Selectively Imaging Cranial Sensory Ganglion Neurons Using AAV-PHP.S.

Because of their ease of use, adeno-associated viruses (AAVs) are indispensable tools for much of neuroscience. Yet AAVs have been used relatively little to study the identities and connectivity of peripheral sensory neurons, principally because methods to selectively target peripheral neurons have been limited. The introduction of the AAV-PHP.S capsid with enhanced tropism for peripheral neurons (Chan et al., 2017) offered a solution, which we further elaborate here. Using AAV-PHP.S with GFP or mScarlet fluorescent proteins, we show that the mouse sensory ganglia for cranial nerves V, VII, IX, and X are targeted. Pseudounipolar neurons of both somatic and visceral origin, but not satellite glia, express the reporters. One week after virus injection, ≈66% of geniculate ganglion neurons were transduced. Fluorescent reporters were transported along the central and peripheral axons of these sensory neurons, permitting visualization of terminals at high resolution, and in intact, cleared brain using light sheet microscopy. Further, using a Cre-dependent reporter, we demonstrate by anatomic and functional criteria, that expression is in a cell type-selective manner. Finally, we integrate earlier neuroanatomical and molecular data with in vivo Ca2+ imaging to demonstrate the sensory characteristics of geniculate ganglion auricular neurons, which were previously undocumented. Our analyses suggest that the AAV-PHP.S serotype will be a powerful tool for anatomically and functionally mapping the receptive fields and circuits of the expanding numbers of molecular subtypes of many somatosensory and viscerosensory neurons that continue to be defined via single-cell RNA sequencing.

The Role of the Anion in Salt (NaCl) Detection by Mouse Taste Buds.

How taste buds detect NaCl remains poorly understood. Among other problems, applying taste-relevant concentrations of NaCl (50-500 mm) onto isolated taste buds or cells exposes them to unphysiological (hypo/hypertonic) conditions. To overcome these limitations, we used the anterior tongue of male and female mice to implement a slice preparation in which fungiform taste buds are in a relatively intact tissue environment and stimuli are limited to the taste pore. Taste-evoked responses were monitored using confocal Ca2+ imaging via GCaMP3 expressed in Type 2 and Type 3 taste bud cells. NaCl evoked intracellular mobilization of Ca2+ in the apical tips of a subset of taste cells. The concentration dependence and rapid adaptation of NaCl-evoked cellular responses closely resembled behavioral and afferent nerve responses to NaCl. Importantly, taste cell responses were not inhibited by the diuretic, amiloride. Post hoc immunostaining revealed that >80% of NaCl-responsive taste bud cells were of Type 2. Many NaCl-responsive cells were also sensitive to stimuli that activate Type 2 cells but never to stimuli for Type 3 cells. Ion substitutions revealed that amiloride-insensitive NaCl responses depended on Cl- rather than Na+ Moreover, choline chloride, an established salt taste enhancer, was equally effective a stimulus as sodium chloride. Although the apical transducer for Cl- remains unknown, blocking known chloride channels and cotransporters had little effect on NaCl responses. Together, our data suggest that chloride, an essential nutrient, is a key determinant of taste transduction for amiloride-insensitive salt taste.SIGNIFICANCE STATEMENT Sodium and chloride are essential nutrients and must be regularly consumed to replace excreted NaCl. Thus, understanding salt taste, which informs salt appetite, is important from a fundamental sensory perspective and forms the basis for interventions to replace/reduce excess Na+ consumption. This study examines responses to NaCl in a semi-intact preparation of mouse taste buds. We identify taste cells that respond to NaCl in the presence of amiloride, which is significant because much of human salt taste also is amiloride-insensitive. Further, we demonstrate that Cl-, not Na+, generates these amiloride-insensitive salt taste responses. Intriguingly, choline chloride, a commercial salt taste enhancer, is also a highly effective stimulus for these cells.

Cell-Based Therapy Restores Olfactory Function in an Inducible Model of Hyposmia.

