Olfactory sensory neurons (OSNs) in the main olfactory epithelium respond to environmental odorants. Recent studies reveal that these OSNs also respond to semiochemicals such as pheromones and that main olfactory input modulates animal reproduction, but the transduction mechanism for these chemosignals is not fully understood. Previously, we determined that responses to putative pheromones in the main olfactory system were reduced but not eliminated in mice defective for the canonical cAMP transduction pathway, and we suggested, on the basis of pharmacology, an involvement of phospholipase C. In the present study, we find that a downstream signaling component of the phospholipase C pathway, the transient receptor potential channel M5 (TRPM5), is coexpressed with the cyclic nucleotide-gated channel subunit A2 in a subset of mature OSNs. These neurons project axons primarily to the ventral olfactory bulb, where information from urine and other socially relevant signals is processed. We find that these chemosignals activate a subset of glomeruli targeted by TRPM5-expressing OSNs. Our data indicate that TRPM5-expressing OSNs that project axons to glomeruli in the ventral area of the main olfactory bulb are involved in processing of information from semiochemicals.signal transduction ͉ pheromone ͉ stimulated emission depletion (STED) microscopy T he olfactory system perceives not only odorants that convey information on the environment but also semiochemicals (chemicals involved in animal communication, from the Greek semeion for ''sign''), including pheromones (1-4). A key step in transmission of information is activation of the canonical cAMP signaling pathway by binding of odorants to olfactory receptors, resulting in influx of Ca 2ϩ through a cyclic nucleotide-gated (CNG) channel and subsequent depolarization (5, 6). Targeted disruption of elements of the cAMP pathway, including the CNG channel subunit A2 (CNGA2) (7), the G protein G olf (8), or adenylyl cyclase (AC) III (9), result in severe deficit of odorantevoked responses to many odorants, demonstrating the dominant role of the cAMP pathway.Semiochemicals, such as pheromones, social attractants, and major histocompatibility complex (MHC)-related odorants, signal social and sexual status, genetic makeup, and species identity important for the survival of the individual and the species (1, 10-13). Recent studies indicate that both the main olfactory epithelium (MOE) and olfactory bulbs respond to these chemosignals (14, 15). Consistent with these findings, sensory information from the main olfactory bulb projects to areas in the hypothalamus housing neurons that produce gonadotropinreleasing hormone and plays an important role in reproduction (16,17). Further, a class of chemosensory receptors that recognize social amines found in urine have been identified recently in the MOE (18). Thus, it now is clear that the main olfactory system is involved in detection of semiochemicals and that the cAMP signaling pathway plays an important role in signal transduction f...

Inhaled airborne irritants elicit sensory responses in trigeminal nerves innervating the nasal epithelium, leading to protective reflexes. The sensory mechanisms involved in the detection of odorous irritants are poorly understood. We identified a large population of solitary chemosensory cells expressing the transient receptor potential channel M5 (TRPM5) using transgenic mice where the promoter of TRPM5 drives the expression of green fluorescent protein (GFP). Most of these solitary chemosensory cells lie in the anterior nasal cavity. These GFP-labeled solitary chemosensory cells exhibited immunoreactivity for synaptobrevin-2, a vesicleassociated membrane protein important for synaptic transmission. Concomitantly, we found trigeminal nerve fibers apposed closely to the solitary chemosensory cells, indicating potential transmission of sensory information to trigeminal fibers. In addition, stimulation of the nasal cavity with high concentrations (0.5-5 mM) of a variety of odorants elicited event-related potentials (ERPs) in areas rich in TRPM5-expressing solitary chemosensory cells. Furthermore, odorous chemicals and trigeminal stimuli induced changes in intracellular Ca 2ϩ levels in isolated TRPM5-expressing solitary chemosensory cells in a concentration-dependent manner. Together, our data show that the TRPM5-expressing cells respond to a variety of chemicals at high exposure levels typical of irritants and are positioned in the nasal cavity appropriately to monitor inhaled air quality.

The mammalian olfactory epithelium is made up of ciliated olfactory sensory neurons (OSNs), supporting cells, basal cells, and microvillous cells. Previously, we reported that a population of nonneuronal microvillous cells expresses transient receptor potential channel M5 (TRPM5). Using transgenic mice and immunocytochemical labeling, we identify that these cells are cholinergic, expressing the signature markers of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter. This result suggests that acetylcholine (ACh) can be synthesized and released locally to modulate activities of neighboring supporting cells and OSNs. In Ca(2+) imaging experiments, ACh induced increases in intracellular Ca(2+) levels in 78% of isolated supporting cells tested in a concentration-dependent manner. Atropine, a muscarinic ACh receptor (mAChR) antagonist suppressed the ACh responses. In contrast, ACh did not induce or potentiate Ca(2+) increases in OSNs. Instead ACh suppressed the Ca(2+) increases induced by the adenylyl cyclase activator forskolin in some OSNs. Supporting these results, we found differential expression of mAChR subtypes in supporting cells and OSNs using subtype-specific antibodies against M(1) through M(5) mAChRs. Furthermore, we found that various chemicals, bacterial lysate, and cold saline induced Ca(2+) increases in TRPM5/ChAT-expressing microvillous cells. Taken together, our data suggest that TRPM5/ChAT-expressing microvillous cells react to certain chemical or thermal stimuli and release ACh to modulate activities of neighboring supporting cells and OSNs via mAChRs. Our studies reveal an intrinsic and potentially potent mechanism linking external stimulation to cholinergic modulation of activities in the olfactory epithelium.

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It is believed that odor transduction in the mammalian main olfactory system only involves the cAMP-signaling pathway. Here, we report on odor responsiveness in mice with a disrupted cyclic nucleotide-gated (CNG) channel subunit A2. Several odorants, including putative pheromones, can be detected and discriminated by these mice behaviorally. These odors elicit responses in the olfactory epithelium, main olfactory bulb, and olfactory (piriform) cortex of CNGA2 knock-out mice. In addition, responses to odors detected by CNGA2 knock-out mice are relatively insensitive to inhibitors of the cAMP pathway. These results provide strong evidence that cAMP-independent pathways in the main olfactory system of mammals participate in detecting a subset of odors.