Wayne L. Silver

Solitary chemoreceptor cells in the nasal cavity serve as sentinels of respiration

Inhalation of irritating substances leads to activation of the trigeminal nerve, triggering protective reflexes that include apnea or sneezing. Receptors for trigeminal irritants are generally assumed to be located exclusively on free nerve endings within the nasal epithelium, requiring that trigeminal irritants diffuse through the junctional barrier at the epithelial surface to activate receptors. We find, in both rats and mice, an extensive population of chemosensory cells that reach the surface of the nasal epithelium and form synaptic contacts with trigeminal afferent nerve fibers. These chemosensory cells express T2R “bitter-taste” receptors and α-gustducin, a G protein involved in chemosensory transduction. Functional studies indicate that bitter substances applied to the nasal epithelium activate the trigeminal nerve and evoke changes in respiratory rate. By extending to the surface of the nasal epithelium, these chemosensory cells serve to expand the repertoire of compounds that can activate trigeminal protective reflexes. The trigeminal chemoreceptor cells are likely to be remnants of the phylogenetically ancient population of solitary chemoreceptor cells found in the epithelium of all anamniote aquatic vertebrates.

Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals

The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl–homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca2+. Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either Gα-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl–homoserine lactones serve as quorum-sensing molecules for Gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms.

Trigeminal collaterals in the nasal epithelium and olfactory bulb: A potential route for direct modulation of olfactory information by trigeminal stimuli

The nasal epithelium is richly invested with peptidergic (substance P and calcitonin gene‐related peptide [CGRP]) trigeminal polymodal nociceptors, which respond to numerous odorants as well as irritants. Peptidergic trigeminal sensory fibers also enter the glomerular layer of the olfactory bulb. To test whether the trigeminal fibers in the olfactory bulb are collaterals of the epithelial trigeminal fibers, we utilized dual retrograde labeling techniques in rats to identify the trigeminal ganglion cells innervating each of these territories. Nuclear Yellow was injected into the dorsal nasal epithelium, and True Blue was injected into the olfactory bulb of the same side. Following a survival period of 3–7 days, the trigeminal ganglion contained double‐labeled, small (11.8 × 8.0 μm), ellipsoid ganglion cells within the ethmoid nerve region of the ganglion. Tracer injections into the spinal trigeminal complex established that these branched trigeminal ganglion cells also extended an axon into the brainstem. These results indicate that some trigeminal ganglion cells with sensory endings in the nasal epithelium also have branches reaching directly into both the olfactory bulb and the spinal trigeminal complex. These trigeminal ganglion cells are unique among primary sensory neurons in having two branches entering the central nervous system at widely distant points. Furthermore, the collateral innervation of the epithelium and bulb may provide an avenue whereby nasal irritants could affect processing of coincident olfactory stimuli. J. Comp. Neurol. 444:221–226, 2002. Erratum: J. Comp. Neurol. 2002;448(4):423.© 2002 Wiley‐Liss, Inc.

A TRPA1-dependent mechanism for the pungent sensation of weak acids

Acetic acid produces an irritating sensation that can be attributed to activation of nociceptors within the trigeminal ganglion that innervate the nasal or oral cavities. These sensory neurons sense a diverse array of noxious agents in the environment, allowing animals to actively avoid tissue damage. Although receptor mechanisms have been identified for many noxious chemicals, the mechanisms by which animals detect weak acids, such as acetic acid, are less well understood. Weak acids are only partially dissociated at neutral pH and, as such, some can cross the cell membrane, acidifying the cell cytosol. The nociceptor ion channel TRPA1 is activated by CO2, through gating of the channel by intracellular protons, making it a candidate to more generally mediate sensory responses to weak acids. To test this possibility, we measured responses to weak acids from heterologously expressed TRPA1 channels and trigeminal neurons with patch clamp recording and Ca2+ microfluorometry. Our results show that heterologously expressed TRPA1 currents can be induced by a series of weak organic acids, including acetic, propionic, formic, and lactic acid, but not by strong acids. Notably, the degree of channel activation was predicted by the degree of intracellular acidification produced by each acid, suggesting that intracellular protons are the proximate stimulus that gates the channel. Responses to weak acids produced a Ca2+-independent inactivation that precluded further activation by weak acids or reactive chemicals, whereas preactivation by reactive electrophiles sensitized TRPA1 channels to weak acids. Importantly, responses of trigeminal neurons to weak acids were highly overrepresented in the subpopulation of TRPA1-expressing neurons and were severely reduced in neurons from TRPA1 knockout mice. We conclude that TRPA1 is a general sensor for weak acids that produce intracellular acidification and suggest that it functions within the pain pathway to mediate sensitivity to cellular acidosis.