Stem cell-based therapies have been proposed as a strategy to replace damaged tissues, especially in the nervous system. A primary sensory modality, olfaction, is impaired in 12% of the US population, but lacks treatment options. We report here the development of a novel mouse model of inducible hyposmia and demonstrate that purified tissue-specific stem cells delivered intranasally engraft to produce olfactory neurons, achieving recovery of function. Adult mice were rendered hyposmic by conditional deletion of the ciliopathy-related IFT88 gene in the olfactory sensory neuron lineage and following experimentally induced olfactory injury, received either vehicle or stem cell infusion intranasally. Engraftment-derived olfactory neurons were identified histologically, and functional improvements were measured via electrophysiology and behavioral assay. We further explored mechanisms in culture that promote expansion of engraftment-competent adult olfactory basal progenitor cells. These findings provide a basis for translational research on propagating adult tissue-specific sensory progenitor cells and testing their therapeutic potential.

Oral thermosensing by murine trigeminal neurons: modulation by capsaicin, menthol and mustard oil.

KEY POINTS: Orosensory thermal trigeminal afferent neurons respond to cool, warm, and nociceptive hot temperatures with the majority activated in the cool range. Many of these thermosensitive trigeminal orosensory afferent neurons also respond to capsaicin, menthol, and/or mustard oil (allyl isothiocyanate) at concentrations found in foods and spices. There is significant but incomplete overlap between afferent trigeminal neurons that respond to oral thermal stimulation and to the above chemesthetic compounds. Capsaicin sensitizes warm trigeminal thermoreceptors and orosensory nociceptors; menthol attenuates cool thermoresponses. ABSTRACT: When consumed with foods, mint, mustard, and chili peppers generate pronounced oral thermosensations. Here we recorded responses in mouse trigeminal ganglion neurons to investigate interactions between thermal sensing and the active ingredients of these plants - menthol, allyl isothiocyanate (AITC), and capsaicin, respectively - at concentrations found in foods and commercial hygiene products. We carried out in vivo confocal calcium imaging of trigeminal ganglia in which neurons express GCaMP3 or GCAMP6s and recorded their responses to oral stimulation with thermal and the above chemesthetic stimuli. In the V3 (oral sensory) region of the ganglion, thermoreceptive neurons accounted for ∼10% of imaged neurons. We categorized them into three distinct classes: cool-responsive and warm-responsive thermosensors, and nociceptors (responsive only to temperatures ≥43-45 °C). Menthol, AITC, and capsaicin also elicited robust calcium responses that differed markedly in their latencies and durations. Most of the neurons that responded to these chemesthetic stimuli were also thermosensitive. Capsaicin and AITC increased the numbers of warm-responding neurons and shifted the nociceptor threshold to lower temperatures. Menthol attenuated the responses in all classes of thermoreceptors. Our data show that while individual neurons may respond to a narrow temperature range (or even bimodally), taken collectively, the population is able to report on graded changes of temperature. Our findings also substantiate an explanation for the thermal sensations experienced when one consumes pungent spices or mint.

Transcriptomes and neurotransmitter profiles of classes of gustatory and somatosensory neurons in the geniculate ganglion.

Taste buds are innervated by neurons whose cell bodies reside in cranial sensory ganglia. Studies on the functional properties and connectivity of these neurons are hindered by the lack of markers to define their molecular identities and classes. The mouse geniculate ganglion contains chemosensory neurons innervating lingual and palatal taste buds and somatosensory neurons innervating the pinna. Here, we report single cell RNA sequencing of geniculate ganglion neurons. Using unbiased transcriptome analyses, we show a pronounced separation between two major clusters which, by anterograde labeling, correspond to gustatory and somatosensory neurons. Among the gustatory neurons, three subclusters are present, each with its own complement of transcription factors and neurotransmitter response profiles. The smallest subcluster expresses both gustatory- and mechanosensory-related genes, suggesting a novel type of sensory neuron. We identify several markers to help dissect the functional distinctions among gustatory neurons and address questions regarding target interactions and taste coding.Characterization of gustatory neural pathways has suffered due to a lack of molecular markers. Here, the authors report single cell RNA sequencing and unbiased transcriptome analyses to reveal major distinctions between gustatory and somatosensory neurons and subclusters of gustatory neurons with unique molecular and functional profiles.

