Microelectrode study of the cellular reactions of the taste bud in the frog Rana temporaria

Microelectrophysiological studies reveal two types of cells in the taste bud of frog which differ by the level of their membrane potential. During vertical implantation of microelectrode through the apical part of the taste bud, the potential difference in the upper layer amounts to 15 mV. Further implantation of the electrode results in a stepwise decrease of the potential difference up to 27 mV. Cells of the deeper layer are located 12-24 micron lower from the apical surface. Stimulation of cells by solutions of chemical substances is accompanied by cell depolarization, its amplitude being proportional to stimulus concentration. The steepness of depolarization depends on the modality of the stimulus, being maximum for salts. The data obtained suggest that cells of the second layer, with a higher resting membrane potential level, are taste ones.     

Quantification and statistical verification of neuronal stimulus responses from noisy spike train data

Usually neuronal responses to short-lasting stimuli are displayed as peri-stimulus time histogram. The function estimated by such a histogram allows to obtain informations about stimulus-induced postsynaptic events as long as the interpretation is restricted to the first response component after the stimulus. The interpretation of secondary response components is much more difficult, as they may be either due to stimulus effects or represent an echo of the primary response. In the present paper two output functions are developed that do not show such an echoing of responses. The first one, the interspike interval change function, represents an ideal way to quantify a neuronal stimulus response as its amplitude was found to be almost independent of the stimulation strategy used during acquisition of the spike train data. The other function, the displaced impulses function, allows to verify the statistical significance of an observed response component. Both functions may be estimated from stimulus-correlated spike train data, even if the neuron under investigation shows considerable interspike-interval variability in the absence of stimulation. The concepts underlying these neuronal output functions are developed on simulated responses of a Hodgkin-Huxley-type model for a mammalian neuron at body temperature that is exposed to a transient excitatory conductance increase. Additionally, estimation of these output functions is also demonstrated on responses of human soleus motoneurons that were exposed to electrical stimuli of the tibial nerve in the popliteal fossa.     

Multiple sensitivity of single taste cells of the frog tongue to four basic taste stimuli

The electrical response of the taste cells of the frog fungiform papillae to four fundamental taste solutions (NaCl, acetic acid, quinine-HCl and sucrose) was studied by using the intracellular recording technique. The average value of resting membrane potential was 22.5 mV, inside negative. Each of the four taste solutions applied to the tongue produced a slow depolarizing potential, the receptor potential, on which no spike potential was superimposed. The amplitude of the receptor potentials increased linearly as a function of the logarithm of the concentration of the stimulus. Amplitudes of depolarizations to a given taste stimulation varied from one cell to another even within a single taste bud. Most of the cells responded to more than two of the four basic taste solutions. Sensitivity patterns in terms of the number of effective solutions and the relative effectiveness of different kinds of solutions were variable among cells. Statistical analysis suggests that at the receptor membranes of the taste cells, the sensitivities for the four basic stimuli are independent and random.     

Improved measures of phase-coupling between spikes and the Local Field Potential

An important tool to study rhythmic neuronal synchronization is provided by relating spiking activity to the Local Field Potential (LFP). Two types of interdependent spike-LFP measures exist. The first approach is to directly quantify the consistency of single spike-LFP phases across spikes, referred to here as point-field phase synchronization measures. We show that conventional point-field phase synchronization measures are sensitive not only to the consistency of spike-LFP phases, but are also affected by statistical dependencies between spike-LFP phases, caused by e.g. non-Poissonian history-effects within spike trains such as bursting and refractoriness. To solve this problem, we develop a new pairwise measure that is not biased by the number of spikes and not affected by statistical dependencies between spike-LFP phases. The second approach is to quantify, similar to EEG-EEG coherence, the consistency of the relative phase between spike train and LFP signals across trials instead of across spikes, referred to here as spike train to field phase synchronization measures. We demonstrate an analytical relationship between point-field and spike train to field phase synchronization measures. Based on this relationship, we prove that the spike train to field pairwise phase consistency (PPC), a quantity closely related to the squared spike-field coherence, is a monotonically increasing function of the number of spikes per trial. This derived relationship is exact and analytic, and takes a linear form for weak phase-coupling. To solve this problem, we introduce a corrected version of the spike train to field PPC that is independent of the number of spikes per trial. Finally, we address the problem that dependencies between spike-LFP phase and the number of spikes per trial can cause spike-LFP phase synchronization measures to be biased by the number of trials. We show how to modify the developed point-field and spike train to field phase synchronization measures in order to make them unbiased by the number of trials.     

