K+ Accumulation and Clearance in the Calyx Synaptic Cleft of Type I Mouse Vestibular Hair Cells.

Vestibular organs of Amniotes contain two types of sensory cells, named Type I and Type II hair cells. While Type II hair cells are contacted by several small bouton nerve terminals, Type I hair cells receive a giant terminal, called a calyx, which encloses their basolateral membrane almost completely. Both hair cell types release glutamate, which depolarizes the afferent terminal by binding to AMPA post-synaptic receptors. However, there is evidence that non-vesicular signal transmission also occurs at the Type I hair cell-calyx synapse, possibly involving direct depolarization of the calyx by K+ exiting the hair cell. To better investigate this aspect, we performed whole-cell patch-clamp recordings from mouse Type I hair cells or their associated calyx. We found that [K+] in the calyceal synaptic cleft is elevated at rest relative to the interstitial (extracellular) solution and can increase or decrease during hair cell depolarization or repolarization, respectively. The change in [K+] was primarily driven by GK,L, the low-voltage-activated, non-inactivating K+ conductance specifically expressed by Type I hair cells. Simple diffusion of K+ between the cleft and the extracellular compartment appeared substantially restricted by the calyx inner membrane, with the ion channels and active transporters playing a crucial role in regulating intercellular [K+]. Calyx recordings were consistent with K+ leaving the synaptic cleft through postsynaptic voltage-gated K+ channels involving KV1 and KV7 subunits. The above scenario is consistent with direct depolarization and hyperpolarization of the calyx membrane potential by intercellular K+.

Late-onset bursts evoked by mossy fibre bundle stimulation in unipolar brush cells: evidence for the involvement of H- and TRP-currents.

Synaptic transmission at central synapses has usually short latency and graded amplitude, thereby regulating threshold crossing and the probability of action potential generation. In the granular layer of the vestibulo-cerebellum, unipolar brush cells (UBCs) receive a giant synapse generating a stereotyped excitatory postsynaptic potential (EPSP)-burst complex with early-onset (∼2 ms) and high reliability. By using patch-clamp recordings in cerebellar slices of the rat vestibulo-cerebellum, we found that mossy fibre bundle stimulation also evoked (in ∼80% of cases) a late-onset burst (after tens to hundreds of milliseconds) independent of EPSP generation. Different from the early-onset, the late-onset burst delay decreased and its duration increased by raising stimulation intensity or the number of impulses. Although depending on synaptic activity, the late-onset response was insensitive to perfusion of APV ((2R)-5-amino-phosphonopentanoate), NBQX (2,3-dioxo-6-nitro-tetrahydrobenzo(f)quinoxaline-7-sulfonamide) and MCPG ((RS)-α-methyl-4-carboxyphenylglycine) and did not therefore depend on conventional glutamatergic transmission mechanisms. The late-onset response was initiated by a slow depolarizing ramp driven by activation of an H-current (sensitive to ZD7288 and Cs(+)) and of a TRP- (transient receptor potential) current (sensitive to SKF96365), while the high voltage-activated and high voltage-activated Ca(2+) currents (sensitive to nimodipine and mibefradil, respectively) played a negligible role. The late-onset burst was occluded by intracellular cAMP. These results indicate that afferent activity can regulate H- and TRP-current gating in UBCs generating synaptically driven EPSP-independent responses, in which the delay rather than amplitude is graded with the intensity of the input pattern. This modality of synaptic transmission may play an important role in regulating UBC activation and granular layer functions in the vestibulo-cerebellum.

Intercellular K⁺ accumulation depolarizes Type I vestibular hair cells and their associated afferent nerve calyx.

Mammalian vestibular organs contain two types of sensory receptors, named Type I and Type II hair cells. While Type II hair cells are contacted by several small afferent nerve terminals, the basolateral surface of Type I hair cells is almost entirely enveloped by a single large afferent nerve terminal, called calyx. Moreover Type I, but not Type II hair cells, express a low-voltage-activated outward K(+) current, I(K,L), which is responsible for their much lower input resistance (Rm) at rest as compared to Type II hair cells. The functional meaning of I(K,L) and associated calyx is still enigmatic. By combining the patch-clamp whole-cell technique with the mouse whole crista preparation, we have recorded the current- and voltage responses of in situ hair cells. Outward K(+) current activation resulted in K(+) accumulation around Type I hair cells, since it induced a rightward shift of the K(+) reversal potential the magnitude of which depended on the amplitude and duration of K(+) current flow. Since this phenomenon was never observed for Type II hair cells, we ascribed it to the presence of a residual calyx limiting K(+) efflux from the synaptic cleft. Intercellular K(+) accumulation added a slow (τ>100ms) depolarizing component to the cell voltage response. In a few cases we were able to record from the calyx and found evidence for intercellular K(+) accumulation as well. The resulting depolarization could trigger a discharge of action potentials in the afferent nerve fiber. Present results support a model where pre- and postsynaptic depolarization produced by intercellular K(+) accumulation cooperates with neurotransmitter exocytosis in sustaining afferent transmission arising from Type I hair cells. While vesicular transmission together with the low Rm of Type I hair cells appears best suited for signaling fast head movements, depolarization produced by intercellular K(+) accumulation could enhance signal transmission during slow head movements.

Elementary properties of Kir2.1, a strong inwardly rectifying K(+) channel expressed by pigeon vestibular type II hair cells.

By using the patch-clamp technique in the cell-attached configuration, we have investigated the single-channel properties of an inward rectifier potassium channel (Kir) