Sour Sensing from the Tongue to the Brain.

The ability to sense sour provides an important sensory signal to prevent the ingestion of unripe, spoiled, or fermented foods. Taste and somatosensory receptors in the oral cavity trigger aversive behaviors in response to acid stimuli. Here, we show that the ion channel Otopetrin-1, a proton-selective channel normally involved in the sensation of gravity in the vestibular system, is essential for sour sensing in the taste system. We demonstrate that knockout of Otop1 eliminates acid responses from sour-sensing taste receptor cells (TRCs). In addition, we show that mice engineered to express otopetrin-1 in sweet TRCs have sweet cells that also respond to sour stimuli. Next, we genetically identified the taste ganglion neurons mediating each of the five basic taste qualities and demonstrate that sour taste uses its own dedicated labeled line from TRCs in the tongue to finely tuned taste neurons in the brain to trigger aversive behaviors.

Chemosensory Cell-Derived Acetylcholine Drives Tracheal Mucociliary Clearance in Response to Virulence-Associated Formyl Peptides.

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.

Molecular and Cellular Mechanisms of Salt Taste.

Salt taste, the taste of sodium chloride (NaCl), is mechanistically one of the most complex and puzzling among basic tastes. Sodium has essential functions in the body but causes harm in excess. Thus, animals use salt taste to ingest the right amount of salt, which fluctuates by physiological needs: typically, attraction to low salt concentrations and rejection of high salt. This concentration-valence relationship is universally observed in terrestrial animals, and research has revealed complex peripheral codes for NaCl involving multiple taste pathways of opposing valence. Sodium-dependent and -independent pathways mediate attraction and aversion to NaCl, respectively. Gustatory sensors and cells that transduce NaCl have been uncovered, along with downstream signal transduction and neurotransmission mechanisms. However, much remains unknown. This article reviews classical and recent advances in our understanding of the molecular and cellular mechanisms underlying salt taste in mammals and insects and discusses perspectives on human salt taste.

EGR4 is critical for cell-fate determination and phenotypic maintenance of geniculate ganglion neurons underlying sweet and umami taste.

The sense of taste starts with activation of receptor cells in taste buds by chemical stimuli which then communicate this signal via innervating oral sensory neurons to the CNS. The cell bodies of oral sensory neurons reside in the geniculate ganglion (GG) and nodose/petrosal/jugular ganglion. The geniculate ganglion contains two main neuronal populations: BRN3A+ somatosensory neurons that innervate the pinna and PHOX2B+ sensory neurons that innervate the oral cavity. While much is known about the different taste bud cell subtypes, considerably less is known about the molecular identities of PHOX2B+ sensory subpopulations. In the GG, as many as 12 different subpopulations have been predicted from electrophysiological studies, while transcriptional identities exist for only 3 to 6. Importantly, the cell fate pathways that diversify PHOX2B+ oral sensory neurons into these subpopulations are unknown. The transcription factor EGR4 was identified as being highly expressed in GG neurons. EGR4 deletion causes GG oral sensory neurons to lose their expression of PHOX2B and other oral sensory genes and up-regulate BRN3A. This is followed by a loss of chemosensory innervation of taste buds, a loss of type II taste cells responsive to bitter, sweet, and umami stimuli, and a concomitant increase in type I glial-like taste bud cells. These deficits culminate in a loss of nerve responses to sweet and umami taste qualities. Taken together, we identify a critical role of EGR4 in cell fate specification and maintenance of subpopulations of GG neurons, which in turn maintain the appropriate sweet and umami taste receptor cells.

Understanding the development of oral epithelial organs through single cell transcriptomic analysis.

During craniofacial development, the oral epithelium begins as a morphologically homogeneous tissue that gives rise to locally complex structures, including the teeth, salivary glands and taste buds. How the epithelium is initially patterned and specified to generate diverse cell types remains largely unknown. To elucidate the genetic programs that direct the formation of distinct oral epithelial populations, we mapped the transcriptional landscape of embryonic day 12 mouse mandibular epithelia at single cell resolution. Our analysis identified key transcription factors and gene regulatory networks that define different epithelial cell types. By examining the spatiotemporal patterning process along the oral-aboral axis, our results propose a model in which the dental field is progressively confined to its position by the formation of the aboral epithelium anteriorly and the non-dental oral epithelium posteriorly. Using our data, we also identified Ntrk2 as a proliferation driver in the forming incisor, contributing to its invagination. Together, our results provide a detailed transcriptional atlas of the embryonic mandibular epithelium, and unveil new genetic markers and regulators that are present during the specification of various oral epithelial structures.

