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Acetylcholine (ACh) is usually considered to be the neurotransmitter of the efferent vestibular system. The nature and the localization of cholinergic receptors have been investigated on frog isolated vestibular hair cells (VHCs), by measuring variations of intracellular calcium concentration ([Ca2+]i), using calcium sensitive dye fura-2. Focal iontophoretic ACh (1 M, 300 nA.40 ms) application induced a rapid increase in [Ca2+]i, reaching a peak in 20 s and representing about 5-fold the resting level (from 61 +/- 6 to 320 +/- 26 nM). Applications of muscarinic agonists as methacholine and carbachol induced weaker calcium responses (from 78 +/- 25 to 238 +/- 53 nM) than the one obtained with ACh applications. These muscarinic agonists were efficient only in precise zones. Desensitization of muscarinic receptors to successive stimulations was significant. Perfusion of nicotine or 1,1-dimethyl-4-phenyl-piperazinium (DMPP), a nicotinic agonist, induced an increase in [Ca2+]i only in some cells (4/28 with DMPP). These results indicated the presence of cholinergic receptors on frog VHCs: muscarinic receptors were more responsive than nicotinic receptors. Presence of muscarinic and nicotinic receptors in the membrane of VHCs could indicate different modulations of VHCs activity mediated by [Ca2+]i and involving an efferent control which represents a central regulation of the vestibular afferent message.

Afferent nerve calyces which surround type I vestibular hair cells (VHCI) have recently been shown to contain synaptic-like vesicles and to be immunoreactive to glutamate antibodies. In order to understand the physiological significance of these observations, the presence of glutamate receptors on type I vestibular sensory cells has been investigated. The effect of excitatory amino acids applied by iontophoresis was examined by spectrofluorimetry using fura-2 sensitive dye. Glutamate application caused a rapid and transient increase in intracellular calcium concentration ([Ca2+]i), in a dose-dependent manner. The ionotropic glutamate receptors agonists N-methyl-D-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and quisqualic acid (QA) induced an increase of [Ca2+]i. The NMDA receptor antagonist 2-amino-5-phosphonovaleric acid and the AMPA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione partially blocked the glutamate response, by 39 +/- 10 and 53 +/- 11% respectively. Metabotropic receptors were also revealed by the specific agonist trans-1-amino-cyclopentyl-1,3-dicarboxylate. The presence of different glutamate receptors on the VHCI membrane suggests two kinds of feedback. (i) At the base of the sensory cell, autoreceptors may locally control the synaptic transmission. (ii) At the apex, postsynaptic receptors may modulate sensory transduction from glutamate release at the upper part of the afferent nerve calyx. These feedbacks suggest presynaptic modulation of the vestibular hair cell response which could affect its sensitivity.

The resting free calcium level was measured in 128 isolated mammalian vestibular sensory cells using the calcium-sensitive dye fura-2. Iontophoresis was used to apply short, localised and limited pulses of K+ which evoked dynamic changes in intracellular free calcium concentration. While most of the type I hair cells tested showed brief reversible and specific calcium responses, some were unresponsive. The changes in intracellular free calcium were also measured by videomicroscopic analysis. Iontophoretic application of K+ ions is shown to be a suitable method for inducing fast, transient changes in intracellular free calcium in vestibular hair cells. This technique could be useful for applying several ions and charged molecules such as amino acids in in-vitro cellular methods.

Living sensory cells were isolated from the cristae ampullaris and macula utriculi of the guinea pig. Enzymatic and mechanical dissociation were used to obtain different populations of hair cells, the most predominant being type I cells. Their form varied: cell body of variable roundness, and neck and cilia of different lengths. The observation of many tilted cuticular plates supports the hypothesis of active mechanisms regulating mechanotransduction at the apex of these cells. Cell viability was verified by double fluorescent labeling (FDA-PI), which indicated that under correct conditions about 90% of the sensory cells could be maintained in vitro for several hours after dissociation. The detection of actin in the cuticular plate and cilia shows that the technique has various potential applications in morphological studies, and can contribute to investigations on the physiology of mammalian vestibular cells.