Contribution of Polycomb group proteins to olfactory basal stem cell self-renewal in a novel c-KIT+ culture model and in vivo.

Olfactory epithelium (OE) has a lifelong capacity for neurogenesis due to the presence of basal stem cells. Despite the ability to generate short-term cultures, the successful in vitro expansion of purified stem cells from adult OE has not been reported. We sought to establish expansion-competent OE stem cell cultures to facilitate further study of the mechanisms and cell populations important in OE renewal. Successful cultures were prepared using adult mouse basal cells selected for expression of c-KIT. We show that c-KIT signaling regulates self-renewal capacity and prevents neurodifferentiation in culture. Inhibition of TGFß family signaling, a known negative regulator of embryonic basal cells, is also necessary for maintenance of the proliferative, undifferentiated state in vitro Characterizing successful cultures, we identified expression of BMI1 and other Polycomb proteins not previously identified in olfactory basal cells but known to be essential for self-renewal in other stem cell populations. Inducible fate mapping demonstrates that BMI1 is expressed in vivo by multipotent OE progenitors, validating our culture model. These findings provide mechanistic insights into the renewal and potency of olfactory stem cells.

Differentiation potential of individual olfactory c-Kit+ progenitors determined via multicolor lineage tracing.

Olfactory tissue undergoes lifelong renewal, due to the presence of basal neural stem cells. Multiple categories of globose basal stem cells have been identified, expressing markers such as Lgr5, Ascl1, GBC-2, and c-Kit. The differentiation potential of individual globose cells has remained unclear. Here, we utilized Cre/loxP lineage tracing with a multicolor reporter system to define c-Kit+ cell contributions at clonal resolution. We determined that reporter expression permitted identification of c-Kit derived progeny with fine cellular detail, and that clones were found to be comprised by neurons only, microvillar cells only, microvillar cells and neurons, or gland/duct cells. Quantification of reporter-labeled cells indicated that c-Kit+ cells behave as transit amplifying or immediate precursors, although we also found evidence for longer-term c-Kit+ cell contributions. Our results from the application of multicolor fate mapping delineate the clonal contributions of c-Kit+ cells to olfactory epithelial renewal, and provide novel insight into tissue maintenance of an adult neuroepithelium.

A permeability barrier surrounds taste buds in lingual epithelia.

Epithelial tissues are characterized by specialized cell-cell junctions, typically localized to the apical regions of cells. These junctions are formed by interacting membrane proteins and by cytoskeletal and extracellular matrix components. Within the lingual epithelium, tight junctions join the apical tips of the gustatory sensory cells in taste buds. These junctions constitute a selective barrier that limits penetration of chemosensory stimuli into taste buds (Michlig et al. J Comp Neurol 502: 1003-1011, 2007). We tested the ability of chemical compounds to permeate into sensory end organs in the lingual epithelium. Our findings reveal a robust barrier that surrounds the entire body of taste buds, not limited to the apical tight junctions. This barrier prevents penetration of many, but not all, compounds, whether they are applied topically, injected into the parenchyma of the tongue, or circulating in the blood supply, into taste buds. Enzymatic treatments indicate that this barrier likely includes glycosaminoglycans, as it was disrupted by chondroitinase but, less effectively, by proteases. The barrier surrounding taste buds could also be disrupted by brief treatment of lingual tissue samples with DMSO. Brief exposure of lingual slices to DMSO did not affect the ability of taste buds within the slice to respond to chemical stimulation. The existence of a highly impermeable barrier surrounding taste buds and methods to break through this barrier may be relevant to basic research and to clinical treatments of taste.

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.

CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes.