Bitter taste receptors in the reproductive system: Function and therapeutic implications.

Type 2 taste receptors (TAS2Rs), traditionally known for their role in bitter taste perception, are present in diverse reproductive tissues of both sexes. This review explores our current understanding of TAS2R functions with a particular focus on reproductive health. In males, TAS2Rs are believed to play potential roles in processes such as sperm chemotaxis and male fertility. Genetic insights from mouse models and human polymorphism studies provide some evidence for their contribution to male infertility. In female reproduction, it is speculated that TAS2Rs influence the ovarian milieu, shaping the functions of granulosa and cumulus cells and their interactions with oocytes. In the uterus, TAS2Rs contribute to uterine relaxation and hold potential as therapeutic targets for preventing preterm birth. In the placenta, they are proposed to function as vigilant sentinels, responding to infection and potentially modulating mechanisms of fetal protection. In the cervix and vagina, their analogous functions to those in other extraoral tissues suggest a potential role in infection defense. In addition, TAS2Rs exhibit altered expression patterns that profoundly affect cancer cell proliferation and apoptosis in reproductive cancers. Notably, TAS2R agonists show promise in inducing apoptosis and overcoming chemoresistance in these malignancies. Despite these advances, challenges remain, including a lack of genetic and functional studies. The application of techniques such as single-cell RNA sequencing and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated endonuclease 9 gene editing could provide deeper insights into TAS2Rs in reproduction, paving the way for novel therapeutic strategies for reproductive disorders.

Effect of salivary gland removal on taste preference in mice.

To evaluate the effect of decreased salivary secretion on taste preference, we investigated taste preference for five basic tastes by a 48 h two-bottle preference test using a mouse model (desalivated mice) that underwent surgical removal of three major salivary glands: the parotid, submandibular, and sublingual glands. In the desalivated mice, the avoidance behaviors for bitter and salty tastes and the attractive behaviors for sweet and umami tastes were significantly decreased. We confirmed that saliva is necessary to maintain normal taste preference. To estimate the cause of the preference changes, we investigated the effects of salivary gland removal on the expression of taste-related molecules in the taste buds. No apparent changes were observed in the expression levels or patterns of taste-related molecules after salivary gland removal. When the protein concentration and composition in the saliva were compared between the control and desalivated mice, the protein concentration decreased and its composition changed after major salivary gland removal. These results suggest that changes in protein concentration and composition in the saliva may be one of the factors responsible for the changes in taste preferences observed in the desalivated mice.

Characteristics of A-type voltage-gated K+ currents expressed on sour-sensing type III taste receptor cells in mice.

Sour taste is detected by type III taste receptor cells that generate membrane depolarization with action potentials in response to HCl applied to the apical membranes. The shape of action potentials in type III cells exhibits larger afterhyperpolarization due to activation of transient A-type voltage-gated K+ currents. Although action potentials play an important role in neurotransmitter release, the electrophysiological features of A-type K+ currents in taste buds remain unclear. Here, we examined the electrophysiological properties of A-type K+ currents in mouse fungiform taste bud cells using in-situ whole-cell patch clamping. Type III cells were identified with SNAP-25 immunoreactivity and/or electrophysiological features of voltage-gated currents. Type III cells expressed A-type K+ currents which were completely inhibited by 10 mM TEA, whereas IP3R3-immunoreactive type II cells did not. The half-maximal activation and steady-state inactivation of A-type K+ currents were 17.9 ± 4.5 (n = 17) and - 11.0 ± 5.7 (n = 17) mV, respectively, which are similar to the features of Kv3.3 and Kv3.4 channels (transient and high voltage-activated K+ channels). The recovery from inactivation was well fitted with a double exponential equation; the fast and slow time constants were 6.4 ± 0.6 ms and 0.76 ± 0.26 s (n = 6), respectively. RT-PCR experiments suggest that Kv3.3 and Kv3.4 mRNAs were detected at the taste bud level, but not at single-cell levels. As the phosphorylation of Kv3.3 and Kv3.4 channels generally leads to the modulation of cell excitability, neuromodulator-mediated A-type K+ channel phosphorylation likely affects the signal transduction of taste.

