RESUMEN
In mammalian taste buds, Type I cells comprise half of all cells. These are termed "glial-like" based on morphologic and molecular features, but there are limited studies describing their function. We tested whether Type I cells sense chemosensory activation of adjacent chemosensory (i.e., Types II and III) taste bud cells, similar to synaptic glia. Using Gad2;;GCaMP3 mice of both sexes, we confirmed by immunostaining that, within taste buds, GCaMP expression is predominantly in Type I cells (with no Type II and ≈28% Type III cells expressing weakly). In dissociated taste buds, GCaMP+ Type I cells responded to bath-applied ATP (10-100 µm) but not to 5-HT (transmitters released by Type II or III cells, respectively). Type I cells also did not respond to taste stimuli (5 µm cycloheximide, 1 mm denatonium). In lingual slice preparations also, Type I cells responded to bath-applied ATP (10-100 µm). However, when taste buds in the slice were stimulated with bitter tastants (cycloheximide, denatonium, quinine), Type I cells responded robustly. Taste-evoked responses of Type I cells in the slice preparation were significantly reduced by desensitizing purinoceptors or by purinoceptor antagonists (suramin, PPADS), and were essentially eliminated by blocking synaptic ATP release (carbenoxolone) or degrading extracellular ATP (apyrase). Thus, taste-evoked release of afferent ATP from type II chemosensory cells, in addition to exciting gustatory afferent fibers, also activates glial-like Type I taste cells. We speculate that Type I cells sense chemosensory activation and that they participate in synaptic signaling, similarly to glial cells at CNS tripartite synapses.SIGNIFICANCE STATEMENT Most studies of taste buds view the chemosensitive excitable cells that express taste receptors as the sole mediators of taste detection and transmission to the CNS. Type I "glial-like" cells, with their ensheathing morphology, are mostly viewed as responsible for clearing neurotransmitters and as the "glue" holding the taste bud together. In the present study, we demonstrate that, when intact taste buds respond to their natural stimuli, Type I cells sense the activation of the chemosensory cells by detecting the afferent transmitter. Because Type I cells synthesize GABA, a known gliotransmitter, and cognate receptors are present on both presynaptic and postsynaptic elements, Type I cells may participate in GABAergic synaptic transmission in the manner of astrocytes at tripartite synapses.
Asunto(s)
Transmisión Sináptica/fisiología , Papilas Gustativas/citología , Papilas Gustativas/fisiología , Animales , Femenino , Ratones , Sinapsis , Gusto/fisiologíaRESUMEN
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.
Asunto(s)
Canales de Calcio/metabolismo , Transmisión Sináptica , Gusto/fisiología , Adenosina Trifosfato/metabolismo , Animales , Canales de Calcio/deficiencia , Canales de Calcio/genética , Femenino , Células HeLa , Humanos , Activación del Canal Iónico , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Receptores Purinérgicos/metabolismo , Análisis de la Célula Individual , Gusto/genética , Papilas Gustativas/citología , Papilas Gustativas/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/metabolismo , Adenosina/fisiología , Receptor de Adenosina A2B/fisiología , Papilas Gustativas/metabolismo , Gusto/fisiología , Adenosina/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Técnicas de Cultivo de Órganos/métodos , Receptor de Adenosina A2B/efectos de los fármacos , Receptor de Adenosina A2B/genética , Edulcorantes/farmacología , Gusto/efectos de los fármacos , Papilas Gustativas/efectos de los fármacosRESUMEN
Taste buds consist of at least three principal cell types that have different functions in processing gustatory signals: glial-like (type I) cells, receptor (type II) cells, and presynaptic (type III) cells. Using a combination of Ca2+ imaging, single-cell reverse transcriptase-PCR and immunostaining, we show that GABA is an inhibitory transmitter in mouse taste buds, acting on GABA(A) and GABA(B) receptors to suppress transmitter (ATP) secretion from receptor cells during taste stimulation. Specifically, receptor cells express GABA(A) receptor subunits ß2, δ, and π, as well as GABA(B) receptors. In contrast, presynaptic cells express the GABA(A) ß3 subunit and only occasionally GABA(B) receptors. In keeping with the distinct expression pattern of GABA receptors in presynaptic cells, we detected no GABAergic suppression of transmitter release from presynaptic cells. We suggest that GABA may serve function(s) in taste buds in addition to synaptic inhibition. Finally, we also defined the source of GABA in taste buds: GABA is synthesized by GAD65 in type I taste cells as well as by GAD67 in presynaptic (type III) taste cells and is stored in both those two cell types. We conclude that GABA is an inhibitory transmitter released during taste stimulation and possibly also during growth and differentiation of taste buds.