Recognition of sweet, bitter and umami tastes requires the non-vesicular release from taste bud cells of ATP, which acts as a neurotransmitter to activate afferent neural gustatory pathways. However, how ATP is released to fulfil this function is not fully understood. Here we show that calcium homeostasis modulator 1 (CALHM1), a voltage-gated ion channel, is indispensable for taste-stimuli-evoked ATP release from sweet-, bitter- and umami-sensing taste bud cells. Calhm1 knockout mice have severely impaired perceptions of sweet, bitter and umami compounds, whereas their recognition of sour and salty tastes remains mostly normal. Calhm1 deficiency affects taste perception without interfering with taste cell development or integrity. CALHM1 is expressed specifically in sweet/bitter/umami-sensing type II taste bud cells. Its heterologous expression induces a novel ATP permeability that releases ATP from cells in response to manipulations that activate the CALHM1 ion channel. Knockout of Calhm1 strongly reduces voltage-gated currents in type II cells and taste-evoked ATP release from taste buds without affecting the excitability of taste cells by taste stimuli. Thus, CALHM1 is a voltage-gated ATP-release channel required for sweet, bitter and umami taste perception.

Adenosine enhances sweet taste through A2B receptors in the taste bud.

Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. This ATP excites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Here we show that extracellular ATP is enzymatically degraded to adenosine within mouse vallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca(2+) mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 µM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 µM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell reverse transcriptase (RT)-PCR on isolated vallate taste cells, we show that many Receptor cells express the adenosine receptor, Adora2b, while Presynaptic (type III) and Glial-like (type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5'-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase. Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry, and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet 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.

Interaction between the second messengers cAMP and Ca2+ in mouse presynaptic taste cells.

The second messenger, 3',5'-cyclic adenosine monophosphate (cAMP), is known to be modulated in taste buds following exposure to gustatory and other stimuli. Which taste cell type(s) (Type I/glial-like cells, Type II/receptor cells, or Type III/presynaptic cells) undergo taste-evoked changes of cAMP and what the functional consequences of such changes are remain unknown. Using Fura-2 imaging of isolated mouse vallate taste cells, we explored how elevating cAMP alters Ca(2+) levels in identified taste cells. Stimulating taste buds with forskolin (Fsk; 1 microm) + isobutylmethylxanthine (IBMX; 100 microm), which elevates cellular cAMP, triggered Ca(2+) transients in 38% of presynaptic cells (n = 128). We used transgenic GAD-GFP mice to show that cAMP-triggered Ca(2+) responses occur only in the subset of presynaptic cells that lack glutamic acid decarboxylase 67 (GAD). We never observed cAMP-stimulated responses in receptor cells, glial-like cells or GAD-expressing presynaptic cells. The response to cAMP was blocked by the protein kinase A inhibitor H89 and by removing extracellular Ca(2+). Thus, the response to elevated cAMP is a PKA-dependent influx of Ca(2+). This Ca(2+) influx was blocked by nifedipine (an inhibitor of L-type voltage-gated Ca(2+) channels) but was unperturbed by omega-agatoxin IVA and omega-conotoxin GVIA (P/Q-type and N-type channel inhibitors, respectively). Single-cell RT-PCR on functionally identified presynaptic cells from GAD-GFP mice confirmed the pharmacological analyses: Ca(v)1.2 (an L-type subunit) is expressed in cells that display cAMP-triggered Ca(2+) influx, while Ca(v)2.1 (a P/Q subunit) is expressed in all presynaptic cells, and underlies depolarization-triggered Ca(2+) influx. Collectively, these data demonstrate cross-talk between cAMP and Ca(2+) signalling in a subclass of taste cells that form synapses with gustatory fibres and may integrate tastant-evoked signals.

Tonic activity of Galpha-gustducin regulates taste cell responsivity.

The taste-selective G protein, alpha-gustducin (alpha-gus) is homologous to alpha-transducin and activates phosphodiesterase (PDE) in vitro. alpha-Gus-knockout mice are compromized to bitter, sweet and umami taste stimuli, suggesting a central role in taste transduction. Here, we suggest a different role for Galpha-gus. In taste buds of alpha-gus-knockout mice, basal (unstimulated) cAMP levels are high compared to those of wild-type mice. Further, H-89, a cAMP-dependent protein kinase inhibitor, dramatically unmasks responses to the bitter tastant denatonium in gus-lineage cells of knockout mice. We propose that an important role of alpha-gus is to maintain cAMP levels tonically low to ensure adequate Ca2+ signaling.