Ultrastructural localization of calcium homeostasis modulator 1 in mouse taste buds.

Taste receptor cells are morphologically classified as types II and III. Type II cells form a unique type of synapses referred to as channel synapses where calcium homeostasis modulator 1 (CALHM1) together with CALHM3 forms voltage-gated channels that release the neurotransmitter, adenosine triphosphate (ATP). To validate the proposed structural model of channel synapses, the ultrastructural localization of CALHM1 in type II cells of both fungiform and circumvallate taste buds was examined. A monoclonal antibody against CALHM1 was developed and its localization was evaluated via immunofluorescence and immunoelectron microscopy using the immunogold-silver labeling technique. CALHM1 was detected as puncta using immunofluorescence and along the presynaptic membrane of channel synapses facing atypical mitochondria, which provide ATP, by immunoelectron microscopy. In addition, it was detected along the plasma membrane lined by subsurface cisternae at sites apposed to afferent nerve fibers. Our results support the validity of a previously proposed structural model for channel synapses and provide insights into the function of subsurface cisternae whose function in taste receptor cells is unknown. We also examined the localization of CALHM1 in hybrid synapses of type III cells, which are conventional chemical synapses accompanied by mitochondria similar to atypical mitochondria of channel synapses. CALHM1 was not detected in the six hybrid synapses examined using immunoelectron microscopy. We further performed double immunolabeling for CALHM1 and Bassoon, which is detected as puncta corresponding to conventional vesicular synapses in type III cells. Our observations suggest that at least some, and probably most, hybrid synapses are not accompanied by CALHM1.

Requirement for an Otopetrin-like protein for acid taste in Drosophila.

Receptors for bitter, sugar, and other tastes have been identified in the fruit fly Drosophila melanogaster, while a broadly tuned receptor for the taste of acid has been elusive. Previous work showed that such a receptor was unlikely to be encoded by a gene within one of the two major families of taste receptors in Drosophila, the "gustatory receptors" and "ionotropic receptors." Here, to identify the acid taste receptor, we tested the contributions of genes encoding proteins distantly related to the mammalian Otopertrin1 (OTOP1) proton channel that functions as a sour receptor in mice. RNA interference (RNAi) knockdown or mutation by CRISPR/Cas9 of one of the genes, Otopetrin-Like A (OtopLA), but not of the others (OtopLB or OtopLC) severely impaired the behavioral rejection to a sweet solution laced with high levels of HCl or carboxylic acids and greatly reduced acid-induced action potentials measured from taste hairs. An isoform of OtopLA that we isolated from the proboscis was sufficient to restore behavioral sensitivity and acid-induced action potential firing in OtopLA mutant flies. At lower concentrations, HCl was attractive to the flies, and this attraction was abolished in the OtopLA mutant. Cell type-specific rescue experiments showed that OtopLA functions in distinct subsets of gustatory receptor neurons for repulsion and attraction to high and low levels of protons, respectively. This work highlights a functional conservation of a sensory receptor in flies and mammals and shows that the same receptor can function in both appetitive and repulsive behaviors.

Effects of temperature on action potentials and ion conductances in type II taste-bud cells.

