Development of planar patch clamp technology and its application in the analysis of cellular electrophysiology

A patch clamp chip, as a novel cell-based chip for electrophysiological recordings, has many prominent advantages such as high resolution, accuracy, high throughput and automation. It can be used to perform multivariate and real-time measurements of cell networks in situ. Therefore, this technology will dramatically promote the research on ionic channels, neuronal networks and the application of this technology in drug screening. This paper reviews the development of planar patch clamp technology and its applications in detail. The latest progress in the research of taste cells electrophysiology and taste transduction is also presented. Finally, this paper analyzes the methodology of neural chips. Based on the current research of our laboratory, the prospective applications of a patch clamp chip in the research of taste sensation and transduction mechanisms at molecular and cellular levels are discussed.     

Possible role for salivary gland protein in taste reception indicated by homology to lipophilic-ligand carrier proteins.

Sensory transduction in taste and olfaction, the principal chemical senses, seems to be mediated by membrane-associated proteins on the apical surfaces of the respective receptor cells. The recent isolation and characterization of soluble 'odorant-binding proteins' secreted from the nasal glands of rat, cow and frog, led to the hypothesis that these proteins function as necessary cofactors in olfactory transduction by concentrating and delivering odorants to the receptors. The primary reception of taste stimuli occurs in specialized neuroepithelial receptor cells bundled in taste buds that are clustered in various types of papillae in the lingual epithelium of the tongue. Small tubulo-alveolar salivary glands, the von Ebner's glands, are located beneath the circumvallate and the foliate papillae. Their ducts open exclusively into the trough at the base of the papillae. Taste buds located in the medial and lateral walls of the papillae open with their taste pores into the trough and consequently are in direct contact with the secretions of von Ebner's glands. Here we report the molecular cloning and characterization of a protein of relative molecular mass 18,000 that is highly expressed in von Ebner's glands. Like the odorant-binding proteins, this protein shows similarity to members of a protein superfamily of hydrophobic molecule transporters, indicating that pre-receptor events could also be necessary for the concentration and delivery of sapid molecules in the gustatory system, and emphasizing the close relationship of taste and olfaction.     

Thermal stimulation of taste

The first electrophysiological recordings from animal and human taste nerves gave clear evidence of thermal sensitivity, and studies have shown that as many as half of the neurons in mammalian taste pathways respond to temperature. Because temperature has never been shown to induce sensations of taste, it has been assumed that thermal stimulation in the gustatory system is somehow nulled. Here we show that heating or cooling small areas of the tongue can in fact cause sensations of taste: warming the anterior edge of the tongue (chorda tympani nerve) from a cold temperature can evoke sweetness, whereas cooling can evoke sourness and/or saltiness. Thermal taste also occurs on the rear of the tongue (glossopharyngeal nerve), but the relationship between temperature and taste is different there than on the front of the tongue. These observations indicate the human gustatory system contains several different types of thermally sensitive neurons that normally contribute to the sensory code for taste.     

Cyclic nucleotides may mediate taste transduction

Taste stimulus adsorption is believed to occur at the taste cell microvillous membrane. But due to technical difficulties of inserting glass electrodes into the mammalian taste cell, little is known about the mechanisms of taste transduction. Reliable intracellular recordings are necessary to determine the characteristics of taste cells. This has been accomplished previously in the mouse and is reported here. Recent experiments indicated that cyclic nucleotides can act on the inner surface of the membranes of a variety of cells to alter their ion-channel activity, and these substances might act as intracellular transmitters in taste cells. But tight junctions found at the apical membrane of mammalian taste cells do not allow stimuli to enter the taste bud, making it difficult to alter the environment of the taste cell by perfusing with chemical solutions. Here we report that cyclic AMP, cyclic GMP, EGTA or tetraethyl-ammonium electrophoretically injected into the mouse taste cell induce membrane depolarization and increased membrane resistance. These results suggest that a cyclic nucleotide enzymatic cascade, modulated by calcium ions, may mediate the potassium permeability that controls taste, in a way analogous to visual and olfactory transduction.     

