Metabotropic glutamate receptors are involved in the detection of IMP and L-amino acids by mouse taste sensory cells.

G-protein-coupled receptors are thought to be involved in the detection of umami and L-amino acid taste. These include the heterodimer taste receptor type 1 member 1 (T1r1)+taste receptor type 1 member 3 (T1r3), taste and brain variants of mGluR4 and mGluR1, and calcium sensors. While several studies suggest T1r1+T1r3 is a broadly tuned lLamino acid receptor, little is known about the function of metabotropic glutamate receptors (mGluRs) in L-amino acid taste transduction. Calcium imaging of isolated taste sensory cells (TSCs) of T1r3-GFP and T1r3 knock-out (T1r3 KO) mice was performed using the ratiometric dye Fura 2 AM to investigate the role of different mGluRs in detecting various L-amino acids and inosine 5' monophosphate (IMP). Using agonists selective for various mGluRs such as (RS)-3,5-dihydroxyphenylglycine (DHPG) (an mGluR1 agonist) and L-(+)-2-amino-4-phosphonobutyric acid (l-AP4) (an mGluR4 agonist), we evaluated TSCs to determine if they might respond to these agonists, IMP, and three L-amino acids (monopotassium L-glutamate, L-serine and L-arginine). Additionally, we used selective antagonists against different mGluRs such as (RS)-L-aminoindan-1,5-dicarboxylic acid (AIDA) (an mGluR1 antagonist), and (RS)-α-methylserine-O-phosphate (MSOP) (an mGluR4 antagonist) to determine if they can block responses elicited by these L-amino acids and IMP. We found that L-amino acid- and IMP-responsive cells also responded to each agonist. Antagonists for mGluR4 and mGluR1 significantly blocked the responses elicited by IMP and each of the L-amino acids. Collectively, these data provide evidence for the involvement of taste and brain variants of mGluR1 and mGluR4 in L-amino acid and IMP taste responses in mice, and support the concept that multiple receptors contribute to IMP and L-amino acid taste.

Estradiol rapidly modulates odor responses in mouse vomeronasal sensory neurons.

In rodents, many social behaviors are driven by the sense of smell. The vomeronasal organ (VNO), part of the accessory olfactory system mediates many of these chemically driven behaviors. The VNO is heavily vascularized, and is readily accessible to circulating peptide or steroid hormones. Potentially, this allows circulating hormones to alter behavior through modulating the output of the primary sensory neurons in the VNO, the vomeronasal sensory neurons (VSNs). Based on this, we hypothesized that steroid hormones, in particular 17ß-estradiol, would modulate activity of VSNs. In this paper, we show that the estrogen receptors, GPR30 and ERα, were present in VSNs and that estradiol may be synthesized locally in the VNO. Our results also showed that 17ß-estradiol decreased responses of isolated VSNs to dilute urine, a potent natural stimulus, with respect to current amplitudes and depolarization. Further, 17ß-estradiol increased the latency of the first action potential (AP) and the AP amplitude. Additionally, calcium responses to sulfated steroids (present in the low molecular weight fraction of urine) that act as ligands for apical vomeronasal receptors were decreased by 17ß-estradiol. In conclusion, we show that estradiol modulates odorant responses mediated by VSNs and hence paves the way for future studies to better understand the mechanisms by which odorant mediated behavior is altered by endocrine status of the animal.

ATP excites mouse vomeronasal sensory neurons through activation of P2X receptors.

Purinergic signaling through activation of P2X and P2Y receptors is critically important in the chemical senses. In the mouse main olfactory epithelium (MOE), adenosine 5'-triphosphate (ATP) elicits an increase in intracellular calcium ([Ca(2+)](I)) and reduces the responsiveness of olfactory sensory neurons to odorants through activation of P2X and P2Y receptors. We investigated the role of purinergic signaling in vomeronasal sensory neuron (VSN)s from the mouse vomeronasal organ (VNO), an olfactory organ distinct from the MOE that responds to many conspecific chemical cues. Using a combination of calcium imaging and patch-clamp electrophysiology with isolated VSNs, we demonstrated that ATP elicits an increase in [Ca(2+)](I) and an inward current with similar EC(50)s. Neither adenosine nor the P2Y receptor ligands adenosine 5'-diphosphate, uridine 5'-triphosphate, and uridine-5'-disphosphate could mimic either effect of ATP. Moreover, the increase in [Ca(2+)](I) required the presence of extracellular calcium and the inward current elicited by ATP was partially blocked by the P2X receptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate and 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate. Consistent with the activation of P2X receptors, we detected gene expression of the P2X1 and 3 receptors in the VNO by Reverse transcription polymerase chain reaction (RT-PCR). When co-delivered with dilute urine, a natural stimulus, ATP significantly increased the inward current above that elicited by dilute urine or ATP alone. Mechanical stimulation of the VNO induced the release of ATP, detected by luciferin-luciferase luminometry, and this release of ATP was completely abolished in the presence of the connexin/pannexin hemichannel blocker, carbenoxolone. We conclude that the release of ATP could occur during the activity of the vasomotor pump that facilitates the movement of chemicals into the VNO for detection by VSNs. This mechanism could lead to a global increase in excitability and the chemosensory response in VSNs through activation of P2X receptors.