Temperature strongly influences the intensity of taste, but it remains understudied despite its physiological, hedonic, and commercial implications. The relative roles of the peripheral gustatory and somatosensory systems innervating the oral cavity in mediating thermal effects on taste sensation and perception are poorly understood. Type II taste-bud cells, responsible for sensing sweet, bitter umami, and appetitive NaCl, release neurotransmitters to gustatory neurons by the generation of action potentials, but the effects of temperature on action potentials and the underlying voltage-gated conductances are unknown. Here, we used patch-clamp electrophysiology to explore the effects of temperature on acutely isolated type II taste-bud cell electrical excitability and whole cell conductances. Our data reveal that temperature strongly affects action potential generation, properties, and frequency and suggest that thermal sensitivities of underlying voltage-gated Na+ and K+ channel conductances provide a mechanism for how and whether voltage-gated Na+ and K+ channels in the peripheral gustatory system contribute to the influence of temperature on taste sensitivity and perception.NEW & NOTEWORTHY The temperature of food affects how it tastes. Nevertheless, the mechanisms involved are not well understood, particularly whether the physiology of taste-bud cells in the mouth is involved. Here we show that the electrical activity of type II taste-bud cells that sense sweet, bitter, and umami substances is strongly influenced by temperature. These results suggest a mechanism for the influence of temperature on the intensity of taste perception that resides in taste buds themselves.

Taste of Fat: A Sixth Taste Modality?

An attraction for palatable foods rich in lipids is shared by rodents and humans. Over the last decade, the mechanisms responsible for this specific eating behavior have been actively studied, and compelling evidence implicates a taste component in the orosensory detection of dietary lipids [i.e., long-chain fatty acids (LCFA)], in addition to textural, olfactory, and postingestive cues. The interactions between LCFA and specific receptors in taste bud cells (TBC) elicit physiological changes that affect both food intake and digestive functions. After a short overview of the gustatory pathway, this review brings together the key findings consistent with the existence of a sixth taste modality devoted to the perception of lipids. The main steps leading to this new paradigm (i.e., chemoreception of LCFA in TBC, cell signaling cascade, transfer of lipid signals throughout the gustatory nervous pathway, and their physiological consequences) will be critically analyzed. The limitations to this concept will also be discussed in the light of our current knowledge of the sense of taste. Finally, we will analyze the recent literature on obesity-related dysfunctions in the orosensory detection of lipids ("fatty" taste?), in relation to the overconsumption of fat-rich foods and the associated health risks.

Synergism, Bifunctionality, and the Evolution of a Gradual Sensory Trade-off in Hummingbird Taste Receptors.

Sensory receptor evolution can imply trade-offs between ligands, but the extent to which such trade-offs occur and the underlying processes shaping their evolution is not well understood. For example, hummingbirds have repurposed their ancestral savory receptor (T1R1-T1R3) to detect sugars, but the impact of this sensory shift on amino acid perception is unclear. Here, we use functional and behavioral approaches to show that the hummingbird T1R1-T1R3 acts as a bifunctional receptor responsive to both sugars and amino acids. Our comparative analyses reveal substantial functional diversity across the hummingbird radiation and suggest an evolutionary timeline for T1R1-T1R3 retuning. Finally, we identify a novel form of synergism between sugars and amino acids in vertebrate taste receptors. This work uncovers an unexplored axis of sensory diversity, suggesting new ways in which nectar chemistry and pollinator preferences can coevolve.

Expression of taste sentinels, T1R, T2R, and PLCß2, on the passageway for olfactory signals in zebrafish.

The senses of taste and smell detect overlapping sets of chemical compounds in fish, e.g. amino acids are detected by both senses. However, so far taste and smell organs appeared morphologically to be very distinct, with a specialized olfactory epithelium for detection of odors and taste buds located in the oral cavity and lip for detection of tastants. Here, we report dense clusters of cells expressing T1R and T2R receptors as well as their signal transduction molecule PLCß2 in nostrils of zebrafish, i.e. on the entrance funnel through which odor molecules must pass to be detected by olfactory sensory neurons. Quantitative evaluation shows the density of these chemosensory cells in the nostrils to be as high or higher than that in the established taste organs oral cavity and lower lip. Hydrodynamic flow is maximal at the nostril rim enabling high throughput chemosensation in this organ. Taken together, our results suggest a sentinel function for these chemosensory cells in the nostril.

Gene expression analyses of TAS1R taste receptors relevant to the treatment of cardiometabolic disease.