Asunto(s)
Antagonistas del GABA/farmacología , Receptores de GABA/fisiología , Papilas Gustativas/efectos de los fármacos , Ácido gamma-Aminobutírico/fisiología , Animales , Células CHO , Calcio/metabolismo , Cricetinae , Cricetulus , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Neurotransmisores/metabolismo , ARN/genética , Receptores de GABA/genética , Receptores Presinapticos/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Gusto/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/metabolismo , Receptores Purinérgicos P2X2/biosíntesis , Receptores Purinérgicos P2X/biosíntesis , Transmisión Sináptica/fisiología , Papilas Gustativas/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Calcio/metabolismo , Conexinas/metabolismo , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Cloruro de Potasio/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos P2X/genética , Transmisión Sináptica/genética , Canales Catiónicos TRPM/metabolismo , Gusto/fisiología , Papilas Gustativas/efectos de los fármacos , Papilas Gustativas/ultraestructuraRESUMEN
A new expression vector containing the 1,944 bp 5'-flanking regulatory region together with exon 1 and intron 1 of the goat alpha-S1-casein gene (CSN1S1), the full-sized human granulocyte colony-stimulating factor gene (hGCSF) and the 3'-flanking sequence of the bovine CSN1S1, was created. The vector DNA was used for generation of four mouse transgenic lines. The transgene was integrated into chromosomes 8 and 12 of two founders as 2 and 5 copies, respectively. Tissue-specific secretion of hG-CSF into the milk of transgenic mice was in the range of 19-40 µg/ml. RT-PCR analysis of various tissues of the transgenic mice demonstrated that expression of hGCSF was detected in only the mammary gland in the progeny of all founders. Moreover, cells were shown to be positive for hG-CSF by immunofluorescent analysis in the mammary glands but not in any other tissues. There were no signs of mosaic expression in the mammary gland. Trace amounts of hG-CSF were detected in the serum of females of two transgenic lines during lactation only. However, no transgenic mice showed any changes in hematopoiesis based on the number of granulocytes in blood. Immunoblotting of hG-CSF in the milk of transgenic mice revealed two forms, presumably the glycosylated and non-glycosylated forms. The hematopoietic activity of hG-CSF in the milk of transgenic females is comparable to that of recombinant G-CSF. In general, the data obtained in this study show that the new expression vector is able to provide correct tissue-specific expression of hG-CSF with high biological activity in transgenic mice.
Asunto(s)
Región de Flanqueo 5' , Caseínas/genética , Cabras/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Glándulas Mamarias Animales/metabolismo , Animales , Recuento de Células Sanguíneas , Caseínas/metabolismo , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Clonación Molecular , Exones , Femenino , Dosificación de Gen , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Glicosilación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Granulocitos/metabolismo , Hematopoyesis , Humanos , Intrones , Lactancia , Glándulas Mamarias Animales/citología , Ratones , Ratones Transgénicos , Leche/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcripción Genética , TransgenesRESUMEN
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.
Asunto(s)
Dependovirus , Ganglios Espinales , Animales , Dependovirus/genética , Ganglios Sensoriales , Ganglios Espinales/metabolismo , Vectores Genéticos , Ratones , Regiones Promotoras Genéticas , Células Receptoras SensorialesRESUMEN
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.