Ionic basis of methacholine-induced shrinkage of dissociated eccrine clear cells

Summary Apical membrane currents were recorded from the taste pore of single taste buds maintained in the tongue of the rat, using a novel approach. Under a dissection microscope, the 150-m opening of a saline-filled glass pipette was positioned onto single fungiform papillae, while the mucosal surface outside the pipette was kept dry. Electrical responses of receptor cells to chemical stimuli, delivered from the pipette, were recorded through the pipette while the cells remained undamaged in their natural environment. We observed monophasic transient currents of 10-msec duration and 10–100 pA amplitude, apparently driven by action potentials arising spontaneously in the receptor cells. When perfusing the pipette with a solution of increased Na but unchanged Cl concentration, a stationary inward current (from pipette to taste cell) of 50–900 pA developed and the collective spike rate of the receptor cells increased. At a mucosal Na concentration of 250mm, the maximal collective spike rate of a bud was in the range of 6–10 sec^–1. In a phasic/tonic response, the high initial rate was followed by an adaptive decrease to 0.5–2 sec^–1. Buds of pure phasic response were also observed. Amiloride (30 m) present in the pipette solution reversibly and completely blocked the increase in spike rate induced by mucosal Na. Amiloride also decreased reversibly the stationary current which depended on the presence of mucosal Na (inhibition constant near 1 m). During washout of amiloride, spike amplitudes were first small, then increased, but always remained smaller than the amiloride-blockable stationary current of the bud. This is understandable since the stationary current of a bud arises from a multitude of taste cells, while each current spike is presumably generated by just one taste cell. We suggest that, in a Na-sensitive receptor cell, (i) the apical amiloride-blockable Na inward current serves as a generator current causing cell depolarization and firing of action potentials, and (ii) each current spike recorded from the taste pore arises mainly from a modulation of the apical Na inward current of this cell, because the action potential generated by the taste cell will transiently decrease or abolish the driving force for the apical Na inward current. The transients are indicators of receptor cell action potentials, which appear to be physiological responses of taste cellsin situ.     

RENEWAL OF CELLS WITHIN TASTE BUDS

Colchicine blocks mitotic division of the epithelial cells surrounding the taste bud of the rat tongue. Response to chemical stimulation decreases 50 per cent 3 hours after colchicine injection as measured by recording the electrical activity from the taste nerve bundle. Radioautography, using tritiated thymidine, shows that those epithelial cells surrounding the taste bud divide and that some of the daughter cells enter the taste bud and slowly move toward the center. The life span of the average cell is about 250 ± 50 hours, although some cells have a much shorter and others a much longer life span. These studies suggest that the cells within the taste bud, as well as the nerves, undergo considerable change with time. Corresponding changes in function are considered.     

Establishment of clonal cell lines of taste buds from a <Emphasis Type="Italic">p53</Emphasis><Superscript>−/−</Superscript> mouse tongue

A taste bud is a sensory organ and consists of 50-100 spindle-shaped cells. The cells function as taste acceptors. They have characteristics of both epithelial and neuronal cells. A taste bud contains four types of cells, type I, type II, type III cells, and basal cells. Taste buds were isolated from a tongue of a p53-deficient mouse at day 12, and 11 clonal taste bud (TBD) cell lines were established. In immunochemical analysis, all cell lines expressed cytokeratin 18, gustducin, T1R3, and neural cellular adhesion molecule, but not GLAST. In RT-PCR analysis, shh was not expressed in any of the cell lines. Further analysis with RT-PCR was conducted on four cell lines. They expressed G protein-coupled taste receptors; T1R3, T2R8 for sweet, bitter, umami. And they also expressed α-ENaC for salty taste. While, a candidate for sour receptor HCN4 was expressed in TBD-a1 and TBD-a7 lines. And another candidate for sour receptor PKD1L3 was slightly expressed in TBD-a1 and TBD-c1.     

The relationship between the Gabor elementary function and a stochastic model of the inter-spike interval distribution in the responses of visual cortex neurons

In a previously reported study (Berger et al. 1990) we analyzed distributions of interspike intervals recorded extracellularly from cat visual cortex under four stimulus conditions. Stimuli were gratings differing in orientation and spatial frequency. The probability density function of first passage time for a random walk with drift process, which is defined by its barrier height and drift coefficient, was used to characterize the generating process of axonal discharge under resting and stimulus conditions. Drift coefficient and barrier height were derived from the sample mean and standard deviation of the measured inter-spike intervals. For cells with simple receptive fields, variations in spatial frequency produced changes only in drift coefficient. Variations in barrier height were produced only by changes in orientation of the stimulus. Currently, the method used to analyze these data was implemented in a simulation which displayed the relationship between the interval distribution of impulses, the random walk which represents the time series characteristic of the spike train model and the Gabor filter function which represents the geometry of the receptive field process.     

