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Transmembrane channel-like protein isoform 1 (TMC1) is a major component of the mechano-electrical transducer (MET) channel in cochlear hair cells and is subject to numerous mutations causing deafness. We report a new dominant human deafness mutation,TMC1p.T422K, and have characterized the homologous mouse mutant,Tmc1p.T416K, which caused deafness and outer hair cell (OHC) loss by the fourth postnatal week. MET channels showed decreased Ca2+permeability and resting open probability, but no change in single-channel conductance or expression. Three adjacent deafness mutations areTMC1p.L416R, p.G417R, and p.M418K, the last homologous to the mouseBeethoventhat exhibits similar channel effects. All substitute a positive for a neutral residue, which could produce charge screening in the channel pore or influence binding of an accessory subunit. Channel properties were compared in mice of both sexes between dominant (Tmc1p.T416K,Tmc1p.D569N) and recessive (Tmc1p.W554L,Tmc1p.D528N) mutations of residues near the putative pore of the channel.Tmc1p.W554L and p.D569N exhibit reduced maximum current with no effect on single-channel conductance, implying a smaller number of channels transported to the stereociliary tips; this may stem from impaired TMC1 binding to LHFPL5.Tmc1p.D528N, located in the pore's narrowest region, uniquely caused large reductions in MET channel conductance and block by dihydrostreptomycin (DHS). ForTmc1p.T416K andTmc1p.D528N, transduction loss occurred between P15 and P20. We propose two mechanisms linking channel mutations and deafness: decreased Ca2+permeability, common to all mutants, and decreased resting open probability in low Ca2+, confined to dominant mutations.SIGNIFICANCE STATEMENTTransmembrane channel-like protein isoform 1 (TMC1) is thought to be a major component of the mechanotransducer channel in auditory hair cells, but the protein organization and channel structure are still uncertain. We made four mouse lines harboringTmc1point mutations that alter channel properties, causing hair cell degeneration and deafness. These include a mouse homolog of a new human deafness mutation pT416K that decreased channel Ca2+permeability by introducing a positively-charged amino acid in the putative pore. All mutations are consistent with the channel structure predicted from modeling, but only one, p.D528N near the external face of the pore, substantially reduced channel conductance and Ca2+permeability and virtually abolished block by dihydrostreptomycin (DHS), strongly endorsing its siting within the pore.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus play a vital role in osmoregulation, but the mechanisms underlying MNC osmosensitivity are not fully understood. We showed previously that high osmolality activates phospholipase C (PLC) in rat MNCs in a Ca2+-dependent manner and that PLC activation is necessary for full osmotic activation of an N-terminal variant of the TRPV1 (ΔN-TRPV1) channel. We therefore hypothesized that the Ca2+-dependent δ1 isoform of PLC contributes to ΔN-TRPV1 activation and tested whether MNC function is defective in a transgenic PLCδ1 KO mouse. Water deprivation for 24 h caused greater increases in serum osmolality and losses in body weight in PLCδ1 KO mice than it did in control mice. Action potentials and ΔN-TRPV1 currents were measured in acutely isolated mouse MNCs using whole-cell patch clamp before and after exposure to hypertonic solutions. This treatment elicited a significant activation of ΔN-TRPV1 currents and an increase in firing rate in MNCs isolated from control mice, but not from PLCδ1 KO mice. Submembranous filamentous actin was measured in isolated MNCs before and after treatment with angiotensin II and hypertonic solution. Both treatments caused an increase in filamentous actin fluorescence in MNCs isolated from control mice, but both responses were significantly attenuated in MNCs from PLCδ1 KO mice. Our data demonstrate that the PLCδ1 isoform plays a key role in the activation of ΔN-TRPV1 channels and in osmosensory transduction in MNCs. This study advances our understanding of the molecular mechanisms underlying mammalian osmoregulation.SIGNIFICANCE STATEMENTMagnocellular neurosecretory cells (MNCs) of the hypothalamus play a central role in osmoregulation. We have identified a key role for the PLCδ1 isoform in the activation of ΔN-TRPV1 channels and osmosensory transduction in MNCs. The data indicate that the PLCδ1 isoform is activated by the Ca2+influx occurring during MNC action potentials and exerts a positive feedback on ΔN-TRPV1 channels to enhance MNC excitability. This study provides evidence that PLCδ1 is a key molecule underlying osmosensory transduction, the regulation of VP release, and osmoregulation.

Sound detection in auditory sensory hair cells depends on the deflection of the stereocilia hair bundle which opens mechano-electric transduction (MET) channels. Adaptation is hypothesized to be a critical property of MET that contributes to the auditory system's wide dynamic range and sharp frequency selectivity. Our recent work using a stiff probe to displace hair bundles showed that the fastest adaptation mechanism (fast adaptation) does not require calcium entry. Using fluid-jet stimuli, others obtained data showing only a calcium-dependent fast adaptation response. Because cochlear stereocilia do not move coherently and the hair cell response depends critically on the magnitude and time course of the hair bundle deflection, we developed a high-speed imaging technique to quantify this deflection in rat cochlear hair cells. The fluid jet delivers a force stimulus, and force steps lead to a complex time course of hair bundle displacement (mechanical creep), which affects the hair cell's macroscopic MET current response by masking the time course of the fast adaptation response. Modifying the fluid-jet stimulus to generate a hair bundle displacement step produced rapidly adapting currents that did not depend on membrane potential, confirming that fast adaptation does not depend on calcium entry. MET current responses differ with stimulus modality and will shape receptor potentials of different hair cell types based on theirin vivostimulus mode. These transformations will directly affect how stimuli are encoded.SIGNIFICANCE STATEMENTMechanotransduction by sensory hair cells represents a key first step for the sound sensing ability in vertebrates. The sharp frequency tuning and wide dynamic range of sound sensation are hypothesized to require a mechanotransduction adaptation mechanism. Recent work indicated that the apparent calcium dependence of the fastest adaptation differs with the method of cochlear hair cell stimulation. Here, we reconcile existing data and show that calcium entry does not drive the fastest adaptation process, independent of the stimulation method. With force stimulation of the hair bundle, adaptation manifests differently than with displacement stimulation, indicating that the stimulation mode of the hair bundle will affect the hair cell receptor current and stimulus coding.

Insect auditory receivers provide an excellent comparative resource to understand general principles of auditory transduction, but analysis of the electrophysiological properties of the auditory neurons has been hampered by their tiny size and inaccessibility. Here we pioneer patch-clamp recordings from the auditory neurons of Müller's organ of the desert locustSchistocerca gregariato characterize dendritic spikes, axonal spikes, and the transduction current. We demonstrate that dendritic spikes, elicited by sound stimuli, trigger axonal spikes, and that both types are sodium and voltage dependent and blocked by TTX. Spontaneous discrete depolarizations summate upon acoustic stimulation to produce a graded transduction potential that in turn elicits the dendritic spikes. The transduction current of Group III neurons of Müller's organ, which are broadly tuned to 3 kHz, is blocked by three ion channel blockers (FM1-43, streptomycin, and 2-APB) that are known to block mechanotransduction channels. We investigated the contribution of the candidate mechanotransduction ion channel Nanchung-Inactive—which is expressed in Müller's organ—to the transduction current. A specific agonist of Nanchung-Inactive, pymetrozine, eliminates the sound-evoked transduction current while inducing a tonic depolarizing current of comparable amplitude. The Nanchung-Inactive ion channels, therefore, have the required conductance to carry the entire transduction current, and sound stimulation appears not to open any additional channels. The application of three mechanotransduction ion channel blockers prevented the pymetrozine-induced depolarizing current. This implies that either Nanchung-Inactive is, or forms part of, the mechanotransduction ion channel or it amplifies a relatively small current (SIGNIFICANCE STATEMENTThe mechanically activated ion channel underpinning hearing is not known. We have pioneered intracellular patch-clamp recordings from locust auditory neurons to unravel the role of the candidate mechanotransduction ion channel Nanchung-Inactive in auditory transduction in insects.