The effects of neonatal capsaicin administration on trigeminal nerve chemoreceptors in the rat nasal cavity

Trigeminal nerve fibers in the nasal cavity respond to a variety of volatile chemical stimuli. Some of these trigeminal nerve fibers have been suggested to be capsaicin-sensitive and thus belong to a class of pain receptor rather than constituting a separate class of chemoreceptor. Our current results confirm this suggestion. Trigeminal nerve responses to volatile chemical stimuli were eliminated in rats which were injected with capsaicin on the second day of life. Animals whose nerves were unresponsive to chemical stimuli also exhibited a loss of intraepithelial peptide-immunoreactive fibers in their nasal cavities. The results of this study suggest that trigeminal nerve fibers in the nasal cavity which respond to chemical stimuli may be polymodal nociceptors which contain substance P, calcitonin gene-related peptide, or perhaps other neuropeptides.

Nasal trigeminal chemoreception: responses to n-aliphatic alcohols

Odorant molecules can stimulate nasal trigeminal receptors, but the properties of such molecules which make them effective stimuli are largely unknown. In the present study, we obtained integrated multiunit responses from the ethmoid branch of the rat trigeminal nerve to a homologous series of aliphatic alcohols. Our aim was to determine whether lipid solubility might correlate with stimulus efficacy. Response thresholds (ranging from 3000 ppm for methanol to 3 ppm for octanol) decreased with increasing carbon chain length, suggesting that lipid solubility is important for stimulus effectiveness. One plausible explanation for the importance of lipophilicity is that the more lipid soluble a substance, the more easily it can penetrate epithelial layers to reach chemoreceptive trigeminal nerve endings. Since all stimuli at vapor saturation elicited responses within 0.5 s, and because diffusion of stimulus molecules through epithelium is slow, we speculate that trigeminal nerve endings lie closer to the epithelial surface than previously thought.

The anatomical and electrophysiological basis of peripheral nasal trigeminal chemoreception.

The trigeminal nerve (TN) provides sensory information from the eyes, nose, and mouth. A subset of trigeminal nerve fibers, particularly those containing the neuropeptides substance P and calcitonin gene‐related peptide (CGRP), responds to chemical irritants in the environment. Axons in the ethmoid and nasopalatine branches of the trigeminal nerve innervate the nasal mucosa where they ramify repeatedly. TN endings extend close to the nasal epithelial surface stopping at the line of tight junctions only a few micrometers from the surface. A single ethmoid nerve axon may send branches to the nasal mucosa, olfactory bulb, and the spinal trigeminal complex. Traditionally, irritants are thought to stimulate free TN endings in the nasal epithelium. Recently, however, solitary chemoreceptor cells (SCCs) have been found scattered throughout the nasal cavity. The SCCs are contacted by TN fibers and may express T2R ‘‘bitter‐taste’’ receptors alpha‐gustducin, and TRPM5. Peripheral trigeminal electrophysiological recordings in response to irritants have been obtained from the mucosa (negative mucosal potential, NMP) and the nerve to analyze characteristics of trigeminal stimuli. Responses to a wide variety of irritants have been recorded from the ethmoid nerve. In general, the more lipid soluble the compound, the lower the threshold. Nerve recordings have also suggested several mechanisms by which irritants elicit responses. Bitter substances elicit responses from the ethmoid nerve and cause a change in respiration indicating stimulation via SCCs. SCCs themselves respond to chemical stimuli and may be contributing to the detection of nasal irritants.