Uteroglobin and other proteins in rabbit blastocyst fluid after development in vivo and in vitro

Summary The taste bud of the human fungiform papilla was examined by electron microscopy. Typical type I, type II, and type III cells were found along with contact sites with nerve endings. Vesicles in nerve fibers contacting type I and type II cells suggest that these cells may receive efferent impulses, whereas vesicles and granules in type III cells adjacent to (afferent) nerve fibers support the view that type III cells are sensory receptors. All of these features are virtually indistinguishable from those previously reported in fungiform taste buds of other mammals.Supported by fellowship from Campbell Institute for Food Research.     

Measuring spike timing distance in the Hindmarsh–Rose neurons

In the present paper, a simple spike timing distance is defined which can be used to measure the degree of synchronization with the information only encoded in the precise timing of the spike trains. Via calculating the spike timing distance defined in this paper, the spike train similarity of uncoupled Hindmarsh–Rose neurons in bursting or spiking states with different initial conditions is investigated and the results are compared with other spike train distance measures. Later, the spike timing distance measure is applied to study the synchronization of coupled or common noise-stimulated neurons. Counterintuitively, the addition of weak coupling or common noise doesn’t enhance the degree of synchronization although after critical values, both of them can induce complete synchronizations. More interestingly, the common noise plays opposite roles for weak and strong enough couplings. Finally, it should be noted that the measure defined in this paper can be extended to measure large neuronal ensembles and the lag synchronization.     

The topography and dynamics of cholinergic transmission in the myenteric plexus-smooth muscle preparation of the guinea-pig ileum; the effect of ketocyclazocine, a kappa opiate ligand

Output of acetylcholine (ACh), neurogenic electromyogram (NEMG) and contractions of guinea-pig ileum preparations were studied during stimulation by high-frequency trains of impulses. Under control conditions the output of ACh per impulse after 2nd to 4th impulses during train stimulation (30 Hz) was higher by 20-40% than the level of ACh output during the first impulse. In the presence of ketocyclazocine (KTZ, 80 nmol x l-1) the output of ACh evoked by the first impulse was more effectively inhibited than that after impulses 2 to 4 so that the increase was higher (80-170%). NEMG, a direct consequence of the localized action of released transmitter (ACh), was recorded in the longitudinal muscle 4 and 10 mm aborally from the focal stimulation site. The incidence of NEMG responses was higher at the proximal than at the distal site and was proportional to the number of impulses in a train (100 Hz). At the distal site KTZ suppressed the appearance of NEMG responses to single impulses whereas at the proximal site its effect was much less; and so was its effect at either site during train stimulation. It is concluded that in the course of train stimulation, sites of transmission more distant from the stimulation focus were recruited, and consequently the secretion of ACh in succeeding impulses was enhanced. KTZ might preferentially inhibit the propagation of excitation by the very first impulse.     

Induction of apoptosis by colchicine in taste bud and epithelial cells of the mouse circumvallate papillae

Apoptotic cells in the taste buds and epithelia of mouse circumvallate papillae after colchicine treatment were examined by the methods of in situ DNA nick-end labeling, immunocytochemistry, and electron microscopy. After colchicine treatment, numerous positive cells appeared in the taste buds by DNA nick-end labeling, and some epithelial cells in the basal and suprabasal layers in and around the circumvallate papillae also revealed positive staining. Condensed and fragmented nuclei with a high density were occasionally found in the taste bud cells and in the basal and suprabasal layer epithelial cells by electron-microscopic observation. An immunocytochemical reaction for tubulin revealed weak staining in taste bud cells, because of the depolymerization of microtubules, and a decrease of the microtubules in the taste bud cells was observed by electron microscopy. These results indicate that colchicine treatment of mice induces the apoptosis of taste bud and epithelial cells in the circumvallate papillae and dorsal epithelial cells around the circumvallate papillae.     

Maintenance of rat taste buds in primary culture

The differentiated taste bud is a complex end organ consisting of multiple cell types with various morphological, immunocytochemical and electrophysiological characteristics. Individual taste cells have a limited lifespan and are regularly replaced by a proliferative basal cell population. The specific factors contributing to the maintenance of a differentiated taste bud are largely unknown. Supporting isolated taste buds in culture would allow controlled investigation of factors relevant to taste bud survival. Here we describe the culture and maintenance of isolated rat taste buds at room temperature and at 37 degrees C. Differentiated taste buds can be sustained for up to 14 days at room temperature and for 3-4 days at 37 degrees C. Over these periods individual cells within the cultured buds maintain an elongated morphology. Further, the taste cells remain electrically excitable and retain various proteins indicative of a differentiated phenotype. Despite the apparent health of differentiated taste cells, cell division occurs for only a short period following plating, suggesting that proliferating cells in the taste bud are quickly affected by isolation and culture.     