Responses in the amiloride-insensitive (AI) pathway, one of the two pathways mediating salty taste in mammals, are modulated by the size of the anion of a salt. This “anion effect” has been hypothesized to result from inhibitory transepithelial potentials (TPs) generated across the lingual epithelium as cations permeate through tight junctions and leave their larger and less permeable anions behind (Ye et al., 1991). We tested directly the necessity of TPs for the anion effect by measuring responses to NaCl and Na-gluconate (small and large anion sodium salts, respectively) in isolated taste cells from mouse circumvallate papillae. Using calcium imaging, we identified AI salt-responsive type III taste cells and demonstrated that they compose a subpopulation of acid-responsive taste cells. Even in the absence of TPs, many (66%) AI salt-responsive type III taste cells still exhibited the anion effect, demonstrating that some component of the transduction machinery for salty taste in type III cells is sensitive to anion size. We hypothesized that osmotic responses could explain why a minority of type III cells (34%) had AI salt responses but lacked anion sensitivity. All AI type III cells had osmotic responses to cellobiose, which were significantly modulated by extracellular sodium concentration, suggesting the presence of a sodium-conducting osmotically sensitive ion channel. However, these responses were significantly larger in AI type III cells that did not exhibit the anion effect. These findings indicate that multiple mechanisms could underlie AI salt responses in type III taste cells, one of which may contribute to the anion effect.SIGNIFICANCE STATEMENTUnderstanding the mechanisms underlying salty taste will help inform strategies to combat the health problems associated with NaCl overconsumption by humans. Of the two pathways underlying salty taste in mammals, the amiloride-insensitive (AI) pathway is the least understood. Using calcium imaging of isolated mouse taste cells, we identify two separate populations of AI salt-responsive type III taste cells distinguished by their sensitivity to anion size and show that these cells compose subpopulations of acid-responsive taste cells. We also find evidence that a sodium-conducting osmotically sensitive mechanism contributes to salt responses in type III taste cells. Our data not only provide new insights into the transduction mechanisms of AI salt taste but also have important implications for general theories of taste encoding.

Temporal limits on perceptual decisions set strict boundaries on the possible underlying neural computations. How odor information is encoded in the olfactory system is still poorly understood. Here, we sought to define the limit on the speed of olfactory processing. To achieve this, we trained mice to discriminate different odor concentrations in a novel behavioral setup with precise odor delivery synchronized to the sniffing cycle. Mice reported their choice by moving a horizontal treadmill with their front limbs. We found that mice reported discriminations of 75% accuracy in 70–90 ms after odor inhalation. For a low concentration and nontrigeminal odorant, this time was 90–140 ms, showing that mice process odor information rapidly even in the absence of trigeminal stimulation. These response times establish, after accounting for odor transduction and motor delays, that olfactory processing can take tens of milliseconds. This study puts a strong limit on the underlying neural computations and suggests that the action potentials forming the neural basis for these decisions are fired in a few tens of milliseconds. SIGNIFICANCE STATEMENT Understanding how sensory information is processed requires different approaches that span multiple levels of investigation from genes to neurons to behavior. Limits on behavioral performance constrain the possible neural mechanisms responsible for specific computations. Using a novel behavioral paradigm, we established that mice can make decisions about odor intensity surprisingly fast. After accounting for sensory and motor delays, the limit on some olfactory neural computations can be as low as a few tens of milliseconds, which suggests that only the first action potentials across a population of neurons contribute to these computations.

In flies and humans, bitter chemicals are known to inhibit sugar detection, but the adaptive role of this inhibition is often overlooked. At best, this inhibition is described as contributing to the rejection of potentially toxic food, but no studies have addressed the relative importance of the direct pathway that involves activating bitter-sensitive cells versus the indirect pathway represented by the inhibition of sugar detection. Using toxins to selectively ablate or inactivate populations of bitter-sensitive cells, we assessed the behavioral responses of flies to sucrose mixed with strychnine (which activates bitter-sensitive cells and inhibits sugar detection) or withl-canavanine (which only activates bitter-sensitive cells). As expected, flies with ablated bitter-sensitive cells failed to detectl-canavanine mixed with sucrose in three different feeding assays (proboscis extension responses, capillary feeding, and two-choice assays). However, such flies were still able to avoid strychnine mixed with sucrose. By means of electrophysiological recordings, we established that bitter molecules differ in their potency to inhibit sucrose detection and that sugar-sensing inhibition affects taste cells on the proboscis and the legs. The optogenetic response of sugar-sensitive cells was not reduced by strychnine, thus suggesting that this inhibition is linked directly to sugar transduction. We postulate that sugar-sensing inhibition represents a mechanism in insects to prevent ingesting harmful substances occurring within mixtures.

Growing evidence suggests that the main olfactory epithelium contains a subset of olfactory sensory neurons (OSNs) responding to pheromones. One candidate subpopulation expresses the calcium activated cation channel TRPM5 (transient receptor potential channel M5). Using GFP driven by the TRPM5 promoter in mice, we show that this subpopulation responds to putative pheromones, urine, and major histocompatibility complex peptides, but not to regular odors or a pheromone detected by other species. In addition, this subpopulation of TRPM5-GFP+OSNs uses novel transduction. In regular OSNs, odorants elicit activation of the cyclic nucleotide-gated (CNG) channel, leading to Ca2+gating of Cl−channels; in TRPM5-GFP+OSNs, the Ca2+-activated Cl−ANO2 (anoctamin 2) channel is not expressed, and pheromones elicit activation of the CNG channel leading to Ca2+gating of TRPM5. In conclusion, we show that OSNs expressing TRPM5 respond to pheromones, but not to regular odors through the opening of CNG channels leading to Ca2+gating of TRPM5.

Chronic itch is a debilitating condition that affects one in 10 people. Little is known about the molecules that mediate chronic itch in primary sensory neurons and skin. We demonstrate that the ion channel TRPA1 is required for chronic itch. Using a mouse model of chronic itch, we show that scratching evoked by impaired skin barrier is abolished in TRPA1-deficient animals. This model recapitulates many of the pathophysiological hallmarks of chronic itch that are observed in prevalent human diseases such as atopic dermatitis and psoriasis, including robust scratching, extensive epidermal hyperplasia, and dramatic changes in gene expression in sensory neurons and skin. Remarkably, TRPA1 is required for both transduction of chronic itch signals to the CNS and for the dramatic skin changes triggered by dry-skin-evoked itch and scratching. These data suggest that TRPA1 regulates both itch transduction and pathophysiological changes in the skin that promote chronic itch.

Afferent nerve fibers in the central zones of vestibular epithelia form calyceal endings around type I hair cells and have phasic response properties that emphasize fast head motions. We investigated how stages from hair-cell transduction to calyceal spiking contribute tuning and timing to central (striolar)-zone afferents of the rat saccular epithelium. In an excised preparation, we deflected individual hair bundles with rigid probes driven with steps and sinusoids (0.5–500 Hz) and recorded whole-cell responses from hair cells and calyces at room temperature and body temperature. In immature hair cells and calyces (postnatal days (P)1–P4), tuning sharpened at each stage. Transducer adaptation and membrane-charging time produced bandpass filtering of the receptor potential with best frequencies of 10–30 Hz and phase leads below 10 Hz. For small stimuli, electrical resonances sharply tuned the hair-cell membrane in the frequency range of 5–40 Hz. The synaptic delay of quantal transmission added a phase lag at frequencies above 10 Hz. The influence of spike thresholds at the calyceal spike initiation stage sharpened tuning and advanced response phase. Two additional mechanisms strongly advanced response phase above 10 Hz when present: (1) maturing (P7–P9) type I hair cells acquired low-voltage-activated channels that shortened the rise time of the receptor potential and (2) some calyces had nonquantal transmission with little synaptic delay. By reducing response time, the identified inner-ear mechanisms (transducer adaptation, low-voltage-activated channels, nonquantal transmission, and spike triggering) may compensate for transmission delays in vestibular reflex pathways and help stabilize posture and gaze during rapid head motions.

