Taste Bud Connectome: Implications for Taste Information Processing.

Taste buds contain multiple cell types, two of which mediate transduction of specific taste qualities: Type III cells transduce sour while Type II cells transduce either sweet, or bitter or umami. In order to discern the degree of interaction between different cell types and specificity of connectivity with the afferent nerve fibers (NFs), we employed serial blockface scanning electron microscopy (sbfSEM) through five circumvallate mouse taste buds. Points of contact between Type II and Type III cells are rare and lack morphologically identifiable synapses, suggesting that interaction between these cell types does not occur via synapses. Of the 127 NFs that make synaptic contacts with taste cells in the sampling volume, ∼70% (n = 91) synapse with only one taste cell while 32 fibers synapse exclusively with multiple Type II cells or multiple Type III cells. Our data do not rule out multimodal fibers innervating Type II cells of separate taste qualities. Notably, four fibers (∼3%) synapse with both Type II and Type III cells, forming both mitochondrial and vesicular synapses on the different cell types. Since Type II and Type III cells transduce different taste qualities, these dual connected fibers are not consistent with a absolute labeled-line encoding system. Further, our data reveal considerable variation in both the number of synapses per cell/nerve pair and the number of innervating NFs per taste cell, both of which likely have consequences for encoding taste quality and concentration. Finally, we identify a subset of Type II cells which may represent an immature stage.SIGNIFICANCE STATEMENT Taste buds, the sensory end organs for the sense of taste, contain multiple types of sensory cells, with each responding to one of the primary tastes: salt, sweet, sour, bitter, and umami. In order to determine the degree of interaction between cell types and specificity of connectivity to afferent nerves, we employed serial blockface electron microscopy (EM) of mouse circumvallate taste buds. We find no synapses between cell types within the taste bud suggesting that any interactions are indirect. While the majority of nerve fibers (NFs) connect to a single type of taste cell, 3.1% of the fibers branch to receive input from taste cells of different specificities. Thus, taste cannot entirely be carried along NFs dedicated to single taste qualities.

Histological and Molecular Characterization of the Inferior Olivary Nucleus and Climbing Fibers in the Goldfish, Carassius auratus.

The cerebellum receives inputs via the climbing fibers originating from the inferior olivary nucleus in the ventral medulla. In mammals, the climbing fibers entwine and terminate onto both major and peripheral branches of dendrites of the Purkinje cells. In this study, the inferior olivary nucleus and climbing fiber in the goldfish were investigated with several histological techniques. By neural tracer application to the hemisphere of the cerebellum, labeled inferior olivary neurons were found in the ventral edge of the contralateral medulla. Kainate stimulated Co + + uptake and gephyrin immunoreactivities were found in inferior olivary neurons, indicating, respectively, that they receive both excitatory (glutamatergic) and inhibitory (GABAergic or glycinergic) inputs. Inferior olivary neurons express vglut2.1 transcripts, suggesting they are glutamatergic. Around 85% of inferior olivary neurons were labeled with anti-calretinin antiserum. Calretinin immunoreactive (ir) climbing fiber terminal-like structures were distributed near the Purkinje cells and in the molecular layer. Double labeling immunofluorescence with anti-calretinin and zebrin II antisera revealed that the calretinin-ir climbing fibers run along and made synaptic-like contacts on the major dendrites of the zebrin II-ir Purkinje cells. In teleost fish, cerebellar efferent neurons, eurydendroid cells, also lie near the Purkinje cells and extend dendrites outward to intermingle with dendrites of the Purkinje cells within the molecular layer. Here we found no contacts between the climbing fiber terminals and the eurydendroid cell dendrites. These results support the idea that Purkinje cells, but not eurydendroid cells, receive strong inputs via the climbing fibers, similar to the mammalian situation.

Sugar causes obesity and metabolic syndrome in mice independently of sweet taste.