Electrophysiological Studies of a Water Receptor Associated With the Taste Sensilla of the Blowfly

Electrophysiological evidence is given that water is the specific stimulus for a fourth sensory cell associated with the taste sensilla of the blowfly. Water elicited impulses from a single cell which responded in two distinct phases: an initial rapid rate of discharge followed by a lesser, sustained steady rate. The latter, in the case of sucrose solutions, was inhibited in direct proportion to the log of the osmotic pressure over a 10⁴ range of pressures. Other non-electrolytes inhibited, but the effect could not be simply correlated with parameters of the solutions. Electrolytes inhibited the water response more sharply and at lower concentrations. The inhibition in all cases was not dependent on impulses in the other sensory cells of the taste sensillum.     

Distribution of vimentin in the developing chick taste bud during the perihatching period.

The tissue environment within which taste bud cells develop has not been wholly elaborated. Previous studies of taste bud development in vertebrates, including the avian chick, have suggested that taste bud cells could arise from one, or several tissue sources (e.g. crest-mesenchyme, local ectoderm or endoderm). Thus, molecular markers which are present in gemmal as well as interfacing (peribud epithelium; mesenchyme-epithelium) regions, and their degree of expression during stages of taste bud development, are of special interest. The intermediate filament protein, vimentin, occurs in mesenchymal and mesodermally-derived (e.g. endothelial, fibroblast) cells as well as highly proliferating epithelium (e.g. tumors). The present study in chick gustatory tissue utilized antibodies against vimentin and the avidin-biotin-peroxidase technique to evaluate vimentin immunoreactivity (IR) within a timeframe which includes: 1) early stages of the taste bud primordium [embryonic days (E)17-E18)]; 2) the beginning of an accelerated bud cell proliferation at the time of initial, taste bud pore opening [around E19]; 3) attaining the adult complement of taste buds [around posthatch (H) day 1], and 4) completed organogenesis (H 17). During this time span, vimentin-IR was characterized in a region including and sometimes bridging taste bud and subepithelial connective tissue, whereas non-gustatory surrounding epithelium and salivary glands were vimentin-immuno-negative. Intragemmally, the proportion of vimentin-IR cells as related to total taste bud cells peaked at E19. These results indicate that vimentin expression, in part, is related to the onset of taste bud cell proliferation and suggest that mesenchyme could be one source of taste bud cells. Secondly, fibronectin, an extracellular matrix component of the epithelial basement membrane interface with mesenchyme, was expressed at or near the apical surfaces of taste bud cells projecting into the bud lumen, and in the basal gemmal region suggesting the possible role of fibronectin as a chemotactic anchor for differentiating and migrating taste bud receptor cells. Lastly, neuron-specific enolase-IR indicates that axonal varicosities are already present intragemmally at E17-E18, that is, during the incipient period of identifiable taste bud primordia.     

On the ultrastructure and permeability of taste buds of the marine teleost Ciliata mustela

The abundant taste buds of the barbels and free fin rays of the five bearded rockling, Ciliata mustela contain an average of 100–150 cells, falling into two types. Tubule-containing cells (‘t-cells’), tentatively identified as receptor cells, and each surrounded by fibril-containing cells (‘f-cells’) in the central part of the bud. t-Cells also occur in two concentric shells separated by indifferent epithelial cells at the periphery of the bud. f-Cells are characterized by their concentrations of fine fibrils, and by granules or vesicles of 180–190 mμ diameter. The 100 or so receptor cells in a taste bud are innervated by some 250 axons.Lanthanum penetrates more deeply into the extracellular space of taste buds than into the extracellular space of the general epithelium, perhaps indicating that a greater area than the mere protruding tip of receptor cells may be accessible to chemical stimulation. Degenerating cells may provide an important route of entry for such external agents.     

