Muscarinic ACh receptor activation causes transmitter release from isolated frog vestibular hair cells.

In the frog, vestibular efferent fibers innervate only type-II vestibular hair cells. Through this direct contact with hair cells, efferent neurons are capable of modifying transmitter release from hair cells onto primary vestibular afferents. The major efferent transmitter, acetylcholine (ACh), is known to produce distinct pharmacological actions involving several ACh receptors. Previous studies have implicated the presence of muscarinic ACh receptors on vestibular hair cells, although, surprisingly, a muscarinic-mediated electrical response has not been demonstrated in solitary vestibular hair cells. This study demonstrates that muscarinic receptors can evoke transmitter release from vestibular hair cells. Detection of this release was obtained through patch-clamp recordings from catfish cone horizontal cells, serving as glutamate detectors after pairing them with isolated frog semicircular canal hair cells in a two-cell preparation. Although horizontal cells alone failed to respond to carbachol, application of 20 microM carbachol to the two-cell preparation resulted in a horizontal cell response that could be mimicked by exogenous application of glutamate. All of the horizontal cells in the two-cell preparation responded to 20 microM CCh. Furthermore, this presumed transmitter release persisted in the presence of d-tubocurarine at concentrations that block all known hair cell nicotinic ACh receptors. The effect on the detector cell, imparted by the carbachol application to the hair cell-horizontal cell preparation, was blocked both by 2-amino-5-phosphonopentanoic acid, a selective N-methyl-D-aspartate antagonist, and the muscarinic antagonist, atropine. Thus vestibular hair cells from the frog semicircular canal can be stimulated to release transmitter by activating their muscarinic receptors.

A pharmacologically distinct nicotinic ACh receptor is found in a subset of frog semicircular canal hair cells.

Frog vestibular organs are endowed with a prominent cholinergic efferent innervation whose stimulation results in several different effects, thereby suggesting diversity in the expression of postsynaptic acetylcholine (ACh) receptors. The application of ACh can mimic efferent stimulation in producing both an inhibition and a facilitation of afferent discharge which are thought to be mediated by at least two distinct ACh receptors present on vestibular hair cells, i.e., alpha9-containing nicotinic receptors (alpha9nAChR) and muscarinic receptors (mAChR), respectively. Using patch-clamp and multiunit vestibular afferent recordings, we demonstrate the presence of an additional excitatory hair cell nicotinic ACh receptor pharmacologically distinct from both alpha9nAChR and mAChR. In order of increasing potency, this distinct receptor was activated by ACh, carbachol, and particularly by the selective nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium (DMPP). This DMPP-sensitive nicotinic receptor (RDMPP) was antagonized by the classic nicotinic antagonist d-tubocurarine, but refractory to strychnine, atropine, and propylbenzilylcholine mustard, at concentrations that completely block alpha9nAChR and/or mAChR. Activation of RDMPP on application of ACh or DMPP to a subpopulation of isolated posterior semicircular canal (SCC) hair cells resulted in a large depolarization (18.0 +/- 1.2 mV). The current underlying this depolarization was typically small (80.1 +/- 21.6 pA) and showed an inward rectification starting around -45 mV. Given their respective EC50s (47 nM vs. 20 microM), RDMPP was nearly 400 times more sensitive to ACh than alpha9nAChR and thus responded to concentrations of ACh considered too low to be effective at stimulating alpha9nAChR. Despite this remarkable sensitivity, exogenous ACh readily stimulated the mAChR in the intact posterior SCC preparation but failed to activate RDMPP unless the acetylcholinesterase inhibitor physostigmine was present, or high concentrations of ACh were used (>3 mM). In frog, RDMPP most likely underlies the rapid excitatory response seen during efferent stimulation.

Localization of the two constitutively expressed nitric oxide synthase isoforms (nNOS and eNOS) in the same cell types in the saccule maculae of the frog Rana pipiens by immunoelectron microscopy: evidence for a back-up system?

There is growing evidence for a nitric oxide/cyclic GMP pathway of signal transduction in the vestibular system. Recently, two isoforms of nitric oxide (NO) synthase (nNOS and eNOS) and NO itself have been identified at the light microscopic level in the vestibulocochlear system of mice using specific antibodies and a new fluorescence indicator. In order to acquire more information about signal transduction and tissue modulation in this neuroepithelium at the cellular and subcellular levels, ultrathin sections of London Resin White-embedded saccule maculae of the frog Rana pipiens were incubated with various concentrations of commercially available antibodies to nNOS and eNOS. The immunoreactivity was visualized by a gold-labelled secondary antibody and the amount of the immunoreactions per microm2 was quantified for the different cell types and subcellular regions. Significant eNOS immunoreactivity was identified in the hair bundles, cuticular plates and the rest of the cytoplasm of the hair cells as well as in different subcellular regions of the supporting cells. Gold-labelled anti-nNOS antibodies stained mainly stereovilli and cuticular structures of hair cells and supporting cells, whereas the number of the immunoreactions in the remaining cytoplasm of both cell types was near the background level. The spatial co-localization of the two NOS isotypes in the same cell regions of hair cells and supporting cells was confirmed in double-labelling experiments. The immunocytochemical findings are suggestive of a redundant system in which one NOS isoform can (partially) replace the other. The different subcellular localization of the NOS isoforms may allow for isoform specific regulation of NOS activity by different Ca2+ currents at the subcellular level, underlining the importance of NO-regulated processes in neuroepithelia of the inner ear.

