Amiloride-sensitive sodium currents in fungiform taste cells of rats chronically exposed to nicotine.

Many studies have demonstrated that chronic exposure to nicotine, one of the main components of tobacco smoke, has profound effects on the functionality of the mammalian taste system. However, the mechanisms underlying nicotine action are poorly understood. In particular no information is available on the chronic effect of nicotine on the functioning of taste cells, the peripheral detectors which transduce food chemicals into electrical signals to the brain. To address this issue, I studied the membrane properties of rat fungiform taste cells and evaluated the effect of long-term exposure to nicotine on the amiloride-sensitive sodium currents (ASSCs). These currents are mediated by the epithelial sodium channels (ENaC) thought to be important, at least in part, in the transduction of salty stimuli. Patch-clamp recording data indicated that ASSCs in taste cells from rats chronically treated with nicotine had a reduced amplitude compared to controls. The pharmacological and biophysical analysis of ASSCs revealed that amplitude reduction was not dependent on changes in amiloride sensitivity or channel ionic permeability, but likely derived from a decrease in the activity of ENaCs. Since these channels are considered to be sodium receptors in taste cells, my results suggest that chronic exposure to nicotine hampers the capability of these cells to respond to sodium ions. This might represent a possible cellular mechanism underlying the reduced taste sensitivity to salt typically found in smokers.

Daytime melatonin treatment influences food carrying (hoarding) behavior in rats.

OBJECTIVE: Three experiments were performed to verify whether melatonin (MEL) may influence hoarding behavior in rats. This hypothesis was supported by the consideration that leptin treatment decreases food hoarding in hamsters and that an inverse relation exists between plasma leptin concentration and MEL treatment. METHODS: Male Sprague-Dawley rats housed individually and kept on 12h:12h light-dark cycle were used. First experiment was performed to check whether a bimodal distribution of food hoarding scores exists in rats, and to select two groups of high (HH-rats) or low (LH-rats) hoarding. Second experiment was designed to verify whether MEL treatment modifies food-hoarding, while the third one was performed to investigate whether MEL treatment was able to modify the reciprocal relation between leptin and MEL plasma concentration. RESULTS: In rats the hoarding tendency fell into a bimodal distribution and the plasma leptin concentration was significantly higher in HH-rats than LH-rats. When MEL was injected, circulating concentration of leptin was decreased in both HH-rats and LH-rats and such MEL treatment significantly increased the number of pellets hoarded by LH-rats but not that hoarded by HH-rats. CONCLUSIONS: MEL influences the food-hoarding in rats either directly, or indirectly by the MEL and leptin reciprocal interaction. Our results support the hypothesis that the endocrine system either directly, by the action of one or more combined hormones (MEL, leptin), or indirectly via its actions on neural substrates determines, at least in part, food-hoarding of rats.

Long-term effects of nicotine on rat fungiform taste buds.

Nicotine, an alkaloid found in tobacco smoke, has been recognized as capable of inducing changes in taste functionality in conditions of chronic exposure. The mechanisms underlying these sensory alterations, however, are currently unknown. We addressed this issue by studying the long-term effects of nicotine on the anatomical features of taste buds, the peripheral end-organs of taste, in rat fungiform papillae. Nicotine was administered to rats via drinking water over a period of 3 weeks, which represents a standard method to achieve chronic drug exposure in laboratory animals. We found that prolonged administration of nicotine induced a significant reduction in the size of fungiform taste buds, without affecting their total number on the rat tongue. Morphometric measurements as well as evaluations of taste cell membrane capacitance suggested that the reduced size of taste organs was determined by a decrease in the number of cells per taste bud. In addition, chronic treatment with nicotine caused an increase in the relative density of cells expressing gustducin, a specific G protein alpha-subunit found in some taste cells and involved in bitter/sweet transduction. Interestingly, changes in the expression pattern of gustducin turned out to be more pronounced in periadolescent/adolescent than in adult rats. As a whole, our data indicate that long-term nicotine administration induces significant changes in the anatomical properties of taste buds in rat fungiform papillae. These changes could have a profound impact on the sensory information relayed to the brain; therefore, they may be responsible, at least in part, for the alterations in taste functionality observed during chronic nicotine exposure, a condition found in regular smokers.