Asunto(s)
Calcio/fisiología , AMP Cíclico/fisiología , Receptores Presinapticos/fisiología , Sistemas de Mensajero Secundario/fisiología , Papilas Gustativas/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/fisiología , Canales de Calcio Tipo P/efectos de los fármacos , Canales de Calcio Tipo P/fisiología , Canales de Calcio Tipo Q/efectos de los fármacos , Canales de Calcio Tipo Q/fisiología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/genética , Señalización del Calcio/fisiología , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Glutamato Descarboxilasa/biosíntesis , Glutamato Descarboxilasa/genética , Procesamiento de Imagen Asistido por Computador , Ratones , Ratones Endogámicos C57BL , Inhibidores de Fosfodiesterasa/farmacología , Receptores Acoplados a Proteínas G/fisiología , Receptores Presinapticos/efectos de los fármacos , Receptores Presinapticos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sistemas de Mensajero Secundario/efectos de los fármacos , Sistemas de Mensajero Secundario/genética , Papilas Gustativas/efectos de los fármacosRESUMEN
Neuronal subtypes show diverse injury responses, but the molecular underpinnings remain elusive. Using transgenic mice that allow reliable visualization of axonal fate, we demonstrate that intrinsically photosensitive retinal ganglion cells (ipRGCs) are both resilient to cell death and highly regenerative. Using RNA sequencing (RNA-seq), we show genes that are differentially expressed in ipRGCs and that associate with their survival and axon regeneration. Strikingly, thrombospondin-1 (Thbs1) ranked as the most differentially expressed gene, along with the well-documented injury-response genes Atf3 and Jun. THBS1 knockdown in RGCs eliminated axon regeneration. Conversely, RGC overexpression of THBS1 enhanced regeneration in both ipRGCs and non-ipRGCs, an effect that was dependent on syndecan-1, a known THBS1-binding protein. All structural domains of the THBS1 were not equally effective; the trimerization and C-terminal domains promoted regeneration, while the THBS type-1 repeats were dispensable. Our results identify cell-type-specific induction of Thbs1 as a novel gene conferring high regenerative capacity.
Asunto(s)
Regeneración Nerviosa/fisiología , Células Ganglionares de la Retina/fisiología , Trombospondina 1/fisiología , Animales , Apoptosis , Axones/metabolismo , Línea Celular , Femenino , Perfilación de la Expresión Génica , Genes Reporteros , Factor I del Crecimiento Similar a la Insulina/deficiencia , Factor I del Crecimiento Similar a la Insulina/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Compresión Nerviosa , Traumatismos del Nervio Óptico/genética , Traumatismos del Nervio Óptico/fisiopatología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Opsinas de Bastones/deficiencia , Opsinas de Bastones/fisiología , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/fisiología , Trombospondina 1/biosíntesis , Trombospondina 1/genética , Transcripción GenéticaRESUMEN
BACKGROUND: "Type II"/Receptor cells express G protein-coupled receptors (GPCRs) for sweet, umami (T1Rs and mGluRs) or bitter (T2Rs), as well as the proteins for downstream signalling cascades. Transduction downstream of T1Rs and T2Rs relies on G-protein and PLCbeta2-mediated release of stored Ca2+. Whereas Galphagus (gustducin) couples to the T2R (bitter) receptors, which Galpha-subunit couples to the sweet (T1R2 + T1R3) receptor is presently not known. We utilized RT-PCR, immunocytochemistry and single-cell gene expression profiling to examine the expression of the Galphaq family (q, 11, 14) in mouse taste buds. RESULTS: By RT-PCR, Galpha14 is expressed strongly and in a taste selective manner in posterior (vallate and foliate), but not anterior (fungiform and palate) taste fields. Galphaq and Galpha11, although detectable, are not expressed in a taste-selective fashion. Further, expression of Galpha14 mRNA is limited to Type II/Receptor cells in taste buds. Immunocytochemistry on vallate papillae using a broad Galphaq family antiserum reveals specific staining only in Type II taste cells (i.e. those expressing TrpM5 and PLCbeta2). This staining persists in Galphaq knockout mice and immunostaining with a Galpha11-specific antiserum shows no immunoreactivity in taste buds. Taken together, these data show that Galpha14 is the dominant Galphaq family member detected. Immunoreactivity for Galpha14 strongly correlates with expression of T1R3, the taste receptor subunit present in taste cells responsive to either umami or sweet. Single cell gene expression profiling confirms a tight correlation between the expression of Galpha14 and both T1R2 and T1R3, the receptor combination that forms sweet taste receptors. CONCLUSION: Galpha14 is co-expressed with the sweet taste receptor in posterior tongue, although not in anterior tongue. Thus, sweet taste transduction may rely on different downstream transduction elements in posterior and anterior taste fields.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/biosíntesis , Papilas Gustativas/metabolismo , Gusto/fisiología , Lengua/fisiología , Animales , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Perfilación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas de Unión al GTP Heterotriméricas/biosíntesis , Proteínas de Unión al GTP Heterotriméricas/genética , Inmunohistoquímica , Ratones , Ratones Noqueados , Ratones Transgénicos , Especificidad de Órganos , Receptores Acoplados a Proteínas G/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Gusto/genética , Papilas Gustativas/citología , Lengua/citologíaRESUMEN