The sweet taste receptor (STR) is a G protein-coupled receptor (GPCR) responsible for mediating cellular responses to sweet stimuli. Early evidence suggests that elements of the STR signaling system are present beyond the tongue in metabolically active tissues, where it may act as an extraoral glucose sensor. This study aimed to delineate expression of the STR in extraoral tissues using publicly available RNA-sequencing repositories. Gene expression data was mined for all genes implicated in the structure and function of the STR, and control genes including highly expressed metabolic genes in relevant tissues, other GPCRs and effector G proteins with physiological roles in metabolism, and other GPCRs with expression exclusively outside the metabolic tissues. Since the physiological role of the STR in extraoral tissues is likely related to glucose sensing, expression was then examined in diseases related to glucose-sensing impairment such as type 2 diabetes. An aggregate co-expression network was then generated to precisely determine co-expression patterns among the STR genes in these tissues. We found that STR gene expression was negligible in human pancreatic and adipose tissues, and low in intestinal tissue. Genes encoding the STR did not show significant co-expression or connectivity with other functional genes in these tissues. In addition, STR expression was higher in mouse pancreatic and adipose tissues, and equivalent to human in intestinal tissue. Our results suggest that STR expression in mice is not representative of expression in humans, and the receptor is unlikely to be a promising extraoral target in human cardiometabolic disease.

Functional and genomic comparative study of the bitter taste receptor family TAS2R: Insight into the role of human TAS2R5.

Bitterness is perceived in humans by 25 subtypes of bitter taste receptors (hTAS2R) that range from broadly tuned to more narrowly tuned receptors. hTAS2R5 is one of the most narrowly tuned bitter taste receptors in humans. In this study, we review the literature on this receptor and show there is no consensus about its role. We then compare the possible role of hTAS2R5 with that of the proteins of the TAS2R family in rat, mouse, and pig. A phylogenetic tree of all mammalian TAS2R domain-containing proteins showed that human hTAS2R5 has no ortholog in pig, mouse, or rat genomes. By comparing the agonists that are common to hTAS2R5 and other members of the family, we observed that hTAS2R39 is the receptor that shares most agonists with hTAS2R5. In mouse, some of these agonists activate mTas2r105 and mTas2r144, which are distant paralogs of hTAS2R5. mTas2r144 seems to be the receptor that is most similar to hTAS2R5 because they are both activated by the same agonists and have affinities in the same range of values. Then, we can conclude that hTAS2R5 has a unique functional specificity in humans as it is activated by selective agonists and that its closest functional homolog in mouse is the phylogenetically distant mTas2r144.

Symptomatic subgemmal neurogenous plaque in patients with COVID-19: Is there an association?

BACKGROUND: Subgemmal neurogenous plaques (SNP) are composed of neural structures found in the posterolateral portion of the tongue, rarely biopsied as most of them are asymptomatic or eventually only clinically managed. We aimed to investigate a case series of possible correlation of symptomatic subgemmal neurogenous plaque (SNP) with coronavirus disease 2019 (COVID-19). METHODS: Eleven formalin-fixed paraffin-embedded cases from patients with previous confirmed COVID-19 (by RT-PCR) were retrieved from two pathology files. Histological sections were morphologically studied, and then submitted to immunohistochemical reactions against S-100 and neurofilament proteins, neuron-specific enolase, Glial fibrillary acidic protein (GFAP), synaptophysin, CD56, Ki67, cytokeratins (7, 8-18, 19, 20), nucleocapsid and spike proteins (SARS-CoV-1; and -2) and epithelial membrane antigen (EMA) antibodies. Clinical data were retrieved from the patients' medical files, including the symptoms and the complete history of the progression of the disease. RESULTS: The patients who had COVID-19 included in this study experienced painful lesions in the tongue that corresponded to prominent or altered SNP. Microscopically, neural structures were positive for S-100, GFAP and neurofilament protein. And the cellular proliferative index (by Ki-67) was very low. CONCLUSION: Thus, based on the current results, we hypothesize that symptomatic SNP may be a late manifestation of COVID-19 infection.

Rewiring the taste system.