The mammalian main olfactory pathway detects volatile chemicals using two families of G protein-coupled receptors—a large repertoire of canonical odorant receptors (ORs) and a much smaller set of Trace Amine-Associated Receptors, or TAARs. The TAARs are evolutionarily conserved in vertebrates, including humans, suggesting an indispensible role in olfaction. However, little is known about the functional properties of TAARs when expressed in native olfactory sensory neurons. Here we describe experiments using gene targeting, electrophysiology and optical imaging to study the response properties of TAAR-expressing sensory neurons and their associated glomeruli in mice. We show that olfactory sensory neurons that express a subset of the TAAR repertoire are preferentially responsive to amines. In addition, neurons expressing one of two specific TAARs, TAAR3 and TAAR4, are highly sensitive and are also broadly tuned—responding to structurally diverse amines at high concentrations. Surprisingly, we find that TAAR4 is exquisitely sensitive, with apparent affinities for a preferred ligand, phenylethylamine, rivaling those seen with mammalian pheromone receptors. We provide evidence that this unprecedented sensitivity is mediated via receptor coupling to the canonical odorant transduction cascade. The data suggest that the TAARs are evolutionarily retained in the olfactory receptor repertoire to mediate high sensitivity detection of a biologically relevant class of odorous stimuli.

Drosophilaphotoreceptor cells use the ubiquitous G-protein-mediated phospholipase C (PLC) cascade to achieve ultimate single-photon sensitivity. This is manifested in the single-photon responses (quantum bumps). In photoreceptor cells, dark activation of Gqα molecules occurs spontaneously and produces unitary dark events (dark bumps). A high rate of spontaneous Gqα activation and dark bump production potentially hampers single-photon detection. We found that in wild-type flies thein vivorate of spontaneous Gqα activation is very high. Nevertheless, this high rate is not manifested in a substantially high rate of dark bumps. Therefore, it is unclear how phototransduction suppresses dark bump production arising from spontaneous Gqα activation, while still maintaining high-fidelity representation of single photons. In this study we show that reduced PLC catalytic activity selectively suppressed production of dark bumps but not light-induced bumps. Manipulations of PLC activity using PLC mutant flies and Ca2+modulations revealed that a critical level of PLC activity is required to induce bump production. The required minimal level of PLC activity selectively suppressed random production of single Gqα-activated dark bumps despite a high rate of spontaneous Gqα activation. This minimal PLC activity level is reliably obtained by photon-induced synchronized activation of several neighboring Gqα molecules activating several PLC molecules, but not by random activation of single Gqα molecules. We thus demonstrate how a G-protein-mediated transduction system, with PLC as its target, selectively suppresses its intrinsic noise while preserving reliable signaling.

Gentle touch sensation inCaenorhabditis elegansis mediated by the MEC-4/MEC-10 channel complex, which is expressed exclusively in six touch receptor neurons (TRNs). The complex contains two pore-forming subunits, MEC-4 and MEC-10, as well as the accessory subunits MEC-2, MEC-6, and UNC-24. MEC-4 is essential for channel function, but beyond its role as a pore-forming subunit, the functional contribution of MEC-10 to the channel complex and to touch sensation is unclear. We addressed this question using behavioral assays,in vivoelectrophysiological recordings from TRNs, and heterologous expression of mutant MEC-10 isoforms. Animals with a deletion inmec-10showed only a partial loss of touch sensitivity and a modest decrease in the size of the mechanoreceptor current (MRC). In contrast, five previously identifiedmec-10alleles acted as recessive gain-of-function alleles that resulted in complete touch insensitivity. Each of these alleles produced a substantial decrease in MRC size and a shift in the reversal potentialin vivo. The latter finding indicates that thesemec-10mutations alter the ionic selectivity of the transduction channelin vivo. Allmec-10mutant animals had properly localized channel complexes, indicating that the loss of MRCs was not attributable to a dramatic mislocalization of transduction channels. Finally, electrophysiological examination of heterologously expressed complexes suggests that mutant MEC-10 proteins may affect channel current via MEC-2.

Auditory and vestibular functions in vertebrates depend on the transduction of sound vibration or head acceleration into electrical responses in inner ear hair cells. Mechanoelectrical transduction occurs at the tip of stereocilia, which are polarized to form an orientational arrangement that determines directional sensitivity. It remains to be clarified when and how premature hair cells acquire their specialized structure and function in living animals. The developmental origin of inner ear hair cells has been studiedin vivoin zebrafish embryos. Tether cells, a small number of ciliated cells associated with an “ear stone” (or otolith) in the embryonic zebrafish inner ear, are believed to be precocious hair cells. However, whether or not tether cells acquire hair bundles and mechanosensitivity remains unknown. In the present study, we investigated the morphological and functional development of tether cells. Immunohistochemical examination revealed that stereocilia appeared on the tether cell apex in a polarized arrangement at 22 h postfertilization (hpf). Labeling with FM1-43, a marker of functional mechanotransduction channels, and thein vivoelectrophysiological recording of mechanotransducer responses in the developing inner ear demonstrated that tether cells acquired direction-selective mechanosensitivity at 23 hpf. These results revealed that tether cells begin to function as hair cells within an hour after the appearance of a polarized array of stereociliary bundles. Thus, the ciliary cells morphologically and functionally differentiate into the first sensory hair cells in the inner ear of the zebrafish.

Vision in dim light, when photons are scarce, requires reliable signaling of the arrival of single photons. Rod photoreceptors accomplish this task through the use of a G-protein-coupled transduction cascade that amplifies the activity of single active rhodopsin molecules. This process is one of the best understood signaling cascades in biology, yet quantitative measurements of the amplitude and kinetics of the rod's response in mice vary by a factor of ∼2 across studies. What accounts for these discrepancies? We used several experimental approaches to reconcile differences in published properties of rod responses. First, we used suction electrode recordings from single rods to compare measurements across a range of recording conditions. Second, we compared measurements of single-cell photocurrents to estimates of rod function fromin vitroelectroretinograms. Third, we assayed the health of the post-receptor retinal tissue in these different conditions. Several salient points emerge from these experiments: (1) recorded responses can be altered dramatically by how the retina is stored; (2) the kinetics of the recovery of responses to bright but not dim flashes are strongly sensitive to the extracellular concentration of magnesium; (3) experimental conditions that produce very different single-photon responses measured in single rods produce near identical derived rod responses from the electroretinogram. The dependence of rod responses on experimental conditions will be a key consideration in efforts to extract general principles of G-protein signaling from studies of phototransduction and to relate these signals to downstream mechanisms that facilitate visual sensitivity.

Tip links are extracellular filaments that connect pairs of hair cell stereocilia and convey tension to mechanosensitive channels. Recent evidence suggests that tip links are formed by calcium-dependent interactions between the N-terminal domains of cadherin-23 (CDH23) and protocadherin-15 (PCDH15). Mutations in either CDH23 or PCDH15 cause deafness in mice and humans, indicating the molecules are required for normal inner ear function. However, there is little physiological evidence to support a direct role for CDH23 and PCDH15 in hair cell mechanotransduction. To investigate the contributions of CDH23 and PCDH15 to mechanotransduction and tip-link formation, we examined outer hair cells of mouse cochleas during development and after chemical disruption of tip links. We found that tip links and mechanotransduction with all the qualitative properties of mature transduction recovered within 24 h after disruption. To probe tip-link formation, we measured transduction currents after extracellular application of recombinant CDH23 and PCDH15 fragments, which included putative interaction domains (EC1). Both fragments inhibited development and regeneration of transduction but did not disrupt transduction in mature cells. PCDH15 fragments that carried a mutation in EC1 that causes deafness in humans did not inhibit transduction development or regeneration. Immunolocalization revealed wild-type fragments bound near the tips of hair cell stereocilia. Scanning electron micrographs revealed that hair bundles exposed to fragments had a reduced number of linkages aligned along the morphological axis of sensitivity of the bundle. Together, the data provide direct evidence implicating CDH23 and PCDH15 proteins in the formation of tip links during development and regeneration of mechanotransduction.