Taste buds are aggregates of 50-100 cells, only a fraction of which express genes for taste receptors and intracellular signaling proteins. We combined functional calcium imaging with single-cell molecular profiling to demonstrate the existence of two distinct cell types in mouse taste buds. Calcium imaging revealed that isolated taste cells responded with a transient elevation of cytoplasmic Ca2+ to either tastants or depolarization with KCl, but never both. Using single-cell reverse transcription (RT)-PCR, we show that individual taste cells express either phospholipase C beta2 (PLCbeta2) (an essential taste transduction effector) or synaptosomal-associated protein 25 (SNAP25) (a key component of calcium-triggered transmitter exocytosis). The two functional classes revealed by calcium imaging mapped onto the two gene expression classes determined by single-cell RT-PCR. Specifically, cells responding to tastants expressed PLCbeta2, whereas cells responding to KCl depolarization expressed SNAP25. We demonstrate this by two methods: first, through sequential calcium imaging and single-cell RT-PCR; second, by performing calcium imaging on taste buds in slices from transgenic mice in which PLCbeta2-expressing taste cells are labeled with green fluorescent protein. To evaluate the significance of the SNAP25-expressing cells, we used RNA amplification from single cells, followed by RT-PCR. We show that SNAP25-positive cells also express typical presynaptic proteins, including a voltage-gated calcium channel (alpha1A), neural cell adhesion molecule, synapsin-II, and the neurotransmitter-synthesizing enzymes glutamic acid decarboxylase and aromatic amino acid decarboxylase. No synaptic markers were detected in PLCbeta2 cells by either amplified RNA profiling or by immunocytochemistry. These data demonstrate the existence of at least two molecularly distinct functional classes of taste cells: receptor cells and synapse-forming cells.
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Terminales Presinápticos/fisiología , Papilas Gustativas/fisiología , Animales , Calcio/fisiología , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Potasio/farmacología , Cloruro de Potasio/farmacología , Terminales Presinápticos/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Receptoras Sensoriales/fisiología , Cloruro de Sodio/farmacologíaRESUMEN
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.
Asunto(s)
Norepinefrina/metabolismo , Serotonina/metabolismo , Papilas Gustativas/metabolismo , Animales , Células Epiteliales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
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.
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Ganglio Geniculado/metabolismo , Neurotransmisores/metabolismo , Células Receptoras Sensoriales/metabolismo , Transcriptoma , Animales , Pabellón Auricular/inervación , Ganglio Geniculado/citología , Proteínas de Homeodominio/genética , Ratones , Proteínas del Tejido Nervioso/genética , Receptores Purinérgicos P2X2/genética , Receptores Purinérgicos P2X3/genética , Análisis de Secuencia de ARN , Análisis de la Célula Individual , ATPasa Intercambiadora de Sodio-Potasio/genética , Proteína 25 Asociada a Sinaptosomas/genética , Gusto , Papilas Gustativas , Lengua/inervación , Tacto , Factores de Transcripción/genéticaRESUMEN
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.
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Canales Iónicos/fisiología , Proteínas de la Membrana , Proteínas del Tejido Nervioso/fisiología , Canales de Sodio , Papilas Gustativas/metabolismo , Gusto/fisiología , Canales Iónicos Sensibles al Ácido , Animales , Calcio/metabolismo , Señalización del Calcio/fisiología , Canales de Sodio Degenerina , Células Epiteliales/metabolismo , Canales Epiteliales de Sodio , Técnicas In Vitro , Canales Iónicos/genética , Canales Iónicos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estimulación Química , Gusto/genética , Papilas Gustativas/citologíaRESUMEN
Gustatory stimuli are detected by taste buds and transmitted to the hindbrain via sensory afferent neurons. Whether each taste quality (sweet, bitter and so on) is encoded by separate neurons ('labelled lines') remains controversial. We used mice expressing GCaMP3 in geniculate ganglion sensory neurons to investigate taste-evoked activity. Using confocal calcium imaging, we recorded responses to oral stimulation with prototypic taste stimuli. Up to 69% of neurons respond to multiple tastants. Moreover, neurons tuned to a single taste quality at low concentration become more broadly tuned when stimuli are presented at higher concentration. Responses to sucrose and monosodium glutamate are most related. Although mice prefer dilute NaCl solutions and avoid concentrated NaCl, we found no evidence for two separate populations of sensory neurons that encode this distinction. Altogether, our data suggest that taste is encoded by activity in patterns of peripheral sensory neurons and challenge the notion of strict labelled line coding.