A dynamic population of excitable cells: the taste receptor cells.

Taste receptor cells (TRCs) represent an unique opportunity to study a dynamic population of excitable cells that undergoes two basic neurobiological processes: postnatal development and cell turnover. We have begun to investigate the functional properties of TRCs and how they mature over time by applying the patch-clamp technique to single cell in taste buds isolated from mouse vallate papilla during postnatal development. We have focussed our attention on a well-defined functional group of taste cells, called Na/OUT cells, and on their voltage-gated K+, and Cl- currents (I(K) and I(Cl), respectively). As in neurons, I(K) and I(Cl) underlie action potential waveform and firing properties in these cells. By analyzing the relative occurrence of I(K) and I(Cl) among cells, we found that in adult mice three different electrophysiological phenotypes of Na/OUT cells could be detected: cells with only I(K) (K cells); cells with both I(K) and I(Cl) (K + Cl cells); and cells with I(Cl) (Cl cells). On the contrary, at early developmental stages (2-4 postnatal day, PD) there were no Cl cells, which appeared at PD 8. The analysis of the changes in current amplitude (which continuously increased in developing cells) during postnatal development suggested that Cl cells and K + Cl cells likely represented a single functional line different from K cells. In addition, electrophysiological data were consistent with the interpretation that Cl cells derived from some K + Cl cells by suppression of I(K). The dynamics of the expression of I(K) and I(Cl) during postnatal development likely reflects a mechanism that could also operate during turnover.

Pheromone reception in mammals.

Pheromonal communication is the most convenient way to transfer information regarding gender and social status in animals of the same species with the holistic goal of sustaining reproduction. This type of information exchange is based on pheromones, molecules often chemically unrelated, that are contained in body fluids like urine, sweat, specialized exocrine glands, and mucous secretions of genitals. So profound is the relevance of pheromones over the evolutionary process that a specific peripheral organ devoted to their recognition, namely the vomeronasal organ of Jacobson, and a related central pathway arose in most vertebrate species. Although the vomeronasal system is well developed in reptiles and amphibians, most mammals strongly rely on pheromonal communication. Humans use pheromones too; evidence on the existence of a specialized organ for their detection, however, is very elusive indeed. In the present review, we will focus our attention on the behavioral, physiological, and molecular aspects of pheromone detection in mammals. We will discuss the responses to pheromonal stimulation in different animal species, emphasizing the complicacy of this type of communication. In the light of the most recent results, we will also discuss the complex organization of the transduction molecules that underlie pheromone detection and signal transmission from vomeronasal neurons to the higher centers of the brain. Communication is a primary feature of living organisms, allowing the coordination of different behavioral paradigms among individuals. Communication has evolved through a variety of different strategies, and each species refined its own preferred communication medium. From a phylogenetic point of view, the most widespread and ancient way of communication is through chemical signals named pheromones: it occurs in all taxa, from prokaryotes to eukaryotes. The release of specific pheromones into the environment is a sensitive and definite way to send messages to other members of the same species. Therefore, the action of an organism can alter the behavior of another organism, thereby increasing the fitness of either or both. Albeit slow in transmission and not easily modulated, pheromones can travel around objects in the dark and over long distances. In addition, they are emitted when necessary and their biosynthesis is usually economic. In essence, they represent the most efficient tool to refine the pattern of social behaviors and reproductive strategies.

Amiloride-sensitive sodium currents in identified taste cells of the frog.