In their natural environment, rodents use their whiskers to locate and distinguish between objects of different textures and shapes. They do so by moving their whiskers actively as well as passively, through body and head movements. To determine the mechanisms by which surface coarseness is translated into neuronal discharges through passive whisker movements, we monitored head movements of awake behaving rats while approaching objects. We then replayed these movements in anesthetized rats, monitored the whiskers' movements across various surfaces, and concurrently recorded the activity of first-order sensory neurons. We found that whiskers, being the first stage of sensory information translation, shape transduction by amplifying small-amplitude high-frequency signals. Thus, surface coarseness is transmitted through high-velocity micromotions. Consistent with this, we find that during surface contact, discrete high-velocity movements, or stick-slip events, evoke first-order neuronal discharge. Transient ringing in whiskers, which primarily represents resonance vibrations, follows these events, but seldom causes neurons to discharge. These sensory transformations are influenced by the whiskers' biomechanical properties. To determine the resemblance of these tactile transformations during passive whisker movements and active whisking, we induced artificial whisking across various surface textures. We found that the processes by which tactile information becomes available to the animal are similar for these different modes of behavior. Together, these findings indicate that the temporal bandpass properties for spike generation in first-order neurons are matched by the biomechanical characteristics of whiskers, which translate surface coarseness into high-frequency whisker micromotions. These properties enable rodents to acquire tactile information through passive and active movements of their whiskers.

On bipolar cells are connected to photoreceptors via a sign-inverting synapse. At this synapse, glutamate binds to a metabotropic receptor which couples to the closure of a cation-selective transduction channel. The molecular identity of both the receptor and the G protein are known, but the identity of the transduction channel has remained elusive. Here, we show that the transduction channel in mouse rod bipolar cells, a subtype of On bipolar cell, is likely to be a member of the TRP family of channels. To evoke a transduction current, the metabotropic receptor antagonist LY341495 was applied to the dendrites of cells that were bathed in a solution containing the mGluR6 agonistsl-AP4 or glutamate. The transduction current was suppressed by ruthenium red and the TRPV1 antagonists capsazepine and SB-366791. Furthermore, focal application of the TRPV1 agonists capsaicin and anandamide evoked a transduction-like current. The capsaicin-evoked and endogenous transduction current displayed prominent outward rectification, a property of the TRPV1 channel. To test the possibility that the transduction channel is TRPV1, we measured rod bipolar cell function in theTRPV1−/−mouse. The ERG b-wave, a measure of On bipolar cell function, as well as the transduction current and the response to TRPV1 agonists were normal, arguing against a role for TRPV1. However, ERG measurements from mice lacking TRPM1 receptors, another TRP channel implicated in retinal function, revealed the absence of a b-wave. Our results suggest that a TRP-like channel, possibly TRPM1, is essential for synaptic function in On bipolar cells.

Osmosensory neurons transduce osmotic signals into a neural spike code that commands behavioral and endocrine responses that mediate body fluid homeostasis. Although changes in osmoregulatory reflex gain are known to occur under physiological and pathological conditions, the basis for this modulation is unknown. Here, we show that angiotensin II amplifies osmosensory transduction by enhancing the proportional relationship between osmolality, receptor potential, and action potential firing in rat supraoptic nucleus neurons. This effect is mediated by a phospholipase C- and protein kinase C-dependent increase in cellular mechanosensitivity that is associated with a rapid increase in cortical actin filament density. Preventing this increase with cytochalasin D eliminated the enhancement of mechanosensitivity, whereas enhancing actin filament density with jasplakinolide potentiated mechanosensitivity and occluded the effects of angiotensin II. These results indicate that a receptor-mediated increase in cortical actin density can enhance osmosensitivity in acutely isolated supraoptic neurons.

Hearing, touch and proprioception are thought to involve direct activation of mechano-electrical transduction (MeT) channels. InCaenorhabditis eleganstouch receptor neurons (TRNs), such channels contain two pore-forming subunits (MEC-4 and MEC-10) and two auxiliary subunits (MEC-2 and MEC-6). MEC-4 and MEC-10 belong to a large superfamily of ion channel proteins (DEG/ENaCs) that form nonvoltage-gated, amiloride-sensitive Na+channels. In TRNs, unique 15-protofilament microtubules and an electron-dense extracellular matrix have been proposed to serve as gating tethers critical for MeT channel activation. We combined high-pressure freezing and serial-section immunoelectron microscopy to determine the position of MeT channels relative to putative gating tethers. MeT channels were visualized using antibodies against MEC-4 and MEC-2. This nanometer-resolution view of a sensory MeT channel establishes structural constraints on the mechanics of channel gating. We show here that MEC-2 and MEC-5 collagen, a putative extracellular tether, occupy overlapping but distinct domains in TRN neurites. Although channels decorate all sides of TRN neurites; they are not associated with the distal endpoints of 15-protofilament microtubules hypothesized to be gating tethers. These specialized microtubules, which are unique to TRNs, assemble into a cross-linked bundle connected by a network of kinked filaments to the neurite membrane. We speculate that the microtubule bundle converts external point loads into membrane stretch which, in turn, facilitates MeT channel activation.

Inner ear mechanosensory hair cells convert mechanical vibrations into electrical signals via the coordinated interaction of multiple proteins precisely positioned within the sensory hair bundle. Present work identifies the time course for the acquisition and maturation of mechanoelectric transduction (MET) in rat cochlea outer hair cells maintained in organotypic cultures. A spatiotemporal developmental progression was observed morphologically and functionally with basal cochlea maturation preceding apical cochlea by 2–3 d in all measured properties. The fraction of mechanosensitive cells increased rapidly, with a midpoint at postnatal day 0 for basal cells, and correlated with myosin IIIa immunoreactivity. MET current magnitude increased over several days. Adaptation lagged the onset of transduction by a day and matured more slowly, overlapping but preceding the rise in myosin Ic immunoreactivity. Less than ∼25% of myosin Ic expression was required for the mature adaptation response, suggesting multiple roles for this protein in hair bundle function. Directional sensitivity, lacking in immature responses, developed rapidly and correlated with the pruning of radial links and an increase in tenting of stereociliary tips. Morphological and electrophysiological data support a hypothesis in which key elements arrive independently at the site of MET, with a mature response occurring as membrane tension increases, likely by the increased tensioning of the tip link with the onset of adaptation. Organotypic cultures developed normal, tonotopically specific, MET response properties, suggesting that maturation was not influenced significantly by external factors such as innervation, endolymph, normal mechanical stimulation, or an intact organ of Corti.

Sensory hair cells of the inner ear express multiple physiologically defined conductances, including mechanotransduction, Ca2+, Na+, and several distinct K+conductances, all of which are critical for normal hearing and balance function. Yet, the molecular underpinnings and their specific contributions to sensory signaling in the inner ear remain obscure. We sought to identify hair-cell conductances mediated by KCNQ4, which, when mutated, causes the dominant progressive hearing loss DFNA2. We used the dominant-negative pore mutation G285S and packaged the coding sequence of KCNQ4 into adenoviral vectors. We transfected auditory and vestibular hair cells of organotypic cultures generated from the postnatal mouse inner ear. Cochlear outer hair cells and vestibular type I cells that expressed the transfection marker, green fluorescent protein, and the dominant-negative KCNQ4 construct lacked the M-like conductances that typify nontransfected control hair cells. As such, we conclude that the M-like conductances in mouse auditory and vestibular hair cells can include KCNQ4 subunits and may also include KCNQ4 coassembly partners. To examine the function of M-like conductances in hair cells, we recorded from cells transfected with mutant KCNQ4 and injected transduction current waveforms in current-clamp mode. Because the M-like conductances were active at rest, they contributed to the very low potassium-selective input resistance, which in turn hyperpolarized the resting potential and significantly attenuated the amplitude of the receptor potential. Modulation of M-like conductances may allow hair cells the ability to control the amplitude of their response to sensory stimuli.

Hearing requires the transduction of vibrational forces by specialized epithelial cells in the cochlea known as hair cells. The human ear contains a finite number of terminally differentiated hair cells that, once lost by noise-induced damage or toxic insult, can never be regenerated. We report here that sphingosine 1-phosphate (S1P) signaling, mainly via activation of its cognate receptor S1P2, is required for the maintenance of vestibular and cochlear hair cellsin vivo. Two S1P receptors, S1P2and S1P3, were found to be expressed in the cochlea by reverse transcription-PCR andin situhybridization. Mice that are null for both these receptors uniformly display progressive cochlear and vestibular defects with hair cell loss, resulting in complete deafness by 4 weeks of age and, with complete penetrance, balance defects of increasing severity. This study reveals the previously unknown role of S1P signaling in the maintenance of cochlear and vestibular integrity and suggests a means for therapeutic intervention in degenerative hearing loss.

Inner hair cells (IHCs) are the true sensory receptors in the cochlea; they transmit auditory information to the brain. IHCs respond to basilar membrane (BM) vibration by producing a transducer current through mechanotransducer (MET) channels located at the tip of their stereocilia when these are deflected. The IHC MET current has not been measured from adult animals. We simultaneously recorded IHC transducer currents and BM motion in a gerbil hemicochlea to examine relationships between these two variables and their variation along the cochlear length. Results show that although maximum transducer currents of IHCs are uniform along the cochlea, their operating range is graded and is narrower in the base. The MET current displays adaptation, which along with response magnitude depends on extracellular calcium concentration. The rate of adaptation is invariant along the cochlear length. We introduce a new method of measuring adaptation using sinusoidal stimuli. There is a phase lead of IHC transducer currents relative to sinusoidal BM displacement, reflecting viscoelastic coupling of their cilia and their adaptation process.

The auditory hair cell resting potential is critical for proper translation of acoustic signals to the CNS, because it determines their filtering properties, their ability to respond to stimuli of both polarities, and, because the hair cell drives afferent firing rates, the resting potential dictates spontaneous transmitter release. In turtle auditory hair cells, the filtering properties are established by the interactions between BK calcium-activated potassium channels and an L-type calcium channel (electrical resonance). However, both theoretical andin vitrorecordings indicate that a third conductance is required to set the resting potential to a point on theICaandIBKactivation curves in which filtering is optimized like that foundin vivo. Present data elucidate a novel mechanism, likely universal among hair cells, in which mechanoelectric transduction (MET) and its calcium-dependent adaptation provide the depolarizing current to establish the hair cell resting potential. First, mechanical block of the MET current hyperpolarized the membrane potential, resulting in broadband asymmetrical resonance. Second, altering steady-state adaptation by altering the [Ca2+] bathing the hair bundle changed the MET current at rest, the magnitude of which resulted in membrane potential changes that encompassed the best resonant voltage. The Ca2+sensitivity of adaptation allowed for the first physiological estimate of endolymphatic Ca2+near the MET channel (56 ± 11 μm), a value similar to bulk endolymph levels. These effects of MET current on resting potential were independently confirmed using a theoretical model of electrical resonance that included the steady-state MET conductance.

Sound stimuli are detected in the cochlea by opening of hair cell mechanotransducer (MT) channels, one of the few ion channels not yet conclusively identified at a molecular level. To define their performancein situ, we measured MT channel properties in inner hair cells (IHCs) and outer hair cells (OHCs) at two locations in the rat cochlea tuned to different characteristic frequencies (CFs). The conductance (in 0.02 mmcalcium) of MT channels from IHCs was estimated as 260 pS at both low-frequency and mid-frequency positions, whereas that from OHCs increased with CFs from 145 to 210 pS. The combination of MT channel conductance and tip link number, assayed from scanning electron micrographs, accounts for variation in whole-cell current amplitude for OHCs and its invariance for IHCs. Channels from apical IHCs and OHCs having a twofold difference in unitary conductance were both highly calcium selective but were distinguishable by a small but significant difference in calcium permeability and in their response to lowering ionic strength. The results imply that the MT channel has properties possessed by few known candidates, and its diversity suggests expression of multiple isoforms.

Drosophilavisual signaling is one of the fastest G-protein-coupled transduction cascades, because effector and modulatory proteins are organized into a macromolecular complex (“transducisome”). Assembly of the complex is orchestrated by inactivation no afterpotential D (INAD), which colocalizes the transient receptor potential (TRP) Ca2+channel, phospholipase Cβ, and eye protein kinase C (eye-PKC), for more efficient signal transduction. Eye-PKC is critical for deactivation of vision. Moreover, deactivation is regulated by the interaction between INAD and TRP, because abrogation of this interaction inInaDp215results in slow deactivation similar to that ofinaCp209lacking eye-PKC. To elucidate the mechanisms whereby eye-PKC modulates deactivation, here we demonstrate that eye-PKC, via tethering to INAD, phosphorylates TRPin vitro. We reveal that Ser982of TRP is phosphorylated by eye-PKCin vitroand, importantly, in the fly eye, as shown by mass spectrometry. Furthermore, transgenic expression of modified TRP bearing an Ala substitution leads to slow deactivation of the visual response similar to that ofInaDp215. These results suggest that the INAD macromolecular complex plays an essential role in termination of the light response by promoting efficient phosphorylation at Ser982of TRP for fast deactivation of the visual signaling.

Molecular mechanisms underlying olfactory signal amplification were investigated by monitoring cAMP dynamics in the intact sensory cilia. We saw that [cAMP]iincreased superlinearly with time during odorant stimuli for >1 s. This time course was remarkably different from that obtained with the rapid quench method previously applied to thein vitropreparation, in which [cAMP]ichange has been reported to be transient. The superlinear increase of [cAMP]iwas attributable to a gradual increase of cAMP production rate that was consistent with the thermodynamical interaction model between elemental molecules, as has been revealed on the rod photoreceptor cell. It thus seems likely that the fundamental mechanism for molecular interactions between olfactory transduction elements is similar to that of the rod. In olfaction, however, cAMP production was extremely small (∼200,000 molecules/s/cell at the maximum), in contrast to the cGMP hydrolysis in the rod (250,000 molecules/photon). The observed numbers indicate that the olfactory receptor cell has lower amplification at the enzymatic cascade. Seemingly, such low amplification is a disadvantage for the signal transduction, but this unique mechanism would be essential to reduce the loss of ATP that is broadly used for the activities of cells. Apparently, transduction by a smaller number of second-messenger formations would be achieved by the fine ciliary structure that has a high surface-volume ratio. In addition, it is speculated that this low amplification at their enzymatic processes may be the reason why the olfactory receptor cell has acquired high amplification at the final stage of transduction channels, using Ca2+as a third messenger.

Calcium buffers are important for shaping and localizing cytoplasmic Ca2+transients in neurons. We measured the concentrations of the four main calcium-buffering proteins (calbindin-D28k, calretinin, parvalbumin-α, and parvalbumin-β) in rat cochlear hair cells in which Ca2+signaling is a central element of fast transduction and synaptic transmission. The proteins were quantified by calibrating immunogold tissue counts against gels containing known amounts of each protein, and the method was verified by application to Purkinje cells in which independent estimates exist for some of the protein concentrations. The results showed that, in animals with fully developed hearing, inner hair cells had\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\frac{1}{10}\) \end{document}of the proteinaceous calcium buffer of outer hair cells in which the cell body contained parvalbumin-β (oncomodulin) and calbindin-D28k at levels equivalent to 5 mmcalcium-binding sites. Both proteins were partially excluded from the hair bundles, which may permit fast unbuffered Ca2+regulation of the mechanotransducer channels. The sum of the calcium buffer concentrations decreased in inner hair cells and increased in outer hair cells as the cells developed their adult properties during cochlear maturation. The results suggest that Ca2+has distinct roles in the two types of hair cell, reflecting their different functions in auditory transduction. Ca2+is used in inner hair cells primarily for fast phase-locked synaptic transmission, whereas Ca2+may be involved in regulating the motor capability underlying cochlear amplification of the outer hair cell. The high concentration of calcium buffer in outer hair cells, similar only to skeletal muscle, may protect against deleterious consequences of Ca2+loading after acoustic overstimulation.

