Patch-clamp study of isolated taste receptor cells of the frog

Summary Taste discs were dissected from the tongue ofR. ridibunda and their cells dissociated by a collagenase/low Ca/mechanical agitation protocol. The resulting cell suspension contained globular epithelial cells and, in smaller number, taste receptor cells. These were identified by staining properties and by their preserved apical process, the tip of which often remained attached to an epithelial (associated) cell. When the patch pipette contained 110mm KCl and the cells were superfused with NaCl Ringer's during whole-cell recording, the mean zero-current potential of 22 taste receptor cells was –65.2 mV and the slope resistance 150 to 750 M. Pulse-depolarization from a holding voltage of –80 mV activated a transient TTX-blockable inward Na current. Activation became noticeable at –25 mV and was half-maximal at –8 mV. Steady-state inactivation was half-maximal at –67 mV and complete at –50 mV. Peak Na current averaged –0.5 nA/cell. The Ca-ionophore A23187 shifted the activation and inactivation curve to more negative voltages. Similar shifts occurred when the pipette Ca was raised. External Ni (5mm) shifted the activation curve towards positive voltages by 10 mV. Pulse depolarization also activated outward K currents. Activation was slower than that of Na current and inactivation slower still. External TEA (7.5mm) and 4-aminopyridine (1mm) did not block, but 5mm Ba blocked the K currents. K-tail currents were seen on termination of depolarizing voltage pulses. A23187 shifted theI _K(V)-curve to more negative voltages. Action potentials were recorded when passing pulses of depolarizing outward current. Of the frog gustatory stimulants, 10mm Ca caused a reversible 5-to 10-mV depolarization in the current-clamp mode. Quinine (0.1mm, bitter) produced a reversible depolarization accompanied by a full block of Na current and, with slower time-course, a partial block of K currents. Cyclic AMP (5mm in the external solution or 0.5 m in the pipette) caused reversible depolarization (to –40 to –20 mV) due to partial blockage of K currents, but only if ATP was added to the pipette solution. Similar responses were elicited by stimulating the adenylate cyclase with forskolin. Blockage of cAMP-phosphodiesterase enhanced the response to cAMP. These results suggest that cAMP may be one of the cytosolic messengers in taste receptor cells. Replacement of ATP by AMP-PNP in the pipette abolished the depolarizing response to cAMP. Inclusion of ATP--S in the pipette caused slow depolarization to –40 to –20 mV, due to partial blockage of K currents. Subsequently, cAMP was without effect. The remaining K currents were blockable by Ba. These results suggest that cAMP initiates phosphorylation of one set of K channels to a nonconducting conformation.     

Gustatory sensitivity of the herbivore Tilapia zillii to amino acids

The gustatory sensitivity of the herbivorous fish Tilapia zillii was investigated using electrophysiological techniques. Integrated responses from facial taste nerves were recorded during stimulation by l l amino acids. All compounds proved to be effective stimuli. The relative stimulatory efficacy (RSE) determined at 1 × 10 ⁻³ m indicated that stimuli can be organized into three groups on the basis of similarity of efficacy. Effects of pH which altered the RSE of some amino acids are documented. Amino acid RSE differed to some extent from those of previously studied carnivorous fish. There was concordance between the composition of preferred food, effective feeding stimuli and the peripheral receptor responses. These observations support the hypothesis that peripheral chemosensitivity might serve as a mechanism of feeding specialization in some fish.     

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.     

Ultrastructural and histochemical observations on the tegument of Gastrodiscoides hominis (Paramphistoma: Digenea).

The tegument of the paramphistome, Gastrodiscoides hominis, is basically similar to that of other digeneans. It is folded into concentrically arranged furrows and ridges bearing numerous tightly packed tubercules, and extends into the oral cavity. An area of specialized tegument is present on the ventral surface, anterior to the disc region. Mitochondria are absent from the tegumental syncytium and underlying tegumental cells, suggesting that the tegument may serve principally as a protective layer rather than in active uptake phenomena. However, extensions of the lymph and parenchyma systems are closely associated with the base of the tegumental syncytium and may provide ATP for active processes. Ciliated and non-ciliated sensory papillae are present, particularly around the oral opening. Numerous lymph channels are present in the sub-tegument and may be involved in osmoregulation.     