Neuromodulatory effects of gonadotropin releasing hormone on olfactory receptor neurons.

The terminal nerve is an anterior cranial nerve that innervates the lamina propria of the chemosensory epithelia of the nasal cavity. The function of the terminal nerve is ambiguous, but it has been suggested to serve a neuromodulatory role. We tested this hypothesis by exposing olfactory receptor neurons from mudpuppies (Necturus maculosus) to a peptide, gonadotropin releasing hormone (GnRH), that is found in cells and fibers of the terminal nerve. We used voltage-clamped whole-cell recordings to examine the effects of 0. 5-50 micrometer GnRH on voltage-activated currents in olfactory receptor neurons from epithelial slices. We found that GnRH increases the magnitude, but does not alter the kinetics, of a tetrodotoxin-sensitive inward current. This increase in magnitude generally begins 5-10 min after initial exposure to GnRH, is sustained for at least 60 min during GnRH exposure, and recovers to baseline within 5 min after GnRH is washed off. This effect occurred in almost 60% of the total number of olfactory receptor neurons examined and appeared to be seasonal: approximately 67% of neurons responded to GnRH during the courtship and mating season, compared with approximately 33% during the summer, when the sexes separate. GnRH also appears to alter an outward current in the same cells. Taken together, these data suggest that GnRH increases the excitability of olfactory receptor neurons and that the terminal nerve functions to modulate the odorant sensitivity of olfactory receptor neurons.

Uptake and release of neurotransmitter candidates, [3H]serotonin, [3H]glutamate, and [3H]gamma-aminobutyric acid, in taste buds of the mudpuppy, Necturus maculosus.

Neurotransmitters in vertebrate taste buds have not yet been identified with confidence. Serotonin, glutamate, and gamma-aminobutyric acid (GABA) have been postulated, but the evidence is incomplete. We undertook an autoradiographic study of [3H]serotonin, [3H]glutamate, and [3H]GABA uptake in lingual epithelium from the amphibian, Necturus maculosus, to determine whether taste bud cells would accumulate and release these substances. Lingual epithelium containing taste buds was incubated in low concentrations (0.4-6 microM) of these tritiated transmitter candidates and the tissue was processed for light microscopic autoradiography. Merkel-like basal taste cells accumulated [3H]serotonin. When the tissue was treated with 40 mM K+ after incubating the tissue in [3H]serotonin, cells released the radiolabelled transmitter. Furthermore, depolarization (KCl)-induced release of [3H]serotonin was Ca-dependent: if Ca2+ was reduced to 0.4 mM and 20 mM Mg2+ added to the high K+ bathing solution, Merkel-like basal cells did not release [3H]serotonin. In contrast, [3H]glutamate was taken up by several cell types, including non-sensory epithelial cells, Schwann cells, and some taste bud cells. [3H]glutamate was not released by depolarizing the tissue with 40 mM K+. [3H]GABA uptake was also widespread, but did not occur in taste bud cells. [3H]GABA accumulated in non-sensory epithelial cells and Schwann cells. These data support the hypothesis that serotonin is a neurotransmitter or neuromodulator released by Merkel-like basal cells in Necturus taste buds. The data do not support (nor rule out) a neurotransmitter role for glutamate or GABA in taste buds.

A cyclic nucleotide-dependent chloride conductance in olfactory receptor neurons.

Whole-cell membrane currents were recorded from olfactory receptor neurons from the neotenic salamander Necturus maculosus. Cyclic nucleotides, released intracellularly by flash photolysis of NPE-caged cAMP or NPE-caged cGMP, activated a transient chloride current. The chloride current could be elicited at constant voltage in the absence of extracellular Ca2+ as well as in the presence of 3 mM intracellular Ca2+, suggesting that the current did not require either voltage or Ca2+ transients for activation. The current could be elicited in the presence of the protein kinase inhibitors H-7 and H-89, and in the absence of intracellular ATP, indicating that activation was independent of protein kinase A activity. These results suggest that Necturus olfactory receptor neurons contain a novel chloride ion channel that may be directly gated by cyclic nucleotides.