Mechanosensory channels of sensory cells mediate the sensations of hearing, touch, and some forms of pain. The TRPA1 (a member of the TRP family of ion channel proteins) channel is activated by pain-producing chemicals, and its inhibition impairs hair cell mechanotransduction. As shown here and previously, TRPA1 is expressed by hair cells as well as by most nociceptors (small neurons of dorsal root, trigeminal, and nodose ganglia) and localizes to their sensory terminals (mechanosensory stereocilia and peripheral free nerves, respectively). Thus, TRPA1 channels are proposed to mediate transduction in both hair cells and nociceptors. Accordingly, we find that heterologously expressed TRPA1 display channel behaviors expected for both auditory and nociceptive transducers. First, TRPA1 and the hair cell transducer share a unique set of pore properties not described for any other channel (block by gadolinium, amiloride, gentamicin, and ruthenium red, a ranging conductance of ∼100 pS that is reduced to 54% by calcium, permeating calcium-induced potentiation followed by closure, and reopening by depolarization), supporting a direct role of TRPA1 as a pore-forming subunit of the hair cell transducer. Second, TRPA1 channels inactivate in hyperpolarized cells but remain open in depolarized cells. This property provides a mechanism for the lack of desensitization, coincidence detection, and allodynia that characterize pain by allowing a sensory neuron to respond constantly to sustained stimulation that is suprathreshold (i.e., noxious) and yet permitting the same cell to ignore sustained stimulation that is subthreshold (i.e., innocuous). Our results support a TRPA1 role in both nociceptor and hair cell transduction.

Calcium is thought to be essential for adaptation of sensory receptor cells. However, the transduction cascade of hyperpolarizing, ciliary photoreceptors of the scallop does not use IP3-mediated Ca release, and the light-sensitive conductance is not measurably permeable to Ca2+. Therefore, two typical mechanisms that couple the light response to [Ca]ichanges seem to be lacking in these photoreceptors. Using fluorescent indicators, we determined that, unlike in their microvillar counterparts, photostimulation of ciliary cells under voltage clamp indeed evokes no detectable change in cytosolic Ca. Notwithstanding, these cells exhibit all of the hallmarks of light adaptation, including response range compression, sensitivity shift, and photoresponse acceleration. A possible mediator of Ca-independent sensory adaptation is cGMP, the second messenger that regulates the light-sensitive conductance; cGMP and 8-bromo cGMP not only activate light-dependent K channels but also reduce the amplitude of the light response to an extent greatly in excess of that expected from simple occlusion between light and chemical stimulation. In addition, these substances accelerate the time course of the photocurrent. Tests with pharmacological antagonists suggest that protein kinase G may be a downstream effector that controls, in part, the cGMP-triggered changes in photoresponse properties during light adaptation. However, additional messengers are likely to be implicated, especially in the regulation of response kinetics. These observations suggest a novel feedback inhibition pathway for signaling sensory adaptation.

Hearing inDrosophiladepends on the transduction of antennal vibration into receptor potentials by ciliated sensory neurons in Johnston's organ, the antennal chordotonal organ. We previously found that aDrosophilaprotein in the vanilloid receptor subfamily (TRPV) channel subunit, Nanchung (NAN), is localized to the chordotonal cilia and required to generate sound-evoked potentials (Kim et al., 2003). Here, we show that the only otherDrosophilaTRPV protein is mutated in the behavioral mutantinactive(iav). The IAV protein forms a hypotonically activated channel when expressed in cultured cells; in flies, it is specifically expressed in the chordotonal neurons, localized to their cilia and required for hearing. IAV and NAN are each undetectable in cilia of mutants lacking the other protein, indicating that they both contribute to a heteromultimeric transduction channelin vivo. A functional green fluorescence protein-IAV fusion protein shows that the channel is restricted to the proximal cilium, constraining models for channel activation.

Recent studies have shown that the T1R3 receptor protein encoded by theTas1r3gene is involved in transduction of sweet taste. To assess ligand specificity of the T1R3 receptor, we analyzed the association ofTas1r3allelic variants with taste responses in mice. In the F2hybrids between the C57BL/6ByJ (B6) and 129P3/J (129) inbred mouse strains, we determined genotypes of markers on chromosome 4, whereTas1r3resides, measured consumption of taste solutions presented in two-bottle preference tests, and recorded integrated responses of the chorda tympani gustatory nerve to lingual application of taste stimuli. For intakes and preferences, significant linkages toTas1r3were found for the sweeteners sucrose, saccharin, andd-phenylalanine but not glycine. For chorda tympani responses, significant linkages toTas1r3were found for the sweeteners sucrose, saccharin,d-phenylalanine,d-tryptophan, and SC-45647 but not glycine,l-proline,l-alanine, orl-glutamine. No linkages to distal chromosome 4 were detected for behavioral or neural responses to non-sweet quinine, citric acid, HCl, NaCl, KCl, monosodium glutamate, inosine 5′-monophosphate, or ammonium glutamate. These results demonstrate that allelic variation of theTas1r3gene affects gustatory neural and behavioral responses to some, but not all, sweeteners. This study describes the range of ligand sensitivity of the T1R3 receptor using anin vivoapproach and, to our knowledge, is the first genetic mapping study of activity in gustatory nerves.

The olfactory epithelium of fish contains three intermingled types of olfactory receptor neurons (ORNs): ciliated, microvillous, and crypt. The present experiments were undertaken to test whether the different types of ORNs respond to different classes of odorants via different families of receptor molecules and G-proteins corresponding to the morphology of the ORN. In catfish, ciliated ORNs express OR-type receptors and Gαolf. Microvillous ORNs are heterogeneous, with many expressing Gαq/11, whereas crypt ORNs express Gαo. Retrograde tracing experiments show that ciliated ORNs project predominantly to regions of the olfactory bulb (OB) that respond to bile salts (medial) and amino acids (ventral) (Nikonov and Caprio, 2001). In contrast, microvillous ORNs project almost entirely to the dorsal surface of the OB, where responses to nucleotides (posterior OB) and amino acids (anterior OB) predominate. These anatomical findings are consistent with our pharmacological results showing that forskolin (which interferes with Gαolf/cAMP signaling) blocks responses to bile salts and markedly reduces responses to amino acids. Conversely, U-73122 and U-73343 (which interfere with Gαq/11/phospholipase C signaling) diminish amino acid responses but leave bile salt and nucleotide responses essentially unchanged. In summary, our results indicate that bile salt odorants are detected predominantly by ciliated ORNs relying on the Gαolf/cAMP transduction cascade. Nucleotides are detected by microvillous ORNs using neither Gαolf/cAMP nor Gαq/11/PLC cascades. Finally, amino acid odorants activate both ciliated and microvillous ORNs but via different transduction pathways in the two types of cells.

We present evidence that resonance properties of rat vibrissae differentially amplify high-frequency and complex tactile signals. Consistent with a model of vibrissa mechanics, optical measurements of vibrissae revealed that their first mechanical resonance frequencies systematically varied from low (60-100 Hz) in longer, posterior vibrissae to high (∼750 Hz) in shorter, anterior vibrissae. Resonance amplification of tactile input was observedin vivoandex vivo, and in a variety of boundary conditions that are likely to occur during perception, including stimulation of the vibrissa with moving complex natural stimuli such as sandpaper. Vibrissae were underdamped, allowing for sharp tuning to resonance frequencies. Vibrissa resonance constitutes a potentially useful mechanism for perception of high-frequency and complex tactile signals. Amplification of small amplitude signals by resonance could facilitate detection of stimuli that would otherwise fail to drive neural activity. The systematic map of frequency sensitivity across the face could facilitate texture discrimination through somatotopic encoding of frequency content. These findings suggest strong parallels between vibrissa tactile processing and auditory encoding, in which the cochlea also uses resonance to amplify low-amplitude signals and to generate a spatial map of frequency sensitivity.