Intake of sugars, especially the fructose component, is strongly associated with the development of obesity and metabolic syndrome, but the relative role of taste versus metabolism in driving preference, intake, and metabolic outcome is not fully understood. We aimed to evaluate the preference for sweet substances and the tendency to develop metabolic syndrome in response to these sugars in mice lacking functional taste signaling [P2X2 (P2X purinoreceptor 2)/P2X3 (P2X purinoreceptor 3) double knockout mice (DKO)] and mice unable to metabolize fructose (fructokinase knockout mice). Of interest, our data indicate that despite their inability to taste sweetness, P2X2/3 DKO mice still prefer caloric sugars (including fructose and glucose) to water in long-term testing, although with diminished preference compared with control mice. Despite reduced intake of caloric sugars by P2X2/3 DKO animals, the DKO mice still show increased levels of the sugar-dependent hormone FGF21 (fibroblast growth factor 21) in plasma and liver. Despite lower sugar intake, taste-blind mice develop severe features of metabolic syndrome due to reduced sensitivity to leptin, reduced ability to mobilize and oxidize fats, and increased hepatic de novo lipogenesis. In contrast to P2X2/3 DKO and wild-type mice, fructokinase knockout mice, which cannot metabolize fructose and are protected against fructose-induced metabolic syndrome, demonstrate reduced preference and intake for all fructose-containing sugars tested but not for glucose or artificial sweeteners. Based on these observations, we conclude that sugar can induce metabolic syndrome in mice independently of its sweet properties. Furthermore, our data demonstrate that the metabolism of fructose is necessary for sugar to drive intake and preference in mice.

Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance.

The integrity of taste buds is intimately dependent on an intact gustatory innervation, yet the molecular nature of this dependency is unknown. Here, we show that differentiation of new taste bud cells, but not progenitor proliferation, is interrupted in mice treated with a hedgehog (Hh) pathway inhibitor (HPI), and that gustatory nerves are a source of sonic hedgehog (Shh) for taste bud renewal. Additionally, epithelial taste precursor cells express Shh transiently, and provide a local supply of Hh ligand that supports taste cell renewal. Taste buds are minimally affected when Shh is lost from either tissue source. However, when both the epithelial and neural supply of Shh are removed, taste buds largely disappear. We conclude Shh supplied by taste nerves and local taste epithelium act in concert to support continued taste bud differentiation. However, although neurally derived Shh is in part responsible for the dependence of taste cell renewal on gustatory innervation, neurotrophic support of taste buds likely involves a complex set of factors.

Genetic Deletion of TrpV1 and TrpA1 Does Not Alter Avoidance of or Patterns of Brainstem Activation to Citric Acid in Mice.

Exposure of the oral cavity to acidic solutions evokes not only a sensation of sour, but also of sharp or tangy. Acidic substances potentially stimulate both taste buds and acid-sensitive mucosal free nerve endings. Mice lacking taste function (P2X2/P2X3 double-KO mice) refuse acidic solutions similar to wildtype (WT) mice and intraoral infusion of acidic solutions in these KO animals evokes substantial c-Fos activity within orosensory trigeminal nuclei as well as of the nucleus of the solitary tract (nTS) (Stratford, Thompson, et al. 2017). This residual acid-evoked, non-taste activity includes areas that receive inputs from trigeminal and glossopharyngeal peptidergic (CGRP-containing) nerve fibers that express TrpA1 and TrpV1 both of which are activated by low pH. We compared avoidance responses in WT and TrpA1/V1 double-KO (TRPA1/V1Dbl-/-) mice in brief-access behavioral assay (lickometer) to 1, 3, 10, and 30 mM citric acid, along with 100 µM SC45647 and H2O. Both WT and TRPA1/V1Dbl-/- show similar avoidance, including to higher concentrations of citric acid (10 and 30 mM; pH 2.62 and pH 2.36, respectively), indicating that neither TrpA1 nor TrpV1 is necessary for the acid-avoidance behavior in animals with an intact taste system. Similarly, induction of c-Fos in the nTS and dorsomedial spinal trigeminal nucleus was similar in the WT and TRPA1/V1Dbl-/- animals. Taken together these results suggest non-TrpV1 and non-TrpA1 receptors underlie the residual responses to acids in mice lacking taste function.