Responses to glutamate in rat taste cells.

We studied taste transduction in sensory receptor cells. Specifically, we examined the actions of glutamate, a significant taste stimulus, on the membrane properties of taste cells by applying whole cell patch-clamp techniques to cells in rat taste buds isolated from foliate and vallate papillae. In 55 of 91 taste cells, bath-applied glutamate, at concentrations that elicit taste responses in the intact animal (10-20 mM), produced one of two different responses when the cell membrane was held near its presumed resting potential, -85 mV. "Sustained" glutamate responses were observed in the majority of taste cells (51 of 55) and consisted of an outward current (reduction of the maintained inward current). Sustained glutamate responses were voltage dependent, were decreased by membrane depolarization, and were accompanied by a reduction in membrane conductance. An analysis of the reversal potential of sustained responses in different ionic conditions and the effect of ion substitutions suggested that the currents were carried by cations. The data suggest that sustained responses are mediated by the closure of nonselective cation channels. Other taste cells (4 of 55) responded to glutamate with a transient inward current--so-called "transient" responses. Transient glutamate responses were voltage dependent and Na+ dependent, and appeared to be generated by nonspecific cation channels activated by glutamate. L(+)-2-amino-4-phosphonobutyric acid (L-AP4), a specific agonist of a metabotropic glutamate receptor (mGluR4) recently identified in rat taste cells and believed to be involved in taste transduction, mimicked the sustained glutamate responses. These findings indicate that glutamate, at concentrations at or slightly above threshold for taste in rats, produces two different membrane currents. The properties of these two responses suggest that there may be two different sets of nonspecific cation channels in taste cells, one closed by glutamate (sustained response) and the other opened (transient response). Our findings on the effect of L-AP4 suggest that the sustained response is the membrane mechanism mediating, at least in part, taste transduction for glutamate.

Serotonin modulates voltage-dependent calcium current in Necturus taste cells.

Necturus taste buds contain two primary cell types: taste receptor cells and basal cells. Merkel-like basal cells are a subset of basal cells that form chemical synapses with taste receptor cells and with innervating nerve fibers. Although Merkel-like basal cells cannot interact directly with taste stimuli, recent studies have shown that Merkel-like basal cells contain serotonin (5-HT), which may be released onto taste receptor cells in response to taste stimulation. With the use of whole cell voltage clamp, we examined whether focal applications of 5-HT to isolated taste receptor cells affected voltage-activated calcium current (I(Ca)). Two different effects were observed. 5-HT at 100 microM increased I(Ca) in 33% of taste receptor cells, whereas it decreased I(Ca) in 67%. Both responses used a 5-HT receptor subtype with a pharmacological profile similar to that of the 5-HT1A receptor, but the potentiation and inhibition of I(Ca) by 5-HT were mediated by two different second-messenger cascades. The results indicate that functional subtypes of taste receptor cells, earlier defined only by their sensitivity to taste stimuli, may also be defined by their response to the neurotransmitter 5-HT and suggest that 5-HT released by Merkel-like basal cells could modulate taste receptor function.

Neuromodulation of transduction and signal processing in the end organs of taste.

Chemical synapses transmit gustatory signals from taste receptor cells to sensory afferent axons. Chemical (and electrical) synapses also provide a lateral pathway for cells within the taste bud to communicate. Lateral synaptic pathways may represent some form of signal processing in the peripheral end organs of taste. Efferent synaptic input may also regulate sensory transduction in taste buds. To date, the synaptic neurotransmitter(s) or neuromodulator(s) released at chemical synapses in taste buds have not been identified unambiguously. This paper summarizes the attempts that have been made over the past 40 years to identify the neuroactive substances acting at taste bud synapses. We review the four traditional criteria for identifying chemical transmitters elsewhere in the nervous system-localization, uptake/degradation, release and physiological actions- and apply these criteria to neuroactive substances in taste buds. The most complete evidence to date implicates serotonin as a neuromodulator of taste transduction in the end organs. However, studies also suggest that adrenergic, cholinergic and peptidergic neurotransmission may be involved in taste buds.

Development of voltage-dependent currents in taste receptor cells.

