Hair cells, the mechanoreceptors of the acoustic and vestibular system, are presynaptic to primary afferent neurons of the eighth nerve and excite neural activity by the release of glutamate. In the present work, the role played by intracellular Ca2+ stores in afferent transmission was investigated, at the presynaptic level, by monitoring changes in the intracellular Ca2+ concentration ([Ca2+]i) in vestibular hair cells, and, at the postsynaptic level, by recording from single posterior canal afferent fibers. Application of 1-10 mm caffeine to hair cells potentiated Ca2+ responses evoked by depolarization at selected Ca2+ hot spots, and also induced a graded increase in cell membrane capacitance (DeltaCm), signaling exocytosis of the transmitter. Ca2+ signals evoked by caffeine peaked in a region located approximately 10 microm from the base of the hair cell. [Ca2+]i increases, similarly localized, were observed after 500 msec depolarizations, but not with 50 msec depolarizations, suggesting the occurrence of calcium-induced calcium release (CICR) from the same stores. Both Ca2+ and DeltaCm responses were inhibited after incubation with ryanodine (40 microm) for 8-10 min. Consistent with these results, afferent transmission was potentiated by caffeine and inhibited by ryanodine both at the level of action potentials and of miniature EPSPs (mEPSPs). Neither caffeine nor ryanodine affected the shape and amplitude of mEPSPs, indicating that both drugs acted at the presynaptic level. These results strongly suggest that endogenous modulators of the CICR process will affect afferent activity elicited by mechanical stimuli in the physiological frequency range.

The presence of a novel voltage-dependent chloride current, active in the subthreshold range of membrane potential, was detected in the mature and intact rat sympathetic neuron in vitro by using the two-microelectrode voltage-clamp technique. Hyperpolarizing voltage steps applied to a neuron held at -40/-50 mV elicited inward currents, whose initial magnitude displayed a linear instantaneous current-voltage (I-V) relationship; afterward, the currents decayed exponentially with a single voltage-dependent time constant (63.5 s at -40 mV; 10.8 s at -130 mV). The cell input conductance decreased during the command step with the same time course as the current. On returning to the holding potential, the ensuing outward currents were accompanied by a slow increase in input conductance toward the initial values; the inward charge movement during the transient ON response (a mean of 76 nC in 8 neurons stepped from -50 to -90 mV) was completely balanced by outward charge displacement during the OFF response. The chloride movements accompanying voltage modifications were studied by estimating the chloride equilibrium potential (E(Cl)) at different holding potentials from the reversal of GABA evoked currents. [Cl(-)](i) was strongly affected by membrane potential, and at steady state it was systematically higher than expected from passive ion distribution. The transient current was blocked by substitution of isethionate for chloride and by Cl(-) channel blockers (9AC and DIDS). It proved insensitive to K(+) channel blockers, external Cd(2+), intracellular Ca(2+) chelators [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)] and reduction of [Na(+)](e). It is concluded that membrane potential shifts elicit a chloride current that reflects readjustment of [Cl(-)](i). The cell input conductance was measured over the -40/-120-mV voltage range, in control medium, and under conditions in which either the chloride or the potassium current was blocked. A mix of chloride, potassium, and leakage conductances was detected at all potentials. The leakage component was voltage independent and constant at approximately 14 nS. Conversely, gCl decreased with hyperpolarization (80 nS at -40 mV, undetectable below -110 mV), whereas gK displayed a maximum at -80 mV (55.3 nS). Thus the ratio gCl/gK continuously varied with membrane polarization (2.72 at -50 mV; 0.33 at -110 mV). These data were forced in a model of the three current components here described, which accurately simulates the behavior observed in the "resting" neuron during membrane migrations in the subthreshold potential range, thereby confirming that active K and Cl conductances contribute to the genesis of membrane potential and possibly to the control of neuronal excitability.