A Subset of Olfactory Sensory Neurons Express Forkhead Box J1-Driven eGFP.

Forkhead box protein J1 (FOXJ1), a member of the forkhead family transcription factors, is a transcriptional regulator of motile ciliogenesis. The nasal respiratory epithelium, but not olfactory epithelium, is lined with FOXJ1-expressing multiciliated epithelial cells with motile cilia. In a transgenic mouse where an enhanced green fluorescent protein (eGFP) transgene is driven by the human FOXJ1 promoter, robust eGFP expression is observed not only in the multiciliated cells of the respiratory epithelium but in a distinctive small subset of olfactory sensory neurons in the olfactory epithelium. These eGFP-positive cells lie at the extreme apical part of the neuronal layer and are most numerous in dorsal-medial regions of olfactory epithelium. Interestingly, we observed a corresponding small number of glomeruli in the olfactory bulb wherein eGFP-labeled axons terminate, suggesting that the population of eGFP+ receptor cells expresses a limited number of olfactory receptors. Similarly, a subset of vomeronasal sensory neurons expresses eGFP and is distributed throughout the full height of the vomeronasal sensory epithelium. In keeping with this broad distribution of labeled vomeronasal receptor cells, eGFP-labeled axons terminate in many glomeruli in both anterior and posterior portions of the accessory olfactory bulb. These findings suggest that Foxj1-driven eGFP marks a specific population of olfactory and vomeronasal sensory neurons, although neither receptor cell population possess motile cilia.

Type III Cells in Anterior Taste Fields Are More Immunohistochemically Diverse Than Those of Posterior Taste Fields in Mice.

Activation of Type III cells in mammalian taste buds is implicated in the transduction of acids (sour) and salty stimuli. Several lines of evidence suggest that function of Type III cells in the anterior taste fields may differ from that of Type III cells in posterior taste fields. Underlying anatomy to support this observation is, however, scant. Most existing immunohistochemical data characterizing this cell type focus on circumvallate taste buds in the posterior tongue. Equivalent data from anterior taste fields-fungiform papillae and soft palate-are lacking. Here, we compare Type III cells in four taste fields: fungiform, soft palate, circumvallate, and foliate in terms of reactivity to four canonical markers of Type III cells: polycystic kidney disease 2-like 1 (PKD2L1), synaptosomal associated protein 25 (SNAP25), serotonin (5-HT), and glutamate decarboxylase 67 (GAD67). Our findings indicate that while PKD2L1, 5-HT, and SNAP25 are highly coincident in posterior taste fields, they diverge in anterior taste fields. In particular, a subset of taste cells expresses PKD2L1 without the synaptic markers, and a subset of SNAP25 cells lacks expression of PKD2L1. In posterior taste fields, GAD67-positive cells are a subset of PKD2L1 expressing taste cells, but anterior taste fields also contain a significant population of GAD67-only expressing cells. These differences in expression patterns may underlie the observed functional differences between anterior and posterior taste fields.

Cholinergic neurotransmission links solitary chemosensory cells to nasal inflammation.