Taste buds, the specialized end organs of gustation, comprise a renewing sensory epithelium. Undifferentiated basal cells become taste receptor cells by elongating and extending processes apically toward the taste pore. Mature taste cells are electrically excitable and express voltage-dependent Na+ Ca2+, and K+ currents, whereas basal stem cells exhibit only slowly activating K+ currents. The question we have addressed in the present study is whether contact with the taste pore is required for expression of voltage-dependent inward currents in Necturus taste cells. Mature taste cells were distinguished from developing cells by labeling the apical surface of the cells with fluorescein-isothiocyanate-conjugated wheat germ agglutinin (FITC-WGA), while the tissue was still intact. Elongate cells lacking FITC-WGA staining were interpreted as developing taste cells that had not yet reached the taste pore. Giga-seal whole-cell recording revealed that most developing taste cells lacked inward currents. Although some developing cells expressed inward currents, they were much smaller than those of mature cells, and the activation kinetics of the K+ currents were slower than in mature cells. Electron microscopy confirmed the identity of labeled and unlabeled cells. All FITC-WGA-labeled cells exhibited the ultrastructural characteristics of mature taste receptor cells, whereas most unlabeled taste cells had a characteristic morphology that was markedly different from mature taste receptor cells or basal stem cells. These data suggest that contact with the taste pore is required for the development of inward currents in taste cells.

Membrane properties of two types of basal cells in Necturus taste buds.

Necturus taste buds contain two types of basal cells: presumptive stem cells and Merkel-like basal cells. Both types of basal cells are small round cells located at the base of the taste bud, indistinguishable from each other with light microscopy. However, with electron microscopy, autoradiography, or immunocytochemistry, these two types of basal cells can be easily distinguished. We isolated basal cells from taste buds, characterized their voltage-dependent currents using gigaseal whole-cell recordings, and processed the cells for electron microscopy or immunocytochemistry. We were able to distinguish two cell types electrophysiologically and to correlate cell type with membrane properties. Isolated Merkel-like basal cells had several voltage-activated currents: transient, TTX-sensitive, inward Na+ current; sustained, saturating outward K+ current; and slowly inactivating inward Ca2+ current. These currents are similar to those observed in taste receptor cells. In contrast, presumptive stem cells from Necturus taste buds only had outward K+ currents.

Merkel-like basal cells in Necturus taste buds contain serotonin.

Several types of cells have been identified in vertebrate taste buds, including dark cells, light cells, intermediate cells, type III cells, and basal cells. The physiological roles of these cell types are not well understood, especially those of basal cells. In this paper we show that there are two types of basal cells in taste buds from Necturus maculosus. One type of basal cell is an undifferentiated cell, presumably a stem cell. By combining light microscopic immunocytochemistry with electron microscopy, we show that the other type of basal cell is positive for serotonin-like immunoreactivity and that these cells have ultrastructural features similar to those found in cutaneous Merkel cells. Based on these findings, and the fact that the Merkel-like taste cells have been shown to make synaptic contacts with adjacent taste cells and with innervating nerve fibers, we conclude that these Merkel-like basal taste cells are serotonergic interneurons.

Ultrastructure of taste cells and synapses in the mudpuppy Necturus maculosus.

Taste buds in the mudpuppy Necturus maculosus were examined with electron microscopy. Three cell types (dark, light, and basal) were identified and reconstructed from serial thick sections. Dark and light cells extend from the basal lamina to the surface of the tongue. The apical process of the dark cells was usually quite lamellar when viewed in cross section, in contrast to light cells, whose apical process appeared more cylindrical. Basal cells are situated at the base of the bud and do not extend processes to the surface of the tongue. The cytoplasm of basal cells contains numerous clear and dense-cored vesicles. Small, spinelike processes (2-3 microns in length) project outward from the basal cells into the cytoplasm of the surrounding tast receptor cells. Morphologically, basal cells in mudpuppy taste buds resemble Merkel cells. Unmyelinated afferent nerve fibers enter the taste bud at the base and course through the lower portion of the bud. Synapses were found between taste receptor cells and nerve fibers, between basal cells and nerve fibers, and between basal cells and taste receptor cells. Over 65% of the synapses observed in the mudpuppy taste bud involved the basal cell. These findings suggest that basal cells play some role in chemosensory signal processing or integration of the taste response.

Ultrastructure of apical specializations of taste cells in the mudpuppy, Necturus maculosus.

The first interaction of taste stimuli with lingual chemoreceptors occurs on the apical membrane of taste cells, since only that portion is exposed to the oral cavity. To gain better insight into this interaction, we examined the pore region of taste buds in Necturus maculosus with scanning electron microscopy (SEM), transmission electron microscopy, and high-voltage electron microscopy. SEM of the pore reveals a patchwork distribution of three morphologically distinct types of apical specializations: long and branched (LB) microvilli, short and unbranched (SU) microvilli, and bundles of stereocilia. As demonstrated in thin and thick sections, LB microvilli are specializations of dark cells, SU microvilli are the apical specializations of light cells, and stereocilia arise from a cell that has the cytoplasmic markers characteristic of light cells. When left in place, the pore mucus completely covers the SU microvilli and partially covers the LB microvilli. However, stereocilia project above the surface and thus are highly exposed to taste stimuli in the oral cavity. These three morphologically distinct types of apical specializations may reveal functional differences among taste cells. The initial interaction between chemical stimulus and taste cell, and possibly chemoreceptor specificity itself, may be influenced by the morphology of the apical ending.