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Potenciales Evocados/fisiología , Ganglio Geniculado/fisiología , Papilas Gustativas/fisiología , Gusto/fisiología , Animales , Señalización del Calcio/fisiología , Proteínas Fluorescentes Verdes , Ratones , Ratones Transgénicos , Microscopía Confocal , Neuronas Aferentes/fisiología , Imagen Óptica , Estimulación Física , Células Receptoras Sensoriales/fisiología , Cloruro de SodioRESUMEN
BACKGROUND: The neuropeptide, oxytocin (OXT), acts on brain circuits to inhibit food intake. Mutant mice lacking OXT (OXT knockout) overconsume salty and sweet (i.e. sucrose, saccharin) solutions. We asked if OXT might also act on taste buds via its receptor, OXTR. METHODOLOGY/PRINCIPAL FINDINGS: Using RT-PCR, we detected the expression of OXTR in taste buds throughout the oral cavity, but not in adjacent non-taste lingual epithelium. By immunostaining tissues from OXTR-YFP knock-in mice, we found that OXTR is expressed in a subset of Glial-like (Type I) taste cells, and also in cells on the periphery of taste buds. Single-cell RT-PCR confirmed this cell-type assignment. Using Ca2+ imaging, we observed that physiologically appropriate concentrations of OXT evoked [Ca2+]i mobilization in a subset of taste cells (EC50 approximately 33 nM). OXT-evoked responses were significantly inhibited by the OXTR antagonist, L-371,257. Isolated OXT-responsive taste cells were neither Receptor (Type II) nor Presynaptic (Type III) cells, consistent with our immunofluorescence observations. We also investigated the source of OXT peptide that may act on taste cells. Both RT-PCR and immunostaining suggest that the OXT peptide is not produced in taste buds or in their associated nerves. Finally, we also examined the morphology of taste buds from mice that lack OXTR. Taste buds and their constituent cell types appeared very similar in mice with two, one or no copies of the OXTR gene. CONCLUSIONS/SIGNIFICANCE: We conclude that OXT elicits Ca2+ signals via OXTR in murine taste buds. OXT-responsive cells are most likely a subset of Glial-like (Type I) taste cells. OXT itself is not produced locally in taste tissue and is likely delivered through the circulation. Loss of OXTR does not grossly alter the morphology of any of the cell types contained in taste buds. Instead, we speculate that OXT-responsive Glial-like (Type I) taste bud cells modulate taste signaling and afferent sensory output. Such modulation would complement central pathways of appetite regulation that employ circulating homeostatic and satiety signals.
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Oxitocina/metabolismo , Receptores de Oxitocina/metabolismo , Transducción de Señal , Papilas Gustativas/metabolismo , Animales , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Ingestión de Alimentos , Regulación de la Expresión Génica , Técnicas de Sustitución del Gen , Ratones , Ratones Transgénicos , Neuroglía/citología , Oxitocina/farmacología , Receptores de Oxitocina/antagonistas & inhibidores , Receptores de Oxitocina/deficiencia , Receptores de Oxitocina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Papilas Gustativas/citología , Papilas Gustativas/efectos de los fármacosRESUMEN
Cells in taste buds are closely packed, with little extracellular space. Tight junctions and other barriers further limit permeability and may result in buildup of extracellular K(+) following action potentials. In many tissues, inwardly rectifying K channels such as the renal outer medullary K (ROMK) channel (also called Kir1.1 and derived from the Kcnj1 gene) help to redistribute K(+). Using reverse-transcription polymerase chain reaction (RT-PCR), we defined ROMK splice variants in mouse kidney and report here the expression of a single one of these, ROMK2, in a subset of mouse taste cells. With quantitative (q)RT-PCR, we show the abundance of ROMK mRNA in taste buds is vallate > foliate > > palate > > fungiform. ROMK protein follows the same pattern of prevalence as mRNA, and is essentially undetectable by immunohistochemistry in fungiform taste buds. ROMK protein is localized to the apical tips of a subset of taste cells. Using tissues from PLCbeta2-GFP and GAD1-GFP transgenic mice, we show that ROMK is not found in PLCbeta2-expressing type II/receptor cells or in GAD1-expressing type III/presynaptic cells. Instead, ROMK is found, by single-cell RT-PCR and immunofluorescence, in most cells that are positive for the taste glial cell marker, Ectonucleotidase2. ROMK is precisely localized to the apical tips of these cells, at and above apical tight junctions. We propose that in taste buds, ROMK in type I/glial-like cells may serve a homeostatic function, excreting excess K(+) through the apical pore, and allowing excitable taste cells to maintain a hyperpolarized resting membrane potential.