Solitary chemosensory cells (SCCs) of the nasal cavity are specialized epithelial chemosensors that respond to irritants through the canonical taste transduction cascade involving Gα-gustducin and transient receptor potential melastatin 5. When stimulated, SCCs trigger peptidergic nociceptive (or pain) nerve fibers, causing an alteration of the respiratory rate indicative of trigeminal activation. Direct chemical excitation of trigeminal pain fibers by capsaicin evokes neurogenic inflammation in the surrounding epithelium. In the current study, we test whether activation of nasal SCCs can trigger similar local inflammatory responses, specifically mast cell degranulation and plasma leakage. The prototypical bitter compound, denatonium, a well-established activator of SCCs, caused significant inflammatory responses in WT mice but not mice with a genetic deletion of elements of the canonical taste transduction cascade, showing that activation of taste signaling components is sufficient to trigger local inflammation. Chemical ablation of peptidergic trigeminal fibers prevented the SCC-induced nasal inflammation, indicating that SCCs evoke inflammation only by neural activity and not by release of local inflammatory mediators. Additionally, blocking nicotinic, but not muscarinic, acetylcholine receptors prevents SCC-mediated neurogenic inflammation for both denatonium and the bacterial signaling molecule 3-oxo-C12-homoserine lactone, showing the necessity for cholinergic transmission. Finally, we show that the neurokinin 1 receptor for substance P is required for SCC-mediated inflammation, suggesting that release of substance P from nerve fibers triggers the inflammatory events. Taken together, these results show that SCCs use cholinergic neurotransmission to trigger peptidergic trigeminal nociceptors, which link SCCs to the neurogenic inflammatory pathway.

The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves.

Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT(3A) promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT(3A) mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 µM 5-HT and this response is blocked by 1 µM ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 µM m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response.

Role of the ectonucleotidase NTPDase2 in taste bud function.

Taste buds are unusual in requiring ATP as a transmitter to activate sensory nerve fibers. In response to taste stimuli, taste cells release ATP, activating purinergic receptors containing the P2X2 and P2X3 subunits on taste nerves. In turn, the released ATP is hydrolyzed to ADP by a plasma membrane nucleoside triphosphate previously identified as nucleoside triphosphate diphosphohydrolase-2 (NTPDase2). In this paper we investigate the role of this ectonucleotidase in the function of taste buds by examining gene-targeted Entpd2-null mice globally lacking NTPDase2. RT-PCR confirmed the absence of NTPDase2, and ATPase enzyme histochemistry reveals no reaction product in taste buds of knockout mice, suggesting that NTPDase2 is the dominant form in taste buds. RT-PCR and immunocytochemistry demonstrated that in knockout mice all cell types are present in taste buds, even those cells normally expressing NTPDase2. In addition, the overall number and size of taste buds are normal in Entpd2-null mice. Luciferin/luciferase assays of circumvallate tissue of knockout mice detected elevated levels of extracellular ATP. Electrophysiological recordings from two taste nerves, the chorda tympani and glossopharyngeal, revealed depressed responses to all taste stimuli in Entpd2-null mice. Responses were more depressed in the glossopharyngeal nerve than in the chorda tympani nerve and involved all taste qualities; responses in the chorda tympani were more depressed to sweet and umami stimuli than to other qualities. We suggest that the excessive levels of extracellular ATP in the Entpd2-knockout animals desensitize the P2X receptors associated with nerve fibers, thereby depressing taste responses.

Postsynaptic P2X3-containing receptors in gustatory nerve fibres mediate responses to all taste qualities in mice.

Taste buds release ATP to activate ionotropic purinoceptors composed of P2X2 and P2X3 subunits, present on the taste nerves. Mice with genetic deletion of P2X2 and P2X3 receptors (double knockout mice) lack responses to all taste stimuli presumably due to the absence of ATP-gated receptors on the afferent nerves. Recent experiments on the double knockout mice showed, however, that their taste buds fail to release ATP, suggesting the possibility of pleiotropic deficits in these global knockouts. To test further the role of postsynaptic P2X receptors in afferent signalling, we used AF-353, a selective antagonist of P2X3-containing receptors to inhibit the receptors acutely during taste nerve recording and behaviour. The specificity of AF-353 for P2X3-containing receptors was tested by recording Ca(2+) transients to exogenously applied ATP in fura-2 loaded isolated geniculate ganglion neurons from wild-type and P2X3 knockout mice. ATP responses were completely inhibited by 10 µm or 100 µm AF-353, but neither concentration blocked responses in P2X3 single knockout mice wherein the ganglion cells express only P2X2-containing receptors. Furthermore, AF-353 had no effect on taste-evoked ATP release from taste buds. In wild-type mice, i.p. injection of AF-353 or simple application of the drug directly to the tongue, inhibited taste nerve responses to all taste qualities in a dose-dependent fashion. A brief access behavioural assay confirmed the electrophysiological results and showed that preference for a synthetic sweetener, SC-45647, was abolished following i.p. injection of AF-353. These data indicate that activation of P2X3-containing receptors is required for transmission of all taste qualities.

