Non-competitive metabotropic glutamate 1 receptor antagonists block activity of slowly adapting type I mechanoreceptor units in the rat sinus hair follicle.

Previous studies suggested that Group I metabotropic glutamate (mGlu) receptors play a role in mechanotransduction processes of slowly adapting type I mechanoreceptors. Using an isolated rat sinus hair follicle preparation we tested a range of compounds. Surprisingly, only non-competitive mGlu1 receptor antagonists produced profound and long-lasting depression of mechanically evoked firing. 6-Amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-alpha]benzimidazole-2-carboxamide hydrochloride (YM-298198) had an IC(50) of 8.7 muM (95% CI 5.7 to 13.2 microM), representing the most potent known blocker of type I mechanoreceptors. The derivative 6-amino-N-cyclohexyl-3-methylthiazolo[3,2-alpha]benzimidazole-2-carboxamide hydrochloride (desmethyl YM-298198) had a comparable potency. Another compound 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) had a similar depressant effect, although it was less potent with an approximate IC(50) of 100 microM. Between three and seven times the concentration of CPCCOEt and YM-298198 respectively was required to produce similar depressions in slowly adapting type II units. No depression, and some weak excitatory effects, were observed using the following ligands: the competitive mGlu1 receptor antagonist alpha-amino-5-carboxy-3-methyl-2-thiopheneacetic acid (3-MATIDA) (300 microM), the phosphoserine phosphatase inhibitor dl-2-amino-3-phosphonopropionic acid (dl-AP3) (2 mM), non-competitive mGlu5 receptor antagonists 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine; (S)-3,5-DHPG, (S)-3,5-dihydroxyphenylglycine (MTEP) (10 microM) and 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) (100 microM), the mGlu1 receptor agonist (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG) (500 microM), and the mGlu5 receptor agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) (1 mM). The results suggest that the non-competitive mGlu1 receptor antagonists are not acting at conventional mGlu1 receptors but at other binding sites, possibly those directly associated with mechanogated channels or on any of a number of indirect biochemical pathways. YM-298198 and related compounds may prove to be useful ligands to identify mechanosensitive channel proteins. The selective interference of type I units may provide further evidence that Merkel cells are mechanotransducers. Finally such compounds may deliver insights or treatments for Merkel cell carcinoma.

A pharmacological study of slowly adapting mechanoreceptors responsive to cold thermal stimulation.

Weber's silver Thaler illusion is the perception that cold objects appear heavier than warm objects. We were interested in studying the pharmacology of mechanoreceptor units that displayed increased spontaneous firing to cold stimuli. An isolated rat sinus hair preparation with intact nerve terminals was used to record the activity of two types of slowly adapting mechanoreceptors (St I and St II) during temperature ramps (0.91-1.73 degrees C/min) from normal bath temperature of 31+/-2 degrees C, cold to 14.5 degrees C and heat to 46 degrees C. Twenty-seven of the 43 mechanoreceptor units displayed marked increases in their spontaneous firing to cold or cooling thermal gradients, and were classified as cold mechanoreceptors. A high proportion (3:1) of St II units were responsive to cold than not, while the ratio was reversed for St I units (1:2). Most cold mechanoreceptor units showed decreases in mechanical responses to cold thermal gradients. Similar to specific cold thermoreceptors, many of the cold mechanoreceptor units briefly displayed increased spontaneous firing at higher (>41 degrees C) temperatures. The spontaneous firing of cold mechanoreceptor units was increased by the transient receptor potential (TRP) channel agonist icilin (30-100 microM) in a dose-dependent manner. Responses to mechanical stimulation were generally unaffected by icilin in these units, although their evoked response latencies were significantly reduced (similar to the effect of K+ channel blocker tetraethylammonium in St II units). TRPM8 channel agonist, (-) menthol 200 microM, had mixed effects on spontaneous firing but consistently enhanced cold responses. Other TRP agonists, cinnamaldehyde 1-2 mM and camphor 0.5-2 microM, reduced spontaneous and evoked responses. TRPA1 agonist allyl isothiocyanate (mustard oil) 50-100 microM and TRPV1 agonist capsaicin 1-3 microM had no effect. A broad spectrum TRP antagonist, Ruthenium Red 30 microM, had no effect. The TRPM8 antagonist, capsazepine 100-200 microM, blocked cold-evoked responses. Although these data generally provide support for the possibility that cooling responses are mediated by TRPM8 channels, the detailed profile of results suggests that another, as yet unidentified TRP channel, is involved. Multiplex coding of mechanical and thermal information by slowly adapting mechanoreceptors may play a functional role in thermal perception, and may explain Weber's silver Thaler illusion.

Group II metabotropic glutamate receptors reduce excitatory but not inhibitory neurotransmission in rat barrel cortex in vivo.

Group II metabotropic (mGlu) receptors are known to play an important role in regulating the release of excitatory transmitter in a number of brain areas. Previous experiments demonstrated that (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) depressed excitatory transmission in the adult rat barrel cortex. Here we show, using in vivo extracellular single unit recordings and iontophoretic application of drugs, that selective activation of Group II mGlu receptors depresses excitatory but not inhibitory transmission. The selective Group II receptor agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC) had similar depressant effects to 1S,3R-ACPD on tactile evoked responses of rapidly adapting neurons. The depressant effects were seen on shorter latency (<12 ms) responses, were most pronounced in layers 3-4 (and 5b for 2R,4R-APDC only), and were reversibly antagonized by the Group II receptor antagonist (2S)-alpha-ethylglutamic acid (EGLU) relative to depressions produced by iontophoretic GABA. Where 1S,3R-ACPD and 2R,4R-APDC depressed excitatory transmission, there was little or no effect on postsynaptic excitations produced by iontophoretic AMPA--a result that supports a presynaptic location of Group II receptors on excitatory terminals. To assess the possible involvement of Group II mGlu receptors in the modulation of inhibition, we studied the effect of iontophoretic 1S,3R-ACPD in a condition-test protocol. The results contrasted markedly from those previously observed using the Group III agonist L(+)-2-amino-4-phosphonobutyric acid in that activation of Group II receptors using 1S,3R-ACPD did not modulate inhibition. Therefore our results show that Group II mGlu receptors play an important role in modulating excitatory, but not inhibitory, transmission. We propose that the Group II mGlu receptors are located on excitatory terminals, and act as autoreceptors. Their role appears to be important in the early stages of cortical processing, by keeping excitatory inputs within specified physiological limits, and possibly by mediating depression evidenced during synaptic plasticity.

Are unconventional NMDA receptors involved in slowly adapting type I mechanoreceptor responses?

Specific immunohistochemical staining for NMDA receptor NR2A/B subunits was found in the outer root sheath layer of rat sinus hair (whisker) follicle. Co-localization with CK 20 confirmed that Merkel cells were stained. The NR2A/B staining seen on Merkel cells was pericellular. In addition it appeared that NF70-positive staining was in close proximity to, but did not colocalise with NR2A/B immunoreactivity, indicating that NR2A/B was only expressed by Merkel cells and not their adjacent nerve terminals. Merkel cells and the nerve terminals have previously been associated with electrophysiological recordings from slowly adapting type I (St I) mechanoreceptor unit activity. Pharmacological experiments with isolated sinus hairs using a wide range of ionotropic glutamate receptor antagonists found that only certain NMDA receptor blockers depressed St I unit responses to mechanical stimuli. AMPA/kainate receptor antagonists (CNQX and NBQX, 100 microM) had no effect, nor did classical competitive NMDA receptor antagonists, D-AP5 (600 microM) and R-CPP (100 microM), nor the NMDA glycine site antagonist 5,7-dichlorokynurenic acid (100 microM). The only effective NMDA receptor blockers were those selective for the polyamine site: ifenprodil (IC50 20 microM) and Ro 25-6981 (IC50 approximately 50 microM), and the associated ion channel: MK 801, ketamine and (+/-)-1-(1,2-diphenylethyl)piperidine (IC50 < 100 microM). The two enantiomers of MK 801 were equipotent. All effects were long lasting, consistent with their non-/uncompetitive actions. The most potent drug tested, ifenprodil, at an effective dose of 30 microM, had a mean recovery time of 74 min. A three-fold increase in drug concentration was required to depress St II units (associated with non-synaptic lanceolate endings). Changes in Zn2+ did not affect St I unit responses. These data suggest that unconventional NMDA receptors are involved in St I unit responses, but question the notion of a glutamatergic synapse between the Merkel cell and nerve terminal.