Sodium ions occurring in food are thought to be detected, at least in part, through specific amiloride-sensitive, sodium channels (ASSCs) localized in taste receptor cells. Cells within taste buds are morphologically heterogeneous, and include taste receptor cells and other cells that could perform a support or even transduction role. It is not known whether subsets of the taste bud cells express ASSCs, and whether the properties of these channels are similar. By applying the patch-clamp technique to morphologically distinct cells, the supporting wing cells, isolated from the frog taste disk, I have found functional ASSCs that are moderately sensitive to amiloride (Ki 3-4 microM) and which are distinctly lower in affinity for amiloride than reported frog taste receptor cells (Ki 0.2 microM). These results support the hypotheses of the existence of distinct, functional ASSCs in different cell morphotypes, at least in frog taste organs.

Modulation of potassium current and calcium influx by somatostatin in rod bipolar cells isolated from the rabbit retina via sst2 receptors.

Somatostatin (somatotropin release-inhibiting factor, SRIF) receptor subtypes are expressed by several retinal neurons, suggesting that SRIF acts at multiple levels of the retinal circuitry, although functional data on this issue are scarce. Of the SRIF receptors, the sst2A isoform is expressed by rod bipolar cells (RBCs) of the rabbit retina, and in isolated RBCs we studied the role of sst2 receptors in modulating both K+ current (IK) and the intracellular free [Ca2+] ([Ca2+]i) using both voltage-clamp and Ca2+-imaging techniques. SRIF and octreotide (a SRIF agonist that binds to sst2 receptors) inhibited that component of IK corresponding to the activation of large-conductance, Ca2+- and voltage-dependent K+ channels (IBK) and reduced the K+-induced [Ca2+]i accumulation, suggesting that SRIF effects on IBK may have been secondary to inhibition of Ca2+ channels. Octreotide effects on IBK or on [Ca2+]i accumulation were prevented by RBC treatment with L-Tyr8-Cyanamid 154806, a novel sst2 receptor antagonist, indicating that SRIF effects were mediated by sst2 receptor activation. The present data indicate that SRIF may modulate the information flow through second-order retinal neurons via an action predominantly at sst2 receptors, contribute to the proposition that SRIF be added to the growing list of retinal neuromodulators, and suggest that one of its possible roles in the retina is to regulate transmitter release from RBCs.

Mouse taste cells with glialike membrane properties.

Taste buds are sensory structures made up by tightly packed, specialized epithelial cells called taste cells. Taste cells are functionally heterogeneous, and a large proportion of them fire action potentials during chemotransduction. In view of the narrow intercellular spaces within the taste bud, it is expected that the ionic composition of the extracellular fluid surrounding taste cells may be altered significantly by activity. This consideration has led to postulate the existence of glialike cells that could control the microenvironment in taste buds. However, the functional identification of such cells has been so far elusive. By using the patch-clamp technique in voltage-clamp conditions, I identified a new type of cells in the taste buds of the mouse vallate papilla. These cells represented about 30% of cells patched in taste buds and were characterized by a large leakage current. Accordingly, I named them "Leaky" cells. The leakage current was carried by K(+), and was blocked by Ba(2+) but not by tetraethylammonium (TEA). Other taste cells, such as those possessing voltage-gated Na(+) currents and thought to be chemosensory in function, did not express any sizeable leakage current. Consistent with the presence of a leakage conductance, Leaky cells had a low input resistance (approximately 0.25 G Omega). In addition, their zero-current ("resting") potential was close to the equilibrium potential for potassium ions. The electrophysiological analysis of the membrane currents remaining after pharmacological block by Ba(2+) revealed that Leaky cells also possessed a Cl(-) conductance. However, in resting conditions the membrane of these cells was about 60 times more permeable to K(+) than to Cl(-). The resting potassium conductance in Leaky cells could be involved in dissipating rapidly the increase in extracellular K(+) during action potential discharge in chemosensory cells. Thus Leaky cells might represent glialike elements in taste buds. These findings support a model in which specific cells control the chemical composition of intercellular fluid in taste buds.