Freeze-fracture study of taste bud pores in the foliate papillae of the rabbit

Summary In freeze-fractured specimens of taste buds from the foliate papillae of rabbits, the intercellular spaces are separated from the pore of the taste bud by zonulae occludentes of the tight-type. Below these tight junctions numerous desmosomes are found at irregular intervals. The epithelial cells adjacent to the pore are also joined by single strands of fusion. The microvilli arising from the neck of the type I cells have a high particle density. The microvilli of type II cells and especially the short microvilli of peripherally situated cells have a lower intramembranous particle density. The single microvillus of type III cells has a very large diameter and is longer than the other microvilli. It contains a few larger intramembranous particles and vesicle-like protrusions of the membrane facing the cytoplasm. Transverse fracturing reveals a filamentous fine structure in all microvilli. The physiological implications of these observations are discussed.Supported by grants from the Deutsche Forschungsgemeinschaft (Ja 205/5+6)     

Postmortem observations on rumen wall histology and gene expression and ruminal and caecal content of beef cattle fattened on barley-based rations

Sub-acute ruminal acidosis (SARA) can reduce the production efficiency and impair the welfare of cattle, potentially in all production systems. The aim of this study was to characterise measurable postmortem observations from divergently managed intensive beef finishing farms with high rates of concentrate feeding. At the time of slaughter, we obtained samples from 19 to 20 animals on each of 6 beef finishing units (119 animals in total) with diverse feeding practices, which had been subjectively classified as being high risk (three farms) or low risk (three farms) for SARA on the basis of the proportions of barley, silage and straw in the ration. We measured the concentrations of histamine, lipopolysaccharide (LPS), lactate and other short-chain fatty acids (SCFAs) in ruminal fluid, LPS and SCFA in caecal fluid. We also took samples of the ventral blind sac of the rumen for histopathology, immunohistopathology and gene expression. Subjective assessments were made of the presence of lesions on the ruminal wall, the colour of the lining of the ruminal wall and the shape of the ruminal papillae. Almost all variables differed significantly and substantially among farms. Very few pathological changes were detected in any of the rumens examined. The animals on the high-risk diets had lower concentrations of SCFA and higher concentrations of lactate and LPS in the ruminal fluid. Higher LPS concentrations were found in the caecum than the rumen but were not related to the risk status of the farm. The diameters of the stratum granulosum, stratum corneum and of the vasculature of the papillae, and the expression of the gene TLR4 in the ruminal epithelium were all increased on the high-risk farms. The expression of IFN-γ and IL-1β and the counts of cluster of differentiation 3 positive and major histocompatibility complex class two positive cells were lower on the high-risk farms. High among-farm variation and the unbalanced design inherent in this type of study in the field prevented confident assignment of variation in the dependent variables to individual dietary components; however, the CP percentage of the total mixed ration DM was the factor that was most consistently associated with the variables of interest. Despite the strong effect of farm on the measured variables, there was wide inter-animal variation.     

Differences in early callose deposition during adapted and non-adapted powdery mildew infection of resistant Arabidopsis lines

The deposition of callose, a (1,3)-β-glucan cell wall polymer, can play an essential role in the defense response to invading pathogens. We could recently show that Arabidopsis thaliana lines with an overexpression of the callose synthase gene PMR4 gained complete penetration resistance to the adapted powdery mildew Golovinomyces cichoracearum and the non-adapted powdery mildew Blumeria graminis f. sp hordei. The penetration resistance is based on the transport of the callose synthase PMR4 to the site of attempted fungal penetration and the subsequent formation of enlarged callose deposits. The deposits differed in their total diameter comparing both types of powdery mildew infection. In this study, further characterization of these callose deposits revealed that size differences were especially pronounced in the core region of the deposits. This suggests that specific, pathogen-dependent factors exist, which might regulate callose synthase transport to the core region of forming deposits.