Immunohistochemical Analysis of Human Vallate Taste Buds.

The morphology of the vallate papillae from postmortem human samples was investigated with immunohistochemistry. Microscopically, taste buds were present along the inner wall of the papilla, and in some cases in the outer wall as well. The typical taste cell markers PLCß2, GNAT3 (gustducin) and the T1R3 receptor stain elongated cells in human taste buds consistent with the Type II cells in rodents. In the human tissue, taste bud cells that stain with Type II cell markers, PLCß2 and GNAT3, also stain with villin antibody. Two typical immunochemical markers for Type III taste cells in rodents, PGP9.5 and SNAP25, fail to stain any taste bud cells in the human postmortem tissue, although these antibodies do stain numerous nerve fibers throughout the specimen. Car4, another Type III cell marker, reacted with only a few taste cells in our samples. Finally, human vallate papillae have a general network of innervation similar to rodents and antibodies directed against SNAP25, PGP9.5, acetylated tubulin and P2X3 all stain free perigemmal nerve endings as well as intragemmal taste fibers. We conclude that with the exception of certain molecular features of Type III cells, human vallate papillae share the structural, morphological, and molecular features observed in rodents.

Chemosensors in the nose: guardians of the airways.

The G-protein-coupled receptor molecules and downstream effectors that are used by taste buds to detect sweet, bitter, and savory tastes are also utilized by chemoresponsive cells of the airways to detect irritants. Here, we describe the different cell types in the airways that utilize taste-receptor signaling to trigger protective epithelial and neural responses to potentially dangerous toxins and bacterial infection.

NaV1.5 sodium channel window currents contribute to spontaneous firing in olfactory sensory neurons.

Olfactory sensory neurons (OSNs) fire spontaneously as well as in response to odor; both forms of firing are physiologically important. We studied voltage-gated Na(+) channels in OSNs to assess their role in spontaneous activity. Whole cell patch-clamp recordings from OSNs demonstrated both tetrodotoxin-sensitive and tetrodotoxin-resistant components of Na(+) current. RT-PCR showed mRNAs for five of the nine different Na(+) channel α-subunits in olfactory tissue; only one was tetrodotoxin resistant, the so-called cardiac subtype NaV1.5. Immunohistochemical analysis indicated that NaV1.5 is present in the apical knob of OSN dendrites but not in the axon. The NaV1.5 channels in OSNs exhibited two important features: 1) a half-inactivation potential near -100 mV, well below the resting potential, and 2) a window current centered near the resting potential. The negative half-inactivation potential renders most NaV1.5 channels in OSNs inactivated at the resting potential, while the window current indicates that the minor fraction of noninactivated NaV1.5 channels have a small probability of opening spontaneously at the resting potential. When the tetrodotoxin-sensitive Na(+) channels were blocked by nanomolar tetrodotoxin at the resting potential, spontaneous firing was suppressed as expected. Furthermore, selectively blocking NaV1.5 channels with Zn(2+) in the absence of tetrodotoxin also suppressed spontaneous firing, indicating that NaV1.5 channels are required for spontaneous activity despite resting inactivation. We propose that window currents produced by noninactivated NaV1.5 channels are one source of the generator potentials that trigger spontaneous firing, while the upstroke and propagation of action potentials in OSNs are borne by the tetrodotoxin-sensitive Na(+) channel subtypes.

