic and glial-like cells, and acetylcholine and ATP used for signalling by receptor cells. Each of these transmitters participates in local negative or positive feedback circuits that target particular cell types. Overall, the role of ATP is the best elucidated. ATP serves as a principal afferent transmitter, and also is the key trigger for autocrine positive feedback and paracrine circuits that result in potentiation (via adenosine) or inhibition (via GABA or 5-HT). While many of the cellular receptors and mechanisms for these circuits are known, their impact on sensory detection and perception remains to be elaborated in most instances. This brief review examines what is known, and some of the open questions and controversies surrounding the transmitters and circuits of the taste periphery.

Phox2b-expressing retrotrapezoid neurons are intrinsically responsive to H+ and CO2.

Central respiratory chemoreceptors sense changes in CO2/H(+) and initiate the adjustments to ventilation required to preserve brain and tissue pH. The cellular nature of the sensors (neurons and/or glia) and their CNS location are not conclusively established but the glutamatergic, Phox2b-expressing neurons located in the retrotrapezoid nucleus (RTN) are strong candidates. However, a direct demonstration that RTN neurons are intrinsically sensitive to CO2/H(+), required for designation as a chemosensor, has been lacking. To address this, we tested the pH sensitivity of RTN neurons that were acutely dissociated from two lines of Phox2b-GFP BAC transgenic mice. All GFP-labeled cells assayed by reverse transcriptase-PCR (n = 40) were Phox2b+, VGlut2+, TH-, and ChAT-, the neurochemical phenotype previously defined for chemosensitive RTN neurons in vivo. We found that most dissociated RTN neurons from both lines of mice were CO2/H(+)-sensitive (∼79%), with discharge increasing during acidification and decreasing during alkalization. The pH-sensitive cells could be grouped into two populations characterized by similar pH sensitivity but different basal firing rates, as previously observed in recordings from GFP-labeled RTN neurons in slice preparations. In conclusion, these data indicate that RTN neurons are inherently pH-sensitive, as expected for a respiratory chemoreceptor.

Central chemoreceptor modulation of breathing via multipath tuning in medullary ventrolateral respiratory column circuits.

Ventrolateral respiratory column (VRC) circuits that modulate breathing in response to changes in central chemoreceptor drive are incompletely understood. We employed multielectrode arrays and spike train correlation methods to test predictions of the hypothesis that pre-Bötzinger complex (pre-BötC) and retrotrapezoid nucleus/parafacial (RTN-pF) circuits cooperate in chemoreceptor-evoked tuning of ventral respiratory group (VRG) inspiratory neurons. Central chemoreceptors were selectively stimulated by injections of CO(2)-saturated saline into the vertebral artery in seven decerebrate, vagotomized, neuromuscularly blocked, and artificially ventilated cats. Among sampled neurons in the Bötzinger complex (BötC)-to-VRG region, 70% (161 of 231) had a significant change in firing rate after chemoreceptor stimulation, as did 70% (101 of 144) of the RTN-pF neurons. Other responsive neurons (24 BötC-VRG; 11 RTN-pF) had a change in the depth of respiratory modulation without a significant change in average firing rate. Seventy BötC-VRG chemoresponsive neurons triggered 189 offset-feature correlograms (96 peaks; 93 troughs) with at least one responsive BötC-VRG cell. Functional input from at least one RTN-pF cell could be inferred for 45 BötC-VRG neurons (19%). Eleven RTN-pF cells were correlated with more than one BötC-VRG target neuron, providing evidence for divergent connectivity. Thirty-seven RTN-pF neurons, 24 of which were chemoresponsive, were correlated with at least one chemoresponsive BötC-VRG neuron. Correlation linkage maps and spike-triggered averages of phrenic nerve signals suggest transmission of chemoreceptor drive via a multipath network architecture: RTN-pF modulation of pre-BötC-VRG rostral-to-caudal excitatory inspiratory neuron chains is tuned by feedforward and recurrent inhibition from other inspiratory neurons and from "tonic" expiratory neurons.

Inhibition of adenylate cyclase attenuates muscarinic Ca²(+) signaling by a PKA-independent mechanism in rat carotid body Type I cells.

Carotid body (CB) Type I cells respond to hypoxia by releasing excitatory and inhibitory neurotransmitters. This mechanism leads to increased firing of the carotid sinus nerve (CSN) which alters breathing to maintain blood gases within the physiological range. Acetylcholine targets both muscarinic and nicotinic receptors in the rat CB, acting postsynaptically on CSN and presynaptically on Type I cells. Muscarinic Ca²(+) signaling is inhibited by the activation of G(i)-coupled receptors including histamine H3 receptors. Here inhibition of adenylate cyclase with SQ22536 mimicked H3 receptor activation. Using Ca²(+) imaging techniques it was observed that inhibition of muscarinic Ca²(+) signaling was independent of protein kinase A (PKA) as PKA inhibitors H89 and KT5720 were without effect on the muscarinic Ca²(+) response. By contrast the Epac (exchange protein activated by cAMP) inhibitor brefeldin A inhibited muscarinic Ca²(+) signaling whereas the Epac activator 8-pCPT-2'-O-Me-cAMP-AM potentiated Ca²(+) signaling. Thus in Type I cells inhibition of adenylate cyclase inhibited muscarinic Ca²(+) signaling via a PKA-independent pathway that may rely upon modulation of Epac.

Neural cell adhesion molecule expression in the human carotid body.

We studied by immunocytochemistry the expression of NCAM in human carotid bodies, sampled at autopsy from 16 adult subjects (mean age +/- SD: 44.3 +/- 3.4 years). No NCAM immunoreactivity was visible in type II cells. An high percentage (78.3 +/- 7.2%) of type I cells showed positive anti-NCAM immunoreaction. Statistically significant differences were not found in anti-NCAM immunostaining of light and dark cells (80.2 +/- 6.2% vs 74.7 +/- 13.4%, P>0.05). The high expression level of NCAM in the carotid body indicates a role in regulating adhesion between type I cells. The ascertained role of NCAM in neural mechanisms of differentiation, survival and cell plasticity suggests a possible involvement in the development/differentiation process of the carotid body and in determining cellular/molecular changes due to chronic hypoxia.