Direct interaction of serotonin type 3 receptor ligands with recombinant and native alpha 9 alpha 10-containing nicotinic cholinergic receptors.

In the present work, we characterized the effects of serotonin type 3 receptor ligands on recombinant and native alpha 9 alpha 10-containing nicotinic acetylcholine receptors (nAChRs). Our results indicate that the recombinant alpha 9 alpha 10 nAChR shares striking pharmacological properties with 5-HT(3) ligand-gated ion channels. Thus, 5-HT(3) receptor antagonists block ACh-evoked currents in alpha 9 alpha 10-injected Xenopus laevis oocytes with a rank order of potency of tropisetron (IC(50), 70.1 +/- 0.9 nM) > ondansetron (IC(50), 0.6 +/- 0.1 microM) = MDL 72222 (IC(50), 0.7 +/- 0.1 microM). Although serotonin does not elicit responses in alpha 9 alpha 10-injected oocytes, it blocks recombinant alpha 9 alpha 10 receptors in a noncompetitive and voltage-dependent manner (IC(50), 5.4 +/- 0.6 microM). On the other hand, we demonstrate an in vivo correlate of these properties of the recombinant receptor, with those of the alpha 9 alpha 10-containing nAChR of frog saccular hair cells. The possibility that the biogenic amine serotonin might act as a neuromodulator of the cholinergic efferent transmission in the vestibular apparatus and in the organ of Corti is discussed.

Signal discrimination in the semicircular canals: a role for group I metabotropic glutamate receptors.

Fluorescence immunocytochemistry indicates that enzymatically isolated semi-circular canal (SCC) hair cells express metabotropic glutamate receptors (mGluRs) 1a and 5. Antibody-antigen preadsorption controls proved entirely negative. Applied while mechanically stimulating the posterior SCC with a piezo-electric bimorph, the non-competitive, mGluR5-selective antagonist MPEP-HCl (1 microM-3 mM) dose-dependently reduces mechanically evoked facilitation of afferent discharge rate (IC50 136 microM; n = 4), while having no effect on tonic, unstimulated afferent discharge. It thus appears that group I mGluRs on SCC hair cells are activated during mechanical stimulation, but are not activated under tonic transmitter release conditions. We conclude that group I mGluRs expressed by SCC hair cells may serve as a mechanism for the selective amplification of mechanically evoked transmitter release, thereby enhancing signal discrimination at the VHC-vestibular afferent synapse.(50)

Transmitter release from Rana pipiens vestibular hair cells via mGluRs: a role for intracellular Ca(++) release.

The response of the semicircular canal (SCC) to the group I mGluR-selective agonist dihydroxyphenylglycine (DHPG; 300 microM) - facilitation of afferent discharge rate - was dose-dependently reduced by the phospholipase C inhibitor U-73122 (1-100 microM; IC(50): 22 microM), the smooth endoplasmic reticulum Ca(++) ATPase inhibitor thapsigargin (100 nM-3 microM; IC(50): 500 nM), and xestospongin C (100 pM-1 microM; IC(50): 11 nM), an inositol trisphosphate receptor (IP(3)R) antagonist. Ryanodine, a modulator of Ca(++)-induced Ca(++) release, biphasically facilitated, then suppressed this response (1 nM-1 mM; approximate IC(50): 50 microM). 5 mM caffeine increased the amplitude (34.6+/-13.4%) and duration (453+/-169.8%; n=4) of the response of the SCC to DHPG, while 50 mM caffeine eliminated this response (n=2). The protein kinase C inhibitor bisindolylmaleimide I-HCl (10-100 microM; n=3) and the cyclic-ADP ribose antagonist 8-Br-cyclic-ADP ribose (1-10 microM; n=3) had no effect on the response of the SCC to DHPG. These data suggest that the increase in transmitter release following activation of group I mGluRs on vestibular hair cells is associated with intracellular Ca(++) release from both IP(3)-sensitive and ryanodine/caffeine-sensitive intracellular Ca(++) stores. Such positive feedback on transmitter release may serve to enhance the contrast between the spontaneous and stimulus-evoked modes of hair cell transmitter release, thereby optimizing signal discrimination at the synapse between hair cells and vestibular afferent fibers.