Olfactory and trigeminal responses to nicotine

Olfactory and trigeminal sensitivities to vapor‐phase nicotine were assessed by using psychophysical studies with normal and anosmic human subjects and using electrophysiological studies with rats and pigeons. This work showed that 1) psychophysical estimates of sensitivity are approximately tenfold higher (i.e., lower thresholds) than those based on neural recordings, with both techniques demonstrating greater olfactory than trigeminal sensitivity for nicotine and other compounds; 2) for both chemosensory inputs, sensitivity to nicotine is at least 30‐fold greater than that to several other compounds; 3) human subjects can discriminate qualitatively between the S‐(−) and the R‐(−) stereoisomers of nicotine, although the relative importance of olfactory and trigeminal inputs in this discriminative ability is unclear; and 4) trigeminal nerve responses in rats show similar thresholds for S‐(−)‐ and R‐(+)‐nicotine but show lower suprathreshold responses to the R‐(+) stereoisomer. The olfactory epithelium and trigeminal ganglion exhibit high‐affinity binding of S‐(−)‐nicotine. In addition, reverse transcriptase‐polymerase chain reaction (RT‐PCR) studies have shown that many of the nicotinic acetylcholine receptor (nAChR) subunits found in other parts of the nervous system are present in the olfactory epithelium and bulb and in the trigeminal ganglion. Collectively, these findings suggest that two or more of the types of nAChRs identified in other parts of the nervous system may serve as receptor proteins that bind nicotine‐like odorants or irritants. Investigation of the pharmacology of chemosensory responses to nicotine may help to establish causal links between specific receptor proteins and the perception of odor and irritation. Drug Dev. Res. 38:160–168

Solitary chemoreceptor cell survival is independent of intact trigeminal innervation

Nasal solitary chemoreceptor cells (SCCs) are a population of specialized chemosensory epithelial cells presumed to broaden trigeminal chemoreceptivity in mammals (Finger et al. [ 2003 ] Proc Natl Acad Sci USA 100:8981–8986). SCCs are innervated by peptidergic trigeminal nerve fibers (Finger et al. [ 2003 ]) but it is currently unknown if intact innervation is necessary for SCC development or survival. We tested the dependence of SCCs on innervation by eliminating trigeminal nerve fibers during development with neurogenin‐1 knockout mice, during early postnatal development with capsaicin desensitization, and during adulthood with trigeminal lesioning. Our results demonstrate that elimination of innervation at any of these times does not result in decreased SCC numbers. In conclusion, neither SCC development nor mature cell maintenance is dependent on intact trigeminal innervation. J. Comp. Neurol. 508:62–71, 2008.

A comparison of the discriminatory ability and sensitivity of the trigeminal and olfactory systems to chemical stimuli in the tiger salamander

SummaryTrigeminal receptors can respond to a wide variety of chemical stimuli, but it is unknown whether these receptors mediate discrimination between chemical stimuli matched for equal perceptual intensity. The present electrophysiological and behavioral experiments address this issue using tiger salamanders, Ambystoma tigrinum, and four compounds (amyl acetate, cyclohexanone, butanol, and d-limonene). In addition, the relative sensitivities of the trigeminaland olfactory systems to these compounds are compared. In electrophysiological cross-adaptation experiments (amyl acetate vs cyclohexanone; butanol vs d-limonene), there was complete cross adaptation such that only concentrations above the background (crossa-dapting) stimulus concentration elicited responses, suggesting that chemical stimuli may stimulate trigeminal receptors nonspecifically. In behavioral experiments (amyl acetate vs cyclohexanone; butanol vs d-limonene), only animals with intact olfactory nerves could discriminate between perceptually equivalent concentrations, that is concentrations that elicited the same level of responding. Both electrophysiologically and behaviorally, the trigeminal system exhibited higher thresholds than the olfactory system. We conclude that trigeminal chemoreceptors, at least in salamanders, are unable to discriminate between these two pairs of compounds when matched for equal perceptual intensity, and that trigeminal chemoreceptors are less sensitive than olfactory receptors.