Heat activation of TRPM5 underlies thermal sensitivity of sweet taste

TRPM5, a cation channel of the TRP superfamily, is highly expressed in taste buds of the tongue, where it has a key role in the perception of sweet, umami and bitter tastes. Activation of TRPM5 occurs downstream of the activation of G-protein-coupled taste receptors and is proposed to generate a depolarizing potential in the taste receptor cells. Factors that modulate TRPM5 activity are therefore expected to influence taste. Here we show that TRPM5 is a highly temperature-sensitive, heat-activated channel: inward TRPM5 currents increase steeply at temperatures between 15 and 35 degrees C. TRPM4, a close homologue of TRPM5, shows similar temperature sensitivity. Heat activation is due to a temperature-dependent shift of the activation curve, in analogy to other thermosensitive TRP channels. Moreover, we show that increasing temperature between 15 and 35 degrees C markedly enhances the gustatory nerve response to sweet compounds in wild-type but not in Trpm5 knockout mice. The strong temperature sensitivity of TRPM5 may underlie known effects of temperature on perceived taste in humans, including enhanced sweetness perception at high temperatures and 'thermal taste', the phenomenon whereby heating or cooling of the tongue evoke sensations of taste in the absence of tastants.     

南方鲇口外味觉系统的外周神经分支和中枢神经系统的组织学研究

应用组织学切片和光镜观察的方法,对南方鲇口外味觉系统的外周神经分支和初级中枢结构的组织构筑进行研究,绘制出南方鲇口外味觉系统图谱,表明南方鲇的外周神经分支与初级中枢之间存在相互对应关系.作为鱼类味觉初级中枢的面叶,其组织结构的复杂性可能与食性及其对食物的好恶有关.     

Mechanisms of sensory transduction in the skin

Sensory neurons innervating the skin encode the familiar sensations of temperature, touch and pain. An explosion of progress has revealed unanticipated cellular and molecular complexity in these senses. It is now clear that perception of a single stimulus, such as heat, requires several transduction mechanisms. Conversely, a given protein may contribute to multiple senses, such as heat and touch. Recent studies have also led to the surprising insight that skin cells might transduce temperature and touch. To break the code underlying somatosensation, we must therefore understand how the skin's sensory functions are divided among signalling molecules and cell types.     

A family of candidate taste receptors in human and mouse

The gustatory system of mammals can sense four basic taste qualities, bitter, sweet, salty and sour, as well as umami, the taste of glutamate. Previous studies suggested that the detection of bitter and sweet tastants by taste receptor cells in the mouth is likely to involve G-protein-coupled receptors. Although two putative G-protein-coupled bitter/sweet taste receptors have been identified, the chemical diversity of bitter and sweet compounds leads one to expect that there is a larger number of different receptors. Here we report the identification of a family of candidate taste receptors (the TRBs) that are members of the G-protein-coupled receptor superfamily and that are specifically expressed by taste receptor cells. A cluster of genes encoding human TRBs is located adjacent to a Prp gene locus, which in mouse is tightly linked to the SOA genetic locus that is involved in detecting the bitter compound sucrose octaacetate. Another TRB gene is found on a human contig assigned to chromosome 5p15, the location of a genetic locus (PROP) that controls the detection of the bitter compound 6-n-propyl-2-thiouracil in humans.     

A TAS1R receptor-based explanation of sweet 'water-taste'

'Water-tastes' are gustatory after-impressions elicited by water following the removal of a chemical solution from the mouth, akin to colour after-images appearing on 'white' paper after fixation on coloured images. Unlike colour after-images, gustatory after-effects are poorly understood. One theory posits that 'water-tastes' are adaptation phenomena, in which adaptation to one taste solution causes the water presented subsequently to act as a taste stimulus. An alternative hypothesis is that removal of the stimulus upon rinsing generates a receptor-based, positive, off-response in taste-receptor cells, ultimately inducing a gustatory perception. Here we show that a sweet 'water-taste' is elicited when sweet-taste inhibitors are rinsed away. Responses of cultured cells expressing the human sweetener receptor directly parallel the psychophysical responses-water rinses remove the inhibitor from the heteromeric sweetener receptor TAS1R2-TAS1R3, which activates cells and results in the perception of strong sweetness from pure water. This 'rebound' activity occurs when equilibrium forces on the two-state allosteric sweet receptors result in their coordinated shift to the activated state upon being released from inhibition by rinsing.     

