Functionally expressed bitter taste receptor TAS2R14 in human epidermal keratinocytes serves as a chemosensory receptor.

Traditionally, it is theorized that skin sensation is initiated when cutaneous sensory afferents and Merkel cells receive sensory stimuli, while epidermal keratinocytes were deemed to have no role. However, mounting evidence has shown that keratinocytes can initiate skin sensation by receiving sensory stimuli and transmitting sensory information to sensory afferents. Knowledge regarding the mechanisms by which keratinocytes receive exogenous stimuli is limited, with TRP channels and olfactory receptors having been proposed to serve as receptors for exogenous stimuli in keratinocytes. Recently, expression analyses have demonstrated the expression of multiple TAS2R genes in human skin. TAS2Rs are chemosensory GPCRs employed by taste cells to detect bitter-tasting substances. However, only subtypes TAS2R1 and TAS2R38 have been characterized in epidermal keratinocytes. We present evidence suggesting that subtype TAS2R14 is functionally expressed in epidermal keratinocytes. TAS2R14 transcripts and protein were detected in primary and N/TERT-1 keratinocytes. Additionally, keratinocytes responded to α-thujone, a TAS2R14 ligand, with an increase in intracellular free Ca2+ concentration. The tastant-evoked Ca2+ signals were found to be mediated by wild-type TAS2R14 and heterotrimeric G proteins. We conclude that TAS2R14 serves as a chemosensory receptor in epidermal keratinocytes and hypothesize that it enables the cells to recognize potentially harmful chemical substances.

Inhibition of TRPM8 channels reduces pain in the cold pressor test in humans.

The transient receptor potential (subfamily M, member 8; TRPM8) is a nonselective cation channel localized in primary sensory neurons, and is a candidate for cold thermosensing, mediation of cold pain, and bladder overactivity. Studies with TRPM8 knockout mice and selective TRPM8 channel blockers demonstrate a lack of cold sensitivity and reduced cold pain in various rodent models. Furthermore, TRPM8 blockers significantly lower body temperature. We have identified a moderately potent (IC50 = 103 nM), selective TRPM8 antagonist, PF-05105679 [(R)-3-[(1-(4-fluorophenyl)ethyl)(quinolin-3-ylcarbonyl)amino]methylbenzoic acid]. It demonstrated activity in vivo in the guinea pig bladder ice water and menthol challenge tests with an IC50 of 200 nM and reduced core body temperature in the rat (at concentrations >1219 nM). PF-05105679 was suitable for acute administration to humans and was evaluated for effects on core body temperature and experimentally induced cold pain, using the cold pressor test. Unbound plasma concentrations greater than the IC50 were achieved with 600- and 900-mg doses. The compound displayed a significant inhibition of pain in the cold pressor test, with efficacy equivalent to oxycodone (20 mg) at 1.5 hours postdose. No effect on core body temperature was observed. An unexpected adverse event (hot feeling) was reported, predominantly periorally, in 23 and 36% of volunteers (600- and 900-mg dose, respectively), which in two volunteers was nontolerable. In conclusion, this study supports a role for TRPM8 in acute cold pain signaling at doses that do not cause hypothermia.

The role of TRPM8 in the Guinea-pig bladder-cooling reflex investigated using a novel TRPM8 antagonist.

Patients with overactive bladder often exhibit abnormal bladder contractions in response to intravesical cold saline (positive ice-water test). The molecular entity involved in cold sensation within the urinary bladder is unknown, but a potential candidate is the ion channel, transient receptor potential (melastatin)-8 (TRPM8). The objective of the present study was to investigate the role of TRPM8 in a bladder-cooling reflex evoked in anaesthetised guinea-pigs that is comparable to the positive ice-water test seen in patients. Guinea-pig TRPM8 was cloned from L6 dorsal root ganglia (DRG) and expressed in HEK293 cells. Functional agonist- and cold-induced Ca2+ influx and electrophysiology assays were performed in these cells, and for comparison in HEK293 cells expressing human TRPM8, using a novel TRPM8 antagonist, the S-enantiomer of 1-phenylethyl 4-(benzyloxy)-3-methoxybenzyl (2-aminoethyl) carbamate hydrochloride (PBMC). Potency data from these assays was used to calculate intravenous infusion protocols for targeted plasma concentrations of PBMC in studies on micturition reflexes evoked by intravesical infusion of menthol or cold saline in anaesthetised guinea-pigs. Tissue expression of TRPM8 in guinea-pig bladder, urethra and in dorsal root ganglia neurones traced from the bladder was also investigated. TRPM8 mRNA and protein were detected in L6 dorsal root ganglia, bladder urothelium and smooth muscle. PBMC antagonised in vitro activation of human and guinea-pig TRPM8 and reversed menthol and cold-induced facilitation of the micturition reflex at plasma concentrations consistent with in vitro potencies. The present data suggest that the bladder-cooling reflex in the guinea-pig involves TRPM8. The potential significance of TRPM8 in bladder disease states deserves future investigation.

NMDAR-mediated EPSCs are maintained and accelerate in time course during maturation of mouse and rat auditory brainstem in vitro.

NMDA receptors (NMDARs) mediate a slow EPSC at excitatory glutamatergic synapses throughout the brain. In many areas the magnitude of the NMDAR-mediated EPSC declines with development and is associated with changes in subunit composition, but the mature channel composition is often unknown. We have employed the calyx of Held terminal with its target, the principal neuron of the medial nucleus of the trapezoid body (MNTB), to examine the NMDAR-mediated EPSC during synapse maturation from P10 to P40. Our data show that the calyx has reached a mature state by around P18. The NMDAR-mediated EPSC amplitude (and dominant decay ) fell from around 5 nA (: 40-50 ms) at P10/11 to 0.3-0.5 nA (: 10-15 ms) by P18. The mature NMDAR-EPSC showed no sensitivity to ifenprodil, indicating lack of NR2B subunits, and no block by submicromolar concentrations of zinc, consistent with NR1-1b subunit expression. Additionally, from P11 to P18 there was a reduction in voltage-dependent block and the apparent dissociation constant for [Mg(2+)](o) (K(o)) changed from 7.5 to 14 mm. Quantitative PCR showed that the relative expression of NR2A and NR2C increased, while immunohistochemistry confirmed the presence of NR2A, NR2B and NR2C protein. Although the mature NMDAR-EPSC is small, it is well coupled to NO signalling, as indicated by DAR-4M imaging. We conclude that native mature NMDAR channels at the calyx of Held have a fast time course and reduced block by [Mg(2+)](o), consistent with dominance of NR2C subunits and functional exclusion of NR2B subunits. The pharmacology suggests a single channel type and we postulate that the mature NMDARs consist of heterotrimers of NR1-1b-NR2A-NR2C.

The impact of synaptic conductance on action potential waveform: evoking realistic action potentials with a simulated synaptic conductance.

Most current clamp studies trigger action potentials (APs) by step current injection through the recording electrode and assume that the resulting APs are essentially identical to those triggered by orthodromic synaptic inputs. However this assumption is not always valid, particularly when the synaptic conductance is of large magnitude and of close proximity to the axon initial segment. We addressed this question of similarity using the Calyx of Held/MNTB synapse; we compared APs evoked by long duration step current injections, short step current injections and orthodromic synaptic stimuli. Neither injected current protocol evoked APs that matched the evoked orthodromic AP waveform, showing differences in AP height, half-width and after-hyperpolarization. We postulated that this 'error' could arise from changes in the instantaneous conductance during the combined synaptic and AP waveforms, since the driving forces for the respective ionic currents are integrating and continually evolving over this time-course. We demonstrate that a simple Ohm's law manipulation of the EPSC waveform, which accounts for the evolving driving force on the synaptic conductance during the AP, produces waveforms that closely mimic those generated by physiological synaptic stimulation. This stimulation paradigm allows supra-threshold physiological stimulation (single stimuli or trains) without the variability caused by quantal fluctuation in transmitter release, and can be implemented without a specialised dynamic clamp system. Combined with pharmacological tools this method provides a reliable means to assess the physiological roles of postsynaptic ion channels without confounding affects from the presynaptic input.

Wide-band information transmission at the calyx of Held.

We use a mathematical model of the calyx of Held to explore information transmission at this giant glutamatergic synapse. The significant depression of the postsynaptic response to repeated stimulation in vitro is a result of various activity-dependent processes in multiple timescales, which can be reproduced by multiexponential functions in this model. When the postsynaptic current is stimulated by Poisson-distributed spike trains, its amplitude varies considerably with the preceding interspike intervals. Here we quantify the information contained in the postsynaptic current amplitude about preceding interspike intervals and determine the impact of different pre- and postsynaptic factors on information transmission. The mutual information between presynaptic spike times and the amplitude of the postsynaptic response in general decreases as the mean stimulation rate increases, but remains high even at frequencies greater than 100 Hz, unlike at many neocortical synapses. The maintenance of information transmission is attributable largely to vesicle recycling rates at low frequencies of stimulation, shifting to vesicle release probability at high frequencies. Also, at higher frequencies, the synapse operates largely in a release-ready mode in which most release sites contain a release-ready vesicle and release probabilities are low.

Interactions between multiple sources of short-term plasticity during evoked and spontaneous activity at the rat calyx of Held.

Sustained activity at most central synapses is accompanied by a number of short-term changes in synaptic strength which act over a range of time scales. Here we examine experimental data and develop a model of synaptic depression at the calyx of Held synaptic terminal that combines many of these mechanisms (acting at differing sites and across a range of time scales). This new model incorporates vesicle recycling, facilitation, activity-dependent vesicle retrieval and multiple mechanisms affecting calcium channel activity and release probability. It can accurately reproduce the time course of experimentally measured short-term depression across different stimulus frequencies and exhibits a slow decay in EPSC amplitude during sustained stimulation. We show that the slow decay is a consequence of vesicle release inhibition by multiple mechanisms and is accompanied by a partial recovery of the releasable vesicle pool. This prediction is supported by patch-clamp data, using long duration repetitive EPSC stimulation at up to 400 Hz. The model also explains the recovery from depression in terms of interaction between these multiple processes, which together generate a stimulus-history-dependent recovery after repetitive stimulation. Given the high rates of spontaneous activity in the auditory pathway, the model also demonstrates how these multiple interactions cause chronic synaptic depression under in vivo conditions. While the magnitude of the depression converges to the same steady state for a given frequency, the time courses of onset and recovery are faster in the presence of spontaneous activity. We conclude that interactions between multiple sources of short-term plasticity can account for the complex kinetics during high frequency stimulation and cause stimulus-history-dependent recovery at this relay synapse.

Evidence for the involvement of G-proteins in the generation of the slow poststimulus afterdepolarisation (sADP) induced by muscarinic receptor activation in rat olfactory cortical neurones in vitro.

The involvement of G-proteins in generating the slow poststimulus afterdepolarising potential (sADP) induced by muscarinic receptor activation in immature (P10-20) rat olfactory cortical brain slice neurones was investigated under whole-cell patch clamp, using GTP-gamma-S (G-protein activator) or GDP-beta-S (G-protein blocker)-filled electrodes. In control experiments using K methylsulphate electrodes, cell resting potential (V(m)) and spike firing properties were unaffected over 10-15 min recording, although input resistance (R(N)) was slightly increased ( approximately 14%). Oxotremorine-M (OXO-M; 10 microM) produced a reversible slow depolarisation, an increase in R(N) ( approximately 90%) and induction of a slow poststimulus inward tail current (I(ADP)) (measured under voltage clamp at -60 mV) that was sustained during drug exposure (up to 15 min); the amplitude of slow inward rectifier (I(h)) currents activated from -50 mV were also apparently increased. By contrast, in GTP-gamma-S-loaded cells, R(N) was consistently decreased ( approximately 22%) and spike firing threshold (V(th)) was raised ( approximately 5 mV) after 10 min recording. In approximately 60% of loaded cells, a persistent muscarinic slow inward current and I(ADP) were induced by OXO-M; I(h) relaxation amplitude was also significantly decreased. The effects of GTP-gamma-S on R(N), V(th) and I(h) were partly counteracted by adding Ba(2+) (100 microM) to the bathing medium or mimicked by adding baclofen (GABA(B) receptor agonist; 100 microM) to normally-recorded cells. Intracellular GDP-beta-S (up to 30 min) had no effect on cell membrane properties or I(h), but irreversibly blocked the muscarinic slow inward current and I(ADP) induced by OXO-M. We conclude that both muscarinic responses require G-protein-linked transduction mechanisms for their generation.