The acetylcholine receptors of frog vestibular endorgans pharmacology, molecular biology, and immunocytochemistry
Description
The impetus for investigating acetylcholine (ACh) and its receptors among inner ear sensory cells began over forty years ago with the demonstration of acetylcholinesterase staining in cochlear and vestibular efferent fibers. A preponderance of evidence has since substantiated that ACh is indeed the major inner ear efferent transmitter among many vertebrates. In frog vestibular organs, efferent neurons exclusively innervate type-II hair cells whereby ACh, acting on acetylcholine receptors of these hair cells, ultimately inhibits and/or facilitates vestibular afferent firing. These two effects are thought to be mediated by at least two pharmacologically distinct ACh receptors that modulate the release of transmitter from the hair cells onto vestibular afferents In multiunit afferent recordings from frog saccule, ACh application results in an inhibition of afferent firing which is antagonized by strychnine, alpha-bungarotoxin, curare, tetraethylammonium, and apamin. In the remaining vestibular organs, the prevailing response to ACh is a facilitation of multiunit afferent firing which is antagonized by atropine and propylbenzilylcholine mustard. A coupling between alpha9-nicotinic acetylcholine receptors (alpha9nAChR) and apamin-sensitive, small-conductance, calcium-dependent potassium channels is thought to drive the inhibition by hyperpolarizing hair cells thereby decreasing their release of transmitter onto afferents. The presence of alpha9nAChR in vestibular cells was demonstrated using pharmacological, immunocytochemical, and molecular biological techniques. Based on the pharmacology, the facilitation of afferent firing is proposed to be mediated by a muscarinic; receptor (mAChR) although an exact molecular description is lacking Vestibular hair cells, dissociated using trypsin, consistently responded to acetylcholine in a manner consistent with alpha9nAChR activation. Under voltage- and current-clamp conditions, ACh routinely produced an increase in outward current with a concomitant hyperpolarization. In agreement with alpha9nAChR pharmacology obtained in multiunit afferent recordings, strychnine, curare, and tetraethylammonium reversibly antagonized this ACh response in saccular hair cells. Both papain and two bacterial proteases (VIII, XXIV), enzymes commonly used to isolate vestibular hair cells, abolish the response to ACh mediated by alpha9nAChR in these same cells. At this present juncture, electrophysiological evidence for mAChR activation in isolated vestibular hair cells is inconclusive. It is hypothesized that ACh may facilitate transmitter release via the mAChR without producing an electrical change in hair cells