Structural comparisons of human and mouse fungiform taste buds.

Taste buds are commonly studied in rodent models, but some differences exist between mice and humans in terms of gustatory mechanisms and sensitivities. Whether these functional differences are reflected in structural differences between species is unclear. Using immunofluorescent image stacks, we compared morphological and molecular characteristics of mouse and human fungiform taste buds. The results suggest that while the general features of fungiform taste buds are similar between mice and humans, several characteristics differ significantly. Human taste buds are larger and taller than those of mice, yet they contain similar numbers of taste cells. Taste buds in humans are more heavily innervated by gustatory nerve fibers expressing the purinergic receptor P2X3 showing a 40% higher innervation density than in mice. Like Type II cells of mice, a subset (about 30%) of cells in human taste buds is immunoreactive for PLCß2. These PLCß2-immunoreactive cells display CALHM1-immunoreactive puncta closely apposed to gustatory nerve fibers suggestive of channel-type synapses described in mice. These puncta, used as a measure of synaptic contact, are however significantly larger in humans compared to mice. Altogether these findings suggest that while many similarities exist in the structural organization of murine and human fungiform taste buds, significant differences do exist in taste bud size, innervation density, and size of synaptic contacts that may impact gustatory signal transmission.

Chemosensory brush cells of the trachea. A stable population in a dynamic epithelium.

Tracheal brush cells (BCs) are specialized epithelial chemosensors that use the canonical taste transduction cascade to detect irritants. To test whether BCs are replaced at the same rate as other cells in the surrounding epithelium of adult mice, we used 5-bromo-2'-deoxyuridine (BrdU) to label dividing cells. Although scattered BrdU-labeled epithelial cells are present 5-20 days after BrdU, no BCs are labeled. These data indicate that BCs comprise a relatively static population. To determine how BCs are generated during development, we injected 5-day-old mice with BrdU and found labeled BCs and non-BC epithelial cells 5 days after BrdU. During the next 60 days, the percentage of labeled BCs increased, whereas the percentage of other labeled cell types decreased. These data suggest that BCs are generated from non-BC progenitor cells during postnatal tracheal growth. To test whether the adult epithelium retains the capacity to generate BCs, tracheal epithelial cells were recovered from adult mice and grown in an air-liquid interface (ALI) culture. After transition to differentiation conditions, BCs are detected, and comprise 1% of the total cell population by Day 14. BrdU added to cultures before the differentiation of BCs was chased into BCs, indicating that the increase in BC density is attributable to the proliferation of a non-BC progenitor. We conclude that: (1) BCs are normally a static population in adult mice; (2) BC progenitors proliferate and differentiate during neonatal development; and (3) BCs can be regenerated from a proliferative population resident in adult epithelium.

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 Ca(2+). Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either G alpha-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.

Variability in P2X receptor composition in human taste nerves: implications for treatment of chronic cough.

Background: Antagonists to the P2X purinergic receptors on airway sensory nerves relieve refractory or unexplained chronic cough (RCC/UCC) but can evoke unwanted dysgeusias because the gustatory nerves innervating taste buds express this same family of receptors. However, the subunit composition of the P2X receptors in these systems may differ, with implications for pharmacological intervention of RCC/UCC. In most species, the extrapulmonary airway nerves involved in cough predominantly express P2X3 subunits that form homotrimeric P2X3 receptors. In contrast, most sensory nerves innervating taste buds in mice express both P2X2 and P2X3 subunits, so the majority of receptors in that system are likely P2X2/P2X3 heteromers. Methods: Since neural P2X subunit composition can differ across species, we used immunohistochemistry to test whether taste nerves in humans and rhesus macaque monkeys express both P2X2 and P2X3 as in mice. Results: In taste bud samples of fungiform papillae and larynx from humans and monkeys, all taste bud samples exhibited P2X3+ nerve fibres, but the majority lacked substantial P2X2+. Of the 35 human subjects, only four (one laryngeal and three fungiform) showed strong P2X2 immunoreactivity in taste nerves; none of the rhesus monkey samples showed immunoreactivity for P2X2. Conclusions: These findings suggest that for most humans, unlike mice, taste buds are innervated by nerve fibres predominantly expressing only P2X3 homomeric receptors and not P2X2/P2X3 heteromers. Thus, antagonists specific for P2X3 homomeric receptors might not be spared from affecting taste function in RCC/UCC patients.