嗅觉与感觉信息整合

 嗅觉作为重要的化学感官,对生命的生存和繁衍起着重要作用。本文阐述了嗅觉感觉的发生,说明了嗅觉在社会信息交流和媒介情绪中发挥独特作用,分析了嗅觉与其他感觉通道进行信息整合,构建了人脑对环境的生动表征。未来仍需进一步研究揭示嗅觉与感觉信息整合更为完整深入的图景。     

黑鲷化学感觉发育和摄食关系

研究了黑鲷（Ｓｐａｒｕｓｍａｃｒｏｃｅｐｈａｌｕｓ）仔、稚、幼鱼嗅觉和味觉器官的基本结构及嗅觉、味觉与摄食的关系，初孵仔鱼的嗅囊很小、很浅，细胞没有分化。５ｄ的嗅囊细胞开始分化，２４ｄ嗅囊分化完毕，嗅觉开始功能化，参与摄食反应。初孵仔鱼味蕾没分化，５ｄ的仔鱼味蕾分化形成，参与摄食反应。随发育的进行，先后从口咽腔、头部、躯干、尾部、鳍等处依次出现味蕾。嗅觉和味觉是鱼类的化学感觉，嗅觉可以对远、近距离的食物发生反应，而味觉只能对接触的或几乎接触的食物发生反应。化学感觉对摄食的作用是对黑暗条件下视觉摄食的补充。     

脱臭蒜素提取机理及脱臭蒜素原汁酒的研制

大蒜中的有效成份是大蒜素 ,它具有强烈的刺激性臭味。但鲜蒜中大蒜素是以稳定无臭的大蒜氨酸的形式存在。当大蒜受到冲击 (切片或捣碎 ) ,大蒜酶接触到空气后活化 ,大蒜氨酸转化大蒜素。根据大蒜素的理化性质 ,采用不同的反应条件进行脱臭实验。开发研制出脱臭蒜素原液、脱臭蒜素保健饮料、脱臭蒜素原汁酒等系列产品     

Receptors and transduction in taste

Taste is the sensory system devoted primarily to a quality check of food to be ingested. Although aided by smell and visual inspection, the final recognition and selection relies on chemoreceptive events in the mouth. Emotional states of acute pleasure or displeasure guide the selection and contribute much to our quality of life. Membrane proteins that serve as receptors for the transduction of taste have for a long time remained elusive. But screening the mass of genome sequence data that have recently become available has provided a new means to identify key receptors for bitter and sweet taste. Molecular biology has also identified receptors for salty, sour and umami taste.     

Insects as chemosensors of humans and crops

Insects transmit disease to hundreds of millions of people a year, and cause enormous losses to the world's agricultural output. Many insects find the human or plant hosts on which they feed, and identify and locate their mates, primarily through olfaction and taste. Major advances have recently been made in understanding insect chemosensation at the molecular and cellular levels. These advances have provided new opportunities to control insects that cause massive damage to health and agriculture across the world.     

味觉传感器的研究──计算机对一个新的液膜体系中酸、甜、苦、咸物质的定性识别

本文用十六烷基三甲基溴化铵十乙醇／含苦味酸的二氯甲烷／蔗糖水溶液为研究体系，在油水界面建立可兴奋性人工膜，观察到甜、酸、苦、咸物质均能对该体系振荡波形有影响。采用模糊数学的方法，用计算机对有味物质实现定性识别，取得了满意的结果。并解释了该味觉电化学传感器产生电位振荡响应的机理和利用模糊数学进行模式识别的规则。     

The detection of carbonation by the Drosophila gustatory system

There are five known taste modalities in humans: sweet, bitter, sour, salty and umami (the taste of monosodium glutamate). Although the fruitfly Drosophila melanogaster tastes sugars, salts and noxious chemicals, the nature and number of taste modalities in this organism is not clear. Previous studies have identified one taste cell population marked by the gustatory receptor gene Gr5a that detects sugars, and a second population marked by Gr66a that detects bitter compounds. Here we identify a novel taste modality in this insect: the taste of carbonated water. We use a combination of anatomical, calcium imaging and behavioural approaches to identify a population of taste neurons that detects CO2 and mediates taste acceptance behaviour. The taste of carbonation may allow Drosophila to detect and obtain nutrients from growing microorganisms. Whereas CO2 detection by the olfactory system mediates avoidance, CO2 detection by the gustatory system mediates acceptance behaviour, demonstrating that the context of CO2 determines appropriate behaviour. This work opens up the possibility that the taste of carbonation may also exist in other organisms.     