Immunohistochemical distribution of CD44 and some of its isoforms during human taste bud development

 Taste buds are accumulations of elongated bipolar cells situated on lingual papillae. The factors that determine the sites where a taste bud may develop are largely obscure, although it is known that the early invasion of nerve fibers plays one of the key roles in taste bud development and maturation. The conditions under which taste bud primordium cells develop are influenced by the interaction between epithelial cells and extracellular matrix molecules of the mesenchyma, such as hyaluronan. Thus, we investigated immunohistochemically the distribution pattern of the receptor for hyaluronan, CD44s, and its epithelial variant isoforms CD44v6 and CD44v9, in taste buds of human embryonic, fetal, perinatal, and adult tongues. Furthermore, we wanted to determine the temporal and spatial relationships of CD44 to sensory innervation of taste bud primordia. In early gestational stages (weeks 7–9), CD44 and its isoforms are expressed on membranes of apical perigemmal (marginal) cells covering taste bud primordia. It seems that CD44 serves as a marker for marginal cells (perigemmal cells) in early developmental stages. The expression of CD44 follows rather than precedes the invasion of sensory nerve fibers and the development of taste bud primordia (weeks 7–8). In new-born and adult taste bud cells, only the standard molecule, CD44s, is expressed; the variant isoforms, CD44v6 and CD44v9, occur only in the adjacent epithelium. From these results it is likely that marginal cells are of the utmost importance for the development and maturation of taste buds. We presume that CD44 is involved in local binding, reuptake, and degradation of hyaluronan in the early stages of taste bud formation. CD44 probably does not induce the transformation of epithelial cells into taste bud primordial cells. What is more, CD44 may change its function in the course of developmental events. Accepted: 13 January 1998     

Fingerprinting taste buds: intermediate filaments and their implication for taste bud formation

Intermediate filaments in taste organs of terrestrial (human and chick) as well as aquatic (Xenopus laevis) species were detected using immunohistochemistry and electron microscopy. During development, the potential importance of the interface between the taste bud primordium and non-gustatory adjacent tissues is evidenced by the distinct immunoreactivity of a subpopulation of taste bud cells for cytokeratins and vimentin. In human foetuses, the selective molecular marker for taste bud primordia, cytokeratin 20, is not detectable prior to the ingrowth of nerve fibres into the epithelium, which supports the hypothesis that nerve fibres are necessary for initiating taste bud development. Another intermediate filament protein, vimentin, occurs in derivatives of mesoderm, but usually not in epithelium. In humans, vimentin immunoreactivity is expressed mainly in border (marginal) epithelial cells of taste bud primordia, while in chick, vimentin expression occurs in most taste bud cells, whereas non-gustatory epithelium is vimentin immunonegative. Our chick data suggest a relationship between the degree of vimentin expression and taste bud cell proliferation especially during the perihatching period. It is suggested that surrounding epithelial cells (human) and mesenchymal cells (chick) may be contributing sources of developing taste buds. The dense perinuclear network of intermediate filaments especially in dark (i.e. non-sensory) taste disc cells of Xenopus indicates that vimentin filaments also might be associated with cells of non-gustatory function. These results indicate that the mechanisms of taste bud differentiation from source tissues may differ among vertebrates of different taxa.     

The Receptor Potential of the Taste Cell of the Rat

The electrical responses of the taste cell of the rat to chemical stimuli were studied by means of microelectrode techniques. Although large positive potential changes in the taste cell were usually elicited by taste stimuli, the response was a small negative potential change with respect to surrounding tissues if the microelectrode was thrust deeply into the taste bud. Both FeCl₃ and cocaine produced a positive change in the steady potential. If this new potential is larger than a certain equilibrium potential, reversal of the polarity of the potential change caused by a taste stimulus is observed. Gamma-aminobutyric acid and acetylcholine had no effect on the receptor steady potential nor on the receptor responses elicited by taste stimuli.     

Taste buds as peripheral chemosensory processors

Taste buds are peripheral chemosensory organs situated in the oral cavity. Each taste bud consists of a community of 50–100 cells that interact synaptically during gustatory stimulation. At least three distinct cell types are found in mammalian taste buds – Type I cells, Receptor (Type II) cells, and Presynaptic (Type III) cells. Type I cells appear to be glial-like cells. Receptor cells express G protein-coupled taste receptors for sweet, bitter, or umami compounds. Presynaptic cells transduce acid stimuli (sour taste). Cells that sense salt (NaCl) taste have not yet been confidently identified in terms of these cell types. During gustatory stimulation, taste bud cells secrete synaptic, autocrine, and paracrine transmitters. These transmitters include ATP, acetylcholine (ACh), serotonin (5-HT), norepinephrine (NE), and GABA. Glutamate is an efferent transmitter that stimulates Presynaptic cells to release 5-HT. This chapter discusses these transmitters, which cells release them, the postsynaptic targets for the transmitters, and how cell–cell communication shapes taste bud signaling via these transmitters.