Imaging cyclic AMP changes in pancreatic islets of transgenic reporter mice.

Cyclic AMP (cAMP) and Ca(2+) are two ubiquitous second messengers in transduction pathways downstream of receptors for hormones, neurotransmitters and local signals. The availability of fluorescent Ca(2+) reporter dyes that are easily introduced into cells and tissues has facilitated analysis of the dynamics and spatial patterns for Ca(2+) signaling pathways. A similar dissection of the role of cAMP has lagged because indicator dyes do not exist. Genetically encoded reporters for cAMP are available but they must be introduced by transient transfection in cell culture, which limits their utility. We report here that we have produced a strain of transgenic mice in which an enhanced cAMP reporter is integrated in the genome and can be expressed in any targeted tissue and with tetracycline induction. We have expressed the cAMP reporter in beta-cells of pancreatic islets and conducted an analysis of intracellular cAMP levels in relation to glucose stimulation, Ca(2+) levels, and membrane depolarization. Pancreatic function in transgenic mice was normal. In induced transgenic islets, glucose evoked an increase in cAMP in beta-cells in a dose-dependent manner. The cAMP response is independent of (in fact, precedes) the Ca(2+) influx that results from glucose stimulation of islets. Glucose-evoked cAMP responses are synchronous in cells throughout the islet and occur in 2 phases suggestive of the time course of insulin secretion. Insofar as cAMP in islets is known to potentiate insulin secretion, the novel transgenic mouse model will for the first time permit detailed analyses of cAMP signals in beta-cells within islets, i.e. in their native physiological context. Reporter expression in other tissues (such as the heart) where cAMP plays a critical regulatory role, will permit novel biomedical approaches.

Biogenic amine synthesis and uptake in rodent taste buds.

Although adenosine triphosphate (ATP) is known to be an afferent transmitter in the peripheral taste system, serotonin (5-HT) and norepinephrine (NE) have also been proposed as candidate neurotransmitters and have been detected immunocytochemically in mammalian taste cells. To understand the significance of biogenic amines in taste, we evaluated the ability of taste cells to synthesize, transport, and package 5-HT and NE. We show by reverse transcriptase-polymerase chain reaction and immunofluorescence microscopy that the enzymes for 5-HT synthesis, tryptophan hydroxylase (TPH) and aromatic amino acid decarboxylase (AADC) are expressed in taste cells. In contrast, enzymes necessary for NE synthesis, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) are absent. Both TH and DBH are expressed in nerve fibers that penetrate taste buds. Taste buds also robustly express plasma membrane transporters for 5-HT and NE. Within the taste bud NET, a specific NE transporter, is expressed in some presynaptic (type III) and some glial-like (type I) cells but not in receptor (type II) cells. By using enzyme immunoassay, we show uptake of NE, probably through NET in taste epithelium. Proteins involved in inactivating and packaging NE, including catechol-O-methyltransferase (COMT), monoamine oxidase-A (MAO-A), vesicular monoamine transporter (VMAT1,2) and chromogranin A (ChrgA), are also expressed in taste buds. Within the taste bud, ChrgA is found only in presynaptic cells and may account for dense-cored vesicles previously seen in some taste cells. In summary, we postulate that aminergic presynaptic taste cells synthesize only 5-HT, whereas NE (perhaps secreted by sympathetic fibers) may be concentrated and repackaged for secretion.

The role of pannexin 1 hemichannels in ATP release and cell-cell communication in mouse taste buds.