The ability to see at night relies on the transduction of single photons by the rod photoreceptors and transmission of the resulting signals through the retina. Using paired patch-clamp recordings, we investigated the properties of the first stage of neural processing of the rod light responses: signal transfer from rods to bipolar and horizontal cells. Bypassing the relatively slow phototransduction process and directly modulating the rod voltage or current allowed us to characterize signal transfer over a wide range of temporal frequencies. We found that the rod to second-order cell synapse acts as a bandpass filter, preferentially transmitting signals with frequencies between 1.5 and 4 Hz while attenuating higher and lower frequency inputs. The similarity of the responses in different types of postsynaptic cell and the properties of miniature EPSCs (mEPSCs) recorded in OFF bipolar cells suggest that most of the bandpass filtering is mediated presynaptically. Modeling of the network of electrically coupled rod photoreceptors suggests that spread of the signal through the network contributed to the observed high-pass filtering but not to the low-pass filtering. Attenuation of low temporal frequencies at the first retinal synapse sharpens the temporal resolution of the light response; attenuation of high temporal frequencies removes voltage noise in the rod that threatens to swamp the light response.

The mammalian peripheral taste system undergoes functional changes during postnatal development. These changes could reflect age-dependent alterations in the membrane properties of taste cells, which use a vast array of ion channels for transduction mechanisms. Yet, scarce information is available on the membrane events in developing taste cells. We have addressed this issue by studying voltage-dependent Na(+), K(+), and Cl(−) currents (I(Na),I(K), andI(Cl), respectively) in a subset of taste cells (the so-called “Na/OUT” cells, which are electrically excitable and thought to be sensory) from mouse vallate papilla. Voltage-dependent currents play a key role during taste transduction, especially in the generation of action potentials. Patch-clamp recordings revealed that I(Na),I(K), andI(Cl) were expressed early in postnatal development. However, only I(K) andI(Cl) densities increased significantly in developing Na/OUT cells. Consistent with the rise ofI(K) density, we found that action potential waveform changed markedly, with an increased speed of repolarization that was accompanied by an enhanced capability of repetitive firing. In addition to membrane excitability changes in putative sensory cells, we observed a concomitant increase in the occurrence of glia-like taste cells (the so called “leaky” cells) among patched cells. Leaky cells are likely involved in dissipating the increase of extracellular K(+) during action potential discharge in chemosensory cells. Thus, developing taste cells of the mouse vallate papilla undergo a significant electrophysiological maturation and diversification. These functional changes may have a profound impact on the transduction capabilities of taste buds during development.

Extrinsic afferent neurons play an essential role in both sensation and reflex control of visceral organs, but their specialized morphological peripheral endings have never been functionally identified. Extracellular recordings were made from fine nerve trunks running between the vagus nerve and esophagus of the guinea pig. Mechanoreceptors, which responded to esophageal distension, fired spontaneously, had low thresholds to circumferential stretch, and were slowly adapting. Calibrated von Frey hairs (0.12 mN) were used to probe the serosal surface at 100-200 sites, which were mapped on a video image of the live preparation. Each stretch-sensitive unit had one to three highly localized receptive fields ("hot spots"), which were marked with Indian ink applied on the tip of the von Frey hair. Recorded nerve trunks were then filled anterogradely, using biotinamide in an artificial intracellular solution. Receptive fields were consistently associated with intraganglionic laminar endings (IGLEs) in myenteric ganglia, but not with other filled neuronal structures. The average distance of receptive fields to IGLEs was 73 +/- 14 microm (24 receptive fields, from 12 units; n = 5), compared to 374 +/- 17 microm for 240 randomly generated sites (n = 5; p0.001). After maintained probing on a single receptive field, spontaneous discharge of units was inhibited, as were responses to distension. During adapted discharge to maintained distension, interspike intervals were distributed in a narrow range. This indicates that multiple receptive fields interact to encode mechanical distortion in a graded manner. IGLEs are specialized transduction sites of mechanosensitive vagal afferent neurons in the guinea pig esophagus.

Recent studies have suggested that myosin Ibeta mediates the adaptation of mechanoelectrical transduction in vestibular hair cells. An important prediction of this hypothesis is that myosin Ibeta should be found in the side insertional plaque, an osmiophilic hair bundle structure that anchors tip links and is thought to house the adaptation motor. To determine whether myosin Ibeta was situated properly to perform adaptation, we used immunofluorescence and immunoelectron microscopy with the monoclonal antibody mT2 to examine the distribution of myosin Ibeta in hair bundles of the bullfrog utricle. Although utricular hair cells differ in their rates and extent of adaptation [Baird RA (1994) Comparative transduction mechanisms of hair cells in the bullfrog utriculus. II. Sensitivity and response dynamics to hair bundle displacement. J Neurophysiol 71:685-705.], myosin Ibeta was present in all hair bundles, regardless of adaptation kinetics. Confirming that, nevertheless, it was positioned properly to mediate adaptation, myosin Ibeta was found at significantly higher levels in the side insertional plaque. Myosin Ibeta was also present at elevated levels at the second tip link anchor of a hair bundle, the tip insertional plaque, found at the tip of a stereocilium. These data support myosin Ibeta as the adaptation motor and are consistent with the suggestion that the motor serves to restore tension applied to transduction channels to an optimal level, albeit with different kinetics in different cell types.

Cochlear outer hair cells are capable of both mechanical-to-electrical and electrical-to-mechanical transduction. Vibration of their stereocilia by sound is believed to stimulate somatic motility via a receptor potential developed across the basolateral membrane, thereby enhancing the mechanical vibration and increasing the sensitivity and frequency selectivity of the ear. Extrinsic electrical currents, applied at the tops of the cells, also appear to activate motilityin vivo, presumably after entering the cell. Earlier experiments suggested such currents might enter through the transduction channels themselves, but an alternative shunt pathway through the membrane capacitance seems more likely on physical grounds. We therefore recorded electrically evoked oto-acoustic emissions while modulating the transduction channels by driving them with low-frequency sound. Recordings of the low-frequency cochlear microphonic provided a measure of the mean electrical conductance through the channels during sound stimulation. Emissions increased during displacement of the basilar membrane toward scala vestibuli, when the channels were biased open, and decreased on the opposite phase, and the modulation of the emission was in direct proportion to the cochlear microphonic. The results are the strongest evidence yet that electrically evoked emissions are generated directly by mechanisms related to cochlear transduction and lead to the surprising conclusion that, for frequencies up to at least 12 kHz, extrinsic electrical currents enter the hair cell predominantly by the resistive pathway through the transduction channels. Alternatively, the results might be consistent with direct modulation of a motility source driven by capacitive currents but whose output depends on the state of the channels.

Behavioral and physiological studies have demonstrated a reduced sensitivity to several taste stimuli early in development. It has been suggested that this reduced sensitivity results from a late maturation of underlying transduction mechanisms. Little is known, however, about maturation of membrane properties of taste cells early in development. We have obtained whole-cell recordings from single fungiform taste cells of rat pups to examine the development of the NaCl transduction system. Although taste buds undergo a considerable increase in size during development, membrane capacitance measurements revealed no change in membrane surface area of individual taste cells, suggesting that the increase in size results from an increase in the total number of cells per bud. Whole-cell recordings showed that taste cells from very young pups [postnatal day 2 (PND2)] already possessed voltage-activated Na+and K+currents with no apparent differences in size or kinetics compared with adults. Surprisingly, amiloride-sensitive Na+responses, important for Na+transduction, were found as early as PND2. The magnitude of responses to amiloride and the percentage of amiloride-sensitive cells remained the same throughout all age groups. Furthermore, the similarity of amiloride inhibition constants suggested that the channel in neonates is the same channel that is expressed in adult taste buds. Our results indicate that taste cells at PND2 already have acquired the transduction elements necessary for signaling NaCl responses to the afferent nerve. We hypothesize that complete functionality of the salt taste transduction system, however, may not be reached until amiloride-sensitive Na+channels become selectively localized at the apical membrane. This would explain previous studies indicating that amiloride sensitivity cannot be detected before PND12 in the intact tongue. Apical clustering of channels along with the opening of the taste pore and an increase in the total number of taste cells per bud likely constitute additional important steps toward a fully functional sensory system.