In mammals, taste buds typically contain 50-100 tightly packed taste-receptor cells (TRCs), representing all five basic qualities: sweet, sour, bitter, salty and umami. Notably, mature taste cells have life spans of only 5-20 days and, consequently, are constantly replenished by differentiation of taste stem cells. Given the importance of establishing and maintaining appropriate connectivity between TRCs and their partner ganglion neurons (that is, ensuring that a labelled line from sweet TRCs connects to sweet neurons, bitter TRCs to bitter neurons, sour to sour, and so on), we examined how new connections are specified to retain fidelity of signal transmission. Here we show that bitter and sweet TRCs provide instructive signals to bitter and sweet target neurons via different guidance molecules (SEMA3A and SEMA7A). We demonstrate that targeted expression of SEMA3A or SEMA7A in different classes of TRCs produces peripheral taste systems with miswired sweet or bitter cells. Indeed, we engineered mice with bitter neurons that now responded to sweet tastants, sweet neurons that responded to bitter or sweet neurons responding to sour stimuli. Together, these results uncover the basic logic of the wiring of the taste system at the periphery, and illustrate how a labelled-line sensory circuit preserves signalling integrity despite rapid and stochastic turnover of receptor cells.

Transient Receptor Potential Ankyrin 1 in Taste Nerve Contributes to the Sense of Sweet Taste in Mice.

Transient receptor potential (TRP) channels play a significant role in taste perception. TRP ankyrin 1 (TRPA1) is present in the afferent sensory neurons and is activated by food-derived ingredients, such as Japanese horseradish, cinnamon, and garlic. The present study aimed to investigate the expression of TRPA1 in taste buds, and determine its functional roles in taste perception using TRPA1-deficient mice. In circumvallate papillae, TRPA1 immunoreactivity colocalised with P2X2 receptor-positive taste nerves but not with type II or III taste cell markers. Behavioural studies showed that TRPA1 deficiency significantly reduced sensitivity to sweet and umami tastes, but not to salty, bitter, and sour tastes, compared to that in wild-type animals. Furthermore, administration of the TRPA1 antagonist HC030031 significantly decreased taste preference to sucrose solution compared to that in the vehicle-treated group in the two-bottle preference tests. TRPA1 deficiency did not affect the structure of circumvallate papillae or the expression of type II or III taste cell and taste nerve markers. Adenosine 5'-O-(3-thio)triphosphate evoked inward currents did not differ between P2X2- and P2X2/TRPA1-expressing human embryonic kidney 293T cells. TRPA1-deficient mice had significantly decreased c-fos expression in the nucleus of the solitary tract in the brain stem following sucrose stimulation than wild-type mice. Taken together, the current study suggested that TRPA1 in the taste nerve contributes to the sense of sweet taste in mice.

A subset of broadly responsive Type III taste cells contribute to the detection of bitter, sweet and umami stimuli.

Taste receptor cells use multiple signaling pathways to detect chemicals in potential food items. These cells are functionally grouped into different types: Type I cells act as support cells and have glial-like properties; Type II cells detect bitter, sweet, and umami taste stimuli; and Type III cells detect sour and salty stimuli. We have identified a new population of taste cells that are broadly tuned to multiple taste stimuli including bitter, sweet, sour, and umami. The goal of this study was to characterize these broadly responsive (BR) taste cells. We used an IP3R3-KO mouse (does not release calcium (Ca2+) from internal stores in Type II cells when stimulated with bitter, sweet, or umami stimuli) to characterize the BR cells without any potentially confounding input from Type II cells. Using live cell Ca2+ imaging in isolated taste cells from the IP3R3-KO mouse, we found that BR cells are a subset of Type III cells that respond to sour stimuli but also use a PLCß signaling pathway to respond to bitter, sweet, and umami stimuli. Unlike Type II cells, individual BR cells are broadly tuned and respond to multiple stimuli across different taste modalities. Live cell imaging in a PLCß3-KO mouse confirmed that BR cells use this signaling pathway to respond to bitter, sweet, and umami stimuli. Short term behavioral assays revealed that BR cells make significant contributions to taste driven behaviors and found that loss of either PLCß3 in BR cells or IP3R3 in Type II cells caused similar behavioral deficits to bitter, sweet, and umami stimuli. Analysis of c-Fos activity in the nucleus of the solitary tract (NTS) also demonstrated that functional Type II and BR cells are required for normal stimulus induced expression.