Ultrastructure of mouse vallate taste buds: II. Cell types and cell lineage.

The lifespan of cells in the mouse taste bud was examined with high-voltage electron microscopic (HVEM) autoradiography (ARG) after giving a single injection of 3H-thymidine. Animals were killed at 1 hour, 6 hours, 12 hours, 24 hours, and then daily up through 10 days postinjection. Lingual tissues were prepared for HVEM ARG so that we could identify and characterize labeled cells. Four categories of taste cells were identified: basal, dark, intermediate, and light cells. Basal cells were polygonal cells located near the basolateral sides of the taste buds and were characterized primarily by the presence of filaments attached to the nuclear envelope. Dark and light cells had the typical features described by previous authors. Intermediate cells had features in between those of dark and light cells. Over 90% of the cells labeled in the first 2 days following injection of 3H-thymidine were basal cells. Labeled dark cells appeared 6 hours after injection, reached their peak incidence at the fourth day postinjection, and then gradually decreased. Labeled intermediate cells were identified after the appearance of dark cells (12 hours) and reached a peak incidence at the fifth day after injection of 3H-thymidine. Lastly, labeled light cells were first observed on the fourth day postinjection and continued to increase until the tenth day, when they constituted 45% of the labeled cells. These data support the hypothesis that there is one cell line in the mouse vallate taste bud that undergoes morphological changes in its lifespan.

Ultrastructure of mouse vallate taste buds. I. Taste cells and their associated synapses.

The ultrastructural features of murine vallate taste bud cells and their associated synapses have been examined in thin and thick sections with conventional transmission electron microscopy and high-voltage electron microscopy. Computer-assisted reconstructions from serial sections were utilized to aid in visualization of taste bud cell-nerve fiber synapses. We have classified taste bud cells on the basis of previously established criteria-namely, size of the nucleus, shape and density of chromatin, density of cytoplasm, and presence or absence of dense-cored or clear vesicles, other cytoplasmic organelles, and synaptic foci. Both dark cells and light cells are present, as well as cells with intermediate morphological characteristics. Synapses were observed from taste bud cells onto nerve fiber processes. In virtually all instances, synapses are associated with the nuclear region of the taste cell. These synapses are characterized by the presence of 40-70 nm clear vesicles embedded in a thickened presynaptic membrane separated from the postsynaptic membrane by a 16-30 nm cleft. Synapses are not unique to any particular cell type. Dark, intermediate, and light cells all synapse onto nerve fibers. Two general types of synapses exist: spot (or macular) and fingerlike. In the latter, the postsynaptic region of the neuronal process protrudes into an invagination of the taste cell membrane. Differences in synaptic morphology are not correlated with taste cell type. In some cases a single taste cell was observed to possess both macular and fingerlike synapses adjacent to one another, forming a synaptic complex onto a single neuronal process. On the basis of the presence of synaptic contacts, we conclude that both "dark" and "light" cells are gustatory receptors.

Calcium in the spine apparatus of dendritic spines in the dentate molecular layer.

With a pyroantimonate precipitation technique, we have demonstrated Ca2+ in the sacs of the dendritic spine apparatus in the SER of dendrites and axon terminals, in synaptic vesicles, multivesicular bodies, mitochondria, and glial processes of the dentate molecular layer. It is speculated that the spine apparatus may be a Ca2+ sequestering organelle which may regulate levels of intraspinal and intradendritic Ca2+ during synaptic activity.

Cytoplasmic actin in neuronal processes as a possible mediator of synaptic plasticity.

We have demonstrated that, after permeation with saponin and decoration with S-1 myosin subfragment, the cytoplasmic actin is organized in filaments in dendritic spines, dendrites, and axon terminals of the dentate molecular layer. The filaments are associated with the plasma membrane and the postsynaptic density with their barbed ends and also in parallel with periodical cross bridges. In the spine stalks and dendrites, the actin filaments are organized in long strands. Given the contractile properties of actin, these results suggest that the cytoplasmic actin may be involved in various forms of experimentally induced synaptic plasticity by changing the shape or volume of the pre- and postsynaptic side and by retracting and sprouting synapses.