ATP has been shown to be a taste bud afferent transmitter, but the cells responsible for, and the mechanism of, its release have not been identified. Using CHO cells expressing high-affinity neurotransmitter receptors as biosensors, we show that gustatory stimuli cause receptor cells to secrete ATP through pannexin 1 hemichannels in mouse taste buds. ATP further stimulates other taste cells to release a second transmitter, serotonin. These results provide a mechanism to link intracellular Ca(2+) release during taste transduction to secretion of afferent transmitter, ATP, from receptor cells. They also indicate a route for cell-cell communication and signal processing within the taste bud.

Tastants evoke cAMP signal in taste buds that is independent of calcium signaling.

We previously showed that rat taste buds express several adenylyl cyclases (ACs) of which only AC8 is known to be stimulated by Ca2+. Here we demonstrate by direct measurements of cAMP levels that AC activity in taste buds is stimulated by treatments that elevate intracellular Ca2+. Specifically, 5 microM thapsigargin or 3 microM A-23187 (calcium ionophore), both of which increase intracellular Ca2+ concentration ([Ca2+]i), lead to a significant elevation of cAMP levels. This calcium stimulation of AC activity requires extracellular Ca2+, suggesting that it is dependent on Ca2+ entry rather than release from stores. With immunofluorescence microscopy, we show that the calcium-stimulated AC8 is principally expressed in taste cells that also express phospholipase Cbeta2 (i.e., cells that elevate [Ca2+]i in response to sweet, bitter, or umami stimuli). Taste transduction for sucrose is known to result in an elevation of both cAMP and calcium in taste buds. Thus we tested whether the cAMP increase in response to sucrose is a downstream consequence of calcium elevation. Even under conditions of depletion of stored and extracellular calcium, the cAMP response to sucrose stimulation persists in taste cells. The cAMP signal in response to monosodium glutamate stimulation is similarly unperturbed by calcium depletion. Our results suggest that tastant-evoked cAMP signals are not simply a secondary consequence of calcium modulation. Instead, cAMP and released Ca2+ may represent independent second messenger signals downstream of taste receptors.

Liposome-mediated transfection of mature taste cells.

The introduction and expression of exogenous DNA in neurons is valuable for analyzing a range of cellular and molecular processes in the periphery, e.g., the roles of transduction-related proteins, the impact of growth factors on development and differentiation, and the function of promoters specific to cell type. However, sensory receptor cells, particularly chemosensory cells, have been difficult to transfect. We have successfully introduced plasmids expressing green and Discosoma Red fluorescent proteins (GFP and DsRed) into rat taste buds in primary culture. Transfection efficiency increased when delaminated taste epithelium was redigested with fresh protease, suggesting that a protective barrier of extracellular matrix surrounding taste cells may normally be present. Because taste buds are heterogeneous aggregates of cells, we used alpha-gustducin, neuronal cell adhesion molecule (NCAM), and neuronal ubiquitin carboxyl terminal hydrolase (PGP9.5), markers for defined subsets of mature taste cells, to demonstrate that liposome-mediated transfection targets multiple taste cell types. After testing eight commercially available lipids, we identified one, Transfast, that is most effective on taste cells. We also demonstrate the effectiveness of two common "promiscuous" promoters and one promoter that taste cells use endogenously. These studies should permit ex vivo strategies for studying development and cellular function in taste cells.

Acid-sensing ion channel-2 is not necessary for sour taste in mice.

The acid-sensitive cation channel acid-sensing ion channel-2 (ASIC2) is widely believed to be a receptor for acid (sour) taste in mammals on the basis of its physiological properties and expression in rat taste bud cells. Using reverse transcriptase-PCR, we detected expression of ASIC1 and ASIC3, but not ASIC4, in mouse and rat taste buds and nonsensory lingual epithelium. Surprisingly, we did not detect mRNA for ASIC2 in mouse taste buds, although we readily observed its expression in rat taste buds. Furthermore, in Ca2+ imaging experiments, ASIC2 knock-out mice exhibited normal physiological responses (increases in intracellular Ca2+ concentrations) to acid taste stimuli. Our results indicate that ASIC2 is not required for acid taste in mice, and that if a universal mammalian acid taste transduction mechanism exists, it likely uses other acid-sensitive receptors or ion channels.