Transient elevations of intracellular Ca2+play an important role in regulating the sensitivity of olfactory transduction, but such elevations have not been demonstrated in the olfactory cilia, which are the site of primary odor transduction. To begin to understand Ca2+signaling in olfactory cilia, we used high-resolution imaging techniques to study the Ca2+transients that occur in salamander olfactory receptor neurons (ORNs) as a result of cyclic nucleotide-gated (CNG) channel activation. To visualize ciliary Ca2+signals, we loaded ORNs with the Ca2+indicator dye Fluo-3 AM and measured fluorescence with a laser scanning confocal microscope. Application of the phosphodiesterase inhibitor IBMX increased fluorescence in the cilia and other neuronal compartments; the ciliary signal occurred first and was more transient. This signal could be abolished by lowering external Ca2+or by applying LY83583, a potent blocker of CNG channels, indicating that Ca2+entry through CNG channels was the primary source of fluorescence increases. Direct activation of CNG channels with low levels of 8-Br-cGMP (1 μm) led to tonic Ca2+signals that were restricted locally to the cilia and the dendritic knob. Elevated external K+, which depolarizes cell membranes, increased fluorescence signals in the cell body and dendrite but failed to increase ciliary Ca2+fluorescence. The results demonstrate the existence and spatiotemporal properties of Ca2+transients in individual olfactory cilia and implicate CNG channels as a major pathway for Ca2+entry into ORN cilia during odor transduction.

The G-protein subunit α-gustducin, which is similar to rod transducin, has been implicated in the transduction of both sweet- and bitter-tasting substances. In rodents, there are differences in sensitivity to sweet and bitter stimuli in different populations of taste buds. Rat fungiform taste buds are more responsive to salts than to sweet stimuli, whereas those on the palate respond predominantly to sweet substances. In contrast, hamster fungiform taste buds are more sensitive to sweet-tasting stimuli. Taste buds in the vallate and foliate papillae of both species are sensitive to bitter compounds. These differences in sensitivity should be reflected in the numbers of gustducin-containing cells in different taste bud populations. We examined taste buds in the rat and hamster for immunoreactivity to an antibody against α-gustducin. Immunofluorescence of labeled taste cells was examined by confocal microscopy, and the cells were counted. Gustducin-positive cells were seen in all taste bud regions; they were spindle-shaped, with circular cross-sections and apical processes that extended to the taste pore. Cells with this characteristic shape in rat vallate taste buds are Type II (light) cells. In the rat, taste buds of the fungiform papillae had fewer gustducin-positive cells (3.1/taste bud) than those of other regions, including the posterior tongue and palate (>8.9/taste bud). Hamster fungiform taste buds contained twice as many gustducin-expressing cells (6.8/taste bud) as those of the rat. These data support the hypothesis that α-gustducin is involved in the transduction of both sweet- and bitter-tasting stimuli by mammalian taste receptor cells.

The mechanism of sensory transduction in chemosensory neurons of the vomeronasal organ (VNO) is not known. Based on molecular data, it is likely to be different from that mediating sensory transduction in the main olfactory system. To begin to understand this system, we have characterized the electrophysiological properties of dissociated mouse VNO neurons with patch-clamp recording. Sensory neurons were distinguished from nonsensory neurons by the presence of a dendrite, by immunoreactivity for olfactory marker protein, and by the firing of action potentials. The resting potential of VNO neurons was approximately −60 mV, and the average input resistance was 3 GΩ. Current injections as small as 1–2 pA elicited steady trains of action potentials that showed no sign of adaptation during a 2 sec stimulus duration. The voltage-gated conductances in VNO neurons are distinct from those in olfactory neurons. The Na+current is composed of two components; the major component was TTX-sensitive (Ki = 3.6 nm). The outward K+current activates at −30 mV with kinetics 10 times slower than for K+currents in olfactory neurons. The Ca2+current is composed of at least two components: an L-type current and a T-type current that activates at −60 mV and is not found in olfactory neurons. We find no evidence for cyclic nucleotide-gated channels in VNO neurons under a variety of experimental conditions, including those that produced large responses in mouse olfactory neurons, which is further evidence for a novel transduction pathway.

Positive deflection of the sensory hair bundle of a vertebrate hair cell opens transduction channels to depolarize the cell. In bullfrog saccular hair cells, there is a subsequent adaptation process, whereby the proportion of transduction channels that are open, and thus the receptor current, declines toward the resting value. This occurs because the sensitivity curve, relating open probability to bundle deflection, shifts along the deflection axis in response to bundle deflections, in a manner consistent with a relaxation of mechanical tension on transduction channels. In this study we determined the extent of adaptation, measured as the shift of the sensitivity curve following deflection of the hair bundle. The shift was determined both by comparison of the receptor current in the adapted state to the resting sensitivity curve, and by comparison of pre- and postadapted sensitivity curves. The adaptive shift approached steady state with a time constant of 20–30 msec, and was at steady state within 150 msec. For all positive and for small negative deflections, both methods showed a shift that was approximately 80% of the deflection. For larger negative deflections, the shift reached a fixed limit that was 100–500 nm negative to the freestanding bundle position. The limited extent of adaptation confers a time-dependent sensitivity: the cell has an instantaneous or phasic sensitivity curve that is steep, and steady- state or tonic sensitivity curve that is about five times broader. It also suggests the existence of two additional structural elements within the transduction apparatus. A revised quantitative theory accommodates these elements.

An expanding knowledge of neuropeptides and their function has led to a profound change in our view of how the PAN contributes to pain. In addition to their expected direct action on postsynaptic cells in the dorsal horn, neuropeptides can modify transmitter release from nearby terminals of other PANs and/or diffuse to act on dorsal horn neurons at a considerable distance from their site of release (Fig. 2). Contrary to early expectations and despite the evidence that several neuropeptides excite central nociceptive neurons, there is no clear correspondence between neuropeptide content and physiologically defined classes of small-diameter primary afferents. There is, however, a tendency for populations of afferents innervating different organs to differ consistently in their peptide content. In fact, the peptide content of primary afferents is, in part, determined by specific factors in the tissues that they innervate. Furthermore, peptide content can change dramatically in response to certain prolonged stimuli or nerve damage. The lack of correspondence of peptide content and physiological response pattern, the plasticity of peptide content, its tissue specificity, and the possibility for action at a distance from the site of their release from central PAN terminals strongly suggest that PAN peptides have functions that are fundamentally different from those of the short-range actions of amino acid neurotransmitters that are also found in the PAN. Finally, nowhere is the plasticity of function of the PAN more evident than at its peripheral terminals. Long-term changes are produced in these terminals by a host of peptides that derive from a variety of cell types. The complexity of this transduction process is augmented by the activity- induced release of peripherally active neuropeptides from the PAN itself. In addition to the variety of fundamental neurobiological issues that recent studies of PANs have raised, they have also generated a great deal of clinical interest, in view of the role of the PAN in inflammation and its accessibility for study and for therapeutic intervention.

Cilia at the tips of dendritic processes of olfactory receptor cells are the sites of initial recognition and transduction events in olfactory reception. We have detached cilia from the olfactory epithelium of the bullfrog, Rana catesbeiana, via a calcium shock and partially purified them in high yield (226 +/- 19 micrograms protein/frog, n = 14) by sucrose gradient centrifugation. The cilia appear to undergo osmotic lysis during the isolation procedure, forming isolated axonemal structures and ciliary plasma membrane vesicles with diameters of 100–500 nm and an internal volume of 2.3 +/- 0.5 microliter/mg protein. PAGE in SDS reveals approximately 30 protein bands, among which cytoskeletal components, such as tubulin and actin, are readily identifiable by immunoblotting. Approximately 15 glycoprotein bands reactive with concanavalin A are discernible with major glycopeptides at apparent molecular weights of 56–65, 95, and 116 kDa. In contrast to olfactory cilia, respiratory cilia, isolated from the palate of the frog, do not contain the prominent glycopeptides observed for olfactory cilia. The 56–65 kDa glycopeptide region reacts with antiserum against chick kidney, Na+/K+-ATPase, and contains the beta subunit of this enzyme. In addition, we have identified the alpha and beta subunits of a guanine nucleotide-binding protein (G-protein) in the olfactory cilia preparation. This preparation of isolated olfactory cilia from Rana catesbeiana represents a readily accessible model system for studies of initial events in chemosensory recognition and signal transduction in the olfactory system.