Electrophysiological characterization of a putative supporting cell isolated from the frog taste disk.

Chemosensory cells in vertebrate taste organs have two obvious specializations: an apical membrane with access to the tastants occurring in food, and synapses with sensory axons. In many species, however, certain differentiated taste cells have access to the tastants but lack any synaptic contacts with axons, and a supportive rather than chemosensory function has been attributed to them. Until now, no functional data are available for these taste cells. To begin to understand their role in taste organ physiology, we have characterized with patch-clamp recording techniques the electrophysiological properties of a putative supporting cell-the so-called wing cell-isolated from frog taste disks. Wing cells were distinguished from chemosensory elements by the presence of a typical, sheet-like apical process. Their resting potential was approximately -52 mV, and the average input resistance was 4.8 GOmega. Wing cells possessed voltage-gated Na+ currents sensitive to TTX, and an inactivating, voltage-gated K+ current sensitive to TEA. Current injections elicited single action potentials but not repetitive firing. We found no evidence for voltage-gated Ca2+ currents under various experimental conditions. Amiloride-sensitive Na+ channels, thought to be involved in Na+ chemotransduction, were present in wing cells. Many of the membrane properties of wing cells have been also reported for chemosensory taste cells. The presence of ion channels in wing cells might be suggestive of a role in controlling the microenvironment inside the taste organs or the functioning of chemosensory cells or both. In addition, they might participate directly in the sensory transduction events by allowing loop currents to flow inside the taste organs during chemostimulation.

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.

Membrane properties and cell ultrastructure of taste receptor cells in Necturus lingual slices.

1. Whole cell patch-clamp recordings and electron micrographs were obtained from cells in Necturus taste buds in lingual slices to study their membrane properties and to correlate these properties with cell ultrastructure. 2. Two different populations of taste receptor cells could be identified: one type possessed voltage-gated Na+ and K+ (noninactivating) currents (group 1 cells); the other type possessed only K+ (inactivating) currents (group 2 cells). 3. The zero-current ("resting") potential (Vo) and whole cell resistance (Ro) of these two types of taste cells differed significantly. For group 1 cells, on average, Vo = -75 mV and Ro = 24.6 G omega, and for group 2 cells, Vo = -49 mV and Ro = 48.9 G omega. The difference in Ro was not explained completely by differences in cell sizes, suggesting that intrinsic membrane properties differed between the populations. 4. Cells injected with biocytin were the electron microscope after tissues were reacted with majority (14 of 16) of cells with voltage-gated Na+ and K+ currents (group 1 cells) were characterized by abundant rough endoplasmic reticulum and dense granular packets in the apical process. These are features of dark cells. All the cells that only possessed K+ currents (group 2 cells) were characterize by well-developed smooth endoplasmic reticulum and an absence granular packets. These features characterize light cells. 5. These findings indicate that there is a good, although not exact, correlation between electrophysiological properties and cell morphotype in Necturus taste bud cells. All dark cells possessed Na+ and K+ currents and thus would be expected to be capable of generating action potentials. Most light cells only possessed outward K+ currents and thus would be incapable of generating action potentials.

Estimation of the junctional resistance between electrically coupled receptor cells in Necturus taste buds.

Junctional resistance between coupled receptor cells in Necturus taste buds was estimated by modeling the results from single patch pipette voltage clamp studies on lingual slices. The membrane capacitance and input resistance of coupled taste receptor cells were measured to monitor electrical coupling and the results compared with those calculated by a simple model of electrically coupled taste cells. Coupled receptor cells were modeled by two identical receptor cells connected via a junctional resistance. On average, the junctional resistance was approximately 200-300 M omega. This was consistent with the electrophysiological recordings. A junctional resistance of 200-300 M omega is close to the threshold for Lucifer yellow dye-coupling detection (approximately 500 M omega). Therefore, the true extent of coupling in taste buds might be somewhat greater than that predicted from Lucifer yellow dye coupling. Due to the high input resistance of single taste receptor cells (> 1 G omega), a junctional resistance of 200-300 M omega assures a substantial electrical communication between coupled taste cells, suggesting that the electrical activity of coupled cells might be synchronized.