五峰夏秋茶主要呈味成分分析研究

为了研究五峰春茶、夏茶和秋茶的主要呈味成分和品质的差异, 以五峰春茶、夏茶和秋茶为样品,利用重量法对五峰春茶、夏茶和秋茶的水分、水浸出物进行测定和比较;利用紫外分光光度法和高效液相色谱法(High Performance Liquid Chromatography, HPLC)测定样品的主要呈味成分,包括茶多酚、儿茶素、表没食子儿茶素没食子酸酯(Epigallocatechin gallate, EGCG)、咖啡因和氨基酸含量.研究结果表明,五峰春茶、夏茶和秋茶的主要呈味成分存在差异.茶多酚、儿茶素、EGCG含量：春茶 < 夏茶 < 秋茶;咖啡因、茶氨酸、总氨基酸含量：春茶 > 夏茶 > 秋茶.不同季节的茶叶呈味成分成分之间存在较大差异,可能是导致五峰夏秋茶苦涩味偏重的主要原因.     

Defect in cyclic AMP phosphodiesterase due to the dunce mutation of learning in Drosophila melanogaster

Cyclic AMP is an intracellular mediator ('second messenger') in the nervous and endocrine control of cellular function, regulating different processes in different cell types. Although evidence is incomplete, it seems that cyclic AMP enhances the calcium-mediated release of neurotransmitter in some neurones. A simple form of memory in the mollusc Aplysia is probably encoded as a cyclic AMP-induced enhancement of neurotransmission at certain synapses of the central nervous system. The possibility that cyclic AMP participates in learning mechanisms may be explored using genetic mutants. For this purpose the fruitfly Drosophila is suitable as it is genetically well characterized and can learn through olfaction, vision or taste. We show here that independent searches for mutations of olfactory learning and of cyclic AMP metabolism, and for mutations causing female infertility have each led to the same gene--the dunce gene. Our evidence indicates that the normal dunce gene may specify a cyclic AMP phosphodiesterase.     

Transduction in taste receptor cells requires cAMP-dependent protein kinase

In taste chemoreception, cyclic adenosine monophosphate (cAMP) appears to be one of the intracellular messengers coupling reception of stimulus to the generation of the response. The recent finding that sweet agents cause a GTP-dependent generation of cAMP poses the question of how this cytosolic messenger acts at the membrane of taste receptor cells. We have shown that cAMP causes a substantial depolarization in these cells. Here we show with whole-cell recordings and inside-out membrane patches that the depolarization caused by cAMP is accounted for by the action of cAMP-dependent protein kinase, which inactivates potassium channels predominantly of 44 pS conductance. Thus, intracellular signalling of the gustatory cells differs from that of olfactory and photoreceptor cells, where cyclic nucleotides control unspecific channels by binding to them rather than by inducing their phosphorylation.     

The cells and logic for mammalian sour taste detection

Mammals taste many compounds yet use a sensory palette consisting of only five basic taste modalities: sweet, bitter, sour, salty and umami (the taste of monosodium glutamate). Although this repertoire may seem modest, it provides animals with critical information about the nature and quality of food. Sour taste detection functions as an important sensory input to warn against the ingestion of acidic (for example, spoiled or unripe) food sources. We have used a combination of bioinformatics, genetic and functional studies to identify PKD2L1, a polycystic-kidney-disease-like ion channel, as a candidate mammalian sour taste sensor. In the tongue, PKD2L1 is expressed in a subset of taste receptor cells distinct from those responsible for sweet, bitter and umami taste. To examine the role of PKD2L1-expressing taste cells in vivo, we engineered mice with targeted genetic ablations of selected populations of taste receptor cells. Animals lacking PKD2L1-expressing cells are completely devoid of taste responses to sour stimuli. Notably, responses to all other tastants remained unaffected, proving that the segregation of taste qualities even extends to ionic stimuli. Our results now establish independent cellular substrates for four of the five basic taste modalities, and support a comprehensive labelled-line mode of taste coding at the periphery. Notably, PKD2L1 is also expressed in specific neurons surrounding the central canal of the spinal cord. Here we demonstrate that these PKD2L1-expressing neurons send projections to the central canal, and selectively trigger action potentials in response to decreases in extracellular pH. We propose that these cells correspond to the long-sought components of the cerebrospinal fluid chemosensory system. Taken together, our results suggest a common basis for acid sensing in disparate physiological settings.