Central representation of postingestive chemosensory cues in mice that lack the ability to taste.

The gustatory nerves of mice lacking P2X2 and P2X3 purinergic receptor subunits (P2X-dblKO) are unresponsive to taste stimulation (Finger et al., 2005). Surprisingly, P2X-dblKO mice show residual behavioral responses to concentrated tastants, presumably via postingestive detection. Therefore, the current study tested whether postingestive signaling is functional in P2X-dblKO mice and if so, whether it activates the primary viscerosensory nucleus of the medulla, the nucleus of the solitary tract (nTS). Like WT animals, P2X-dblKO mice learned to prefer a flavor paired with 150 mm monosodium glutamate (MSG) over a flavor paired with water. This preference shows that, even in the absence of taste sensory input, postingestive cues are detected and associated with a flavor in P2X-dblKO mice. MSG-evoked neuronal activation in the nTS was measured by expression of the immediate early gene c-Fos [c-Fos-like immunoreactivity (Fos-LI)]. In rostral, gustatory nTS, P2X-dblKO animals, unlike WT animals, showed no taste quality-specific labeling of neurons. Furthermore, MSG-evoked Fos-LI was significantly less in P2X-dblKO mice compared with WT animals. In contrast, in more posterior, viscerosensory nTS, MSG-induced Fos-LI was similar in WT and P2X-dblKO mice. Together, these results suggest that P2X-dblKO mice can form preferences based on postingestive cues and that postingestive detection of MSG does not rely on the same purinergic signaling that is crucial for taste.

Knocking out P2X receptors reduces transmitter secretion in taste buds.

In response to gustatory stimulation, taste bud cells release a transmitter, ATP, that activates P2X2 and P2X3 receptors on gustatory afferent fibers. Taste behavior and gustatory neural responses are largely abolished in mice lacking P2X2 and P2X3 receptors [P2X2 and P2X3 double knock-out (DKO) mice]. The assumption has been that eliminating P2X2 and P2X3 receptors only removes postsynaptic targets but that transmitter secretion in mice is normal. Using functional imaging, ATP biosensor cells, and a cell-free assay for ATP, we tested this assumption. Surprisingly, although gustatory stimulation mobilizes Ca(2+) in taste Receptor (Type II) cells from DKO mice, as from wild-type (WT) mice, taste cells from DKO mice fail to release ATP when stimulated with tastants. ATP release could be elicited by depolarizing DKO Receptor cells with KCl, suggesting that ATP-release machinery remains functional in DKO taste buds. To explore the difference in ATP release across genotypes, we used reverse transcriptase (RT)-PCR, immunostaining, and histochemistry for key proteins underlying ATP secretion and degradation: Pannexin1, TRPM5, and NTPDase2 (ecto-ATPase) are indistinguishable between WT and DKO mice. The ultrastructure of contacts between taste cells and nerve fibers is also normal in the DKO mice. Finally, quantitative RT-PCR show that P2X4 and P2X7, potential modulators of ATP secretion, are similarly expressed in taste buds in WT and DKO taste buds. Importantly, we find that P2X2 is expressed in WT taste buds and appears to function as an autocrine, positive feedback signal to amplify taste-evoked ATP secretion.