Reduction of electrical coupling between Necturus taste receptor cells, a possible role in acid taste.

Cytoplasmic acidification in taste receptor cells is thought to be involved, at least in part, in acid taste transduction. Since in taste buds about 20% of the receptor cells are electrically coupled, we have tested whether reduction in intracellular pH affects these lateral synaptic interactions. By applying the patch clamp technique to a slice preparation of Necturus lingual epithelium, we found that electrical coupling between taste receptor cells was strongly reduced by cytoplasmic acidification. Therefore, electrical coupling in taste buds might be modified during acid stimulation.

Identification of electrophysiologically distinct cell subpopulations in Necturus taste buds.

We used the patch clamp technique to record from taste cells in thin transverse slices of lingual epithelium from Necturus maculosus. In this preparation, the epithelial polarity and the cellular organization of the taste buds, as well as the interrelationships among cells within the taste bud, were preserved. Whole-cell recording, combined with cell identification using Lucifer yellow, allowed us to identify distinct subpopulations of taste cells based on their electrophysiological properties. Receptor cells could be divided in two groups: one group was characterized by the presence of voltage-gated Na+, K+, and Ca2+ currents; the other group was characterized by the presence of K+ currents only. Therefore, receptor cells in the first group would be expected to be capable of generating action potentials, whereas receptor cells in the second group would not. Basal taste cells could also be divided into two different groups. Some basal cells possessed voltage-gated Na+, K+, and Ca2+ conductances, whereas other basal cells only had K+ conductance. In addition to single taste cells, we were able to identify electrically coupled taste cells. We monitored cell-cell coupling by measuring membrane capacitance and by observing Lucifer yellow dye coupling. Electrical coupling in pairs of dye-coupled taste receptor cells was strong, as indicated by experiments with the uncoupling agent 1-octanol. Electrically coupled receptor cells possessed voltage-gated currents, including Na+ and K+ currents. The electrophysiological differentiation among taste cells presumably is related to functional diversifications, such as different chemosensitivities.

Mediation of responses to calcium in taste cells by modulation of a potassium conductance.

Calcium salts are strong taste stimuli in vertebrate animals. However, the chemosensory transduction mechanisms for calcium are not known. In taste buds of Necturus maculosus (mud puppy), calcium evokes depolarizing receptor potentials by acting extracellularly on the apical ends of taste cells to block a resting potassium conductance. Therefore, divalent cations elicit receptor potentials in taste cells by modulating a potassium conductance rather than by permeating the cell membrane, the mechanism utilized by monovalent cations such as sodium and potassium ions.

Reduction in extrasynaptic acetylcholine sensitivity of axotomized anterior pagoda neurones in the leech.

1. The effects of axotomy on the sensitivity of the leech anterior pagoda (AP) neurone to acetylcholine (ACh) and carbamylcholine (CCh) have been studied 1-5 days after axon interruption. 2. Hyperpolarizing responses to ionophoretically applied ACh and CCh have been recorded intracellularly from desheathed cell bodies of normal and axotomized neurones. The electrical properties of the membrane have also been measured in the same neurones. 3. Axotomy produced a progressive loss of sensitivity to both ACh and CCh with a similar percentage reduction. 4. No significant changes have been found in the time to peak and in the reversal potential of the responses to agonists, or in the number of drug molecules needed to combine with a single receptor to produce a response. 5. Interruption of nerve roots and connectives which do not contain the AP axon did not induce the alterations of ACh sensitivity observed after axotomy. 6. It is concluded that the loss of ACh sensitivity following axotomy is due to a reduction in density of functional ACh receptors (AChRs).