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Neuroglía/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Papilas Gustativas/citología , Papilas Gustativas/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Uniones Comunicantes/metabolismo , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Inmunohistoquímica , Riñón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosfolipasa C beta/genética , Fosfolipasa C beta/metabolismo , Canales de Potasio de Rectificación Interna/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Uniones Estrechas/metabolismoRESUMEN
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.
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Adenosina Trifosfato/metabolismo , Comunicación Celular/fisiología , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal/fisiología , Papilas Gustativas/fisiología , Animales , Células CHO , Calcio/metabolismo , Conexinas , Cricetinae , Cricetulus , Hibridación in Situ , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serotonina/metabolismoRESUMEN
The spectrum and features of neurological disorders have been changed due to the Chernobyl catastrophe in the Republic of Belarus. More recently neurologists in Belarus have noted a significant increase in the frequency of myasthenia gravis (MG) with concomitant rise in the thymomas. There is some evidence suggesting that retroviruses play a key role in the development and pathogenesis of autoimmune diseases. This study analyzed thymomas from 45 MG patients from the Republic of Belarus by using PCR and primers for two regions of FV--gag and bel-2 genes. The results showed that none of the varied thymuses from the 45 MG patients contained FV genome. No relationship can be confirmed between FV and this disease and the results suggest that no pathological link between FV and MG exists.
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Genoma Viral , Miastenia Gravis/complicaciones , Virus Espumoso de los Simios/genética , Timoma/complicaciones , Timoma/virología , ADN Viral/análisis , Proteínas de Unión al ADN/genética , Productos del Gen gag/genética , Humanos , Miastenia Gravis/diagnóstico , Reacción en Cadena de la Polimerasa , República de Belarús , Timoma/diagnóstico , Transactivadores/genéticaRESUMEN
Sour (acid) taste is postulated to result from intracellular acidification that modulates one or more acid-sensitive ion channels in taste receptor cells. The identity of such channel(s) remains uncertain. Potassium channels, by regulating the excitability of taste cells, are candidates for acid transducers. Several 2-pore domain potassium leak conductance channels (K(2)P family) are sensitive to intracellular acidification. We examined their expression in mouse vallate and foliate taste buds using RT-PCR, and detected TWIK-1 and -2, TREK-1 and -2, and TASK-1. Of these, TWIK-1 and TASK-1 were preferentially expressed in taste cells relative to surrounding nonsensory epithelium. The related TRESK channel was not detected, whereas the acid-insensitive TASK-2 was. Using confocal imaging with pH-, Ca(2+)-, and voltage-sensitive dyes, we tested pharmacological agents that are diagnostic for these channels. Riluzole (500 microM), selective for TREK-1 and -2 channels, enhanced acid taste responses. In contrast, halothane (< or = approximately 17 mM), which acts on TREK-1 and TASK-1 channels, blocked acid taste responses. Agents diagnostic for other 2-pore domain and voltage-gated potassium channels (anandamide, 10 microM; Gd(3+), 1 mM; arachidonic acid, 100 microM; quinidine, 200 microM; quinine, 100 mM; 4-AP, 10 mM; and TEA, 1 mM) did not affect acid responses. The expression of 2-pore domain channels and our pharmacological characterization suggest that a matrix of ion channels, including one or more acid-sensitive 2-pore domain K channels, could play a role in sour taste transduction. However, our results do not unambiguously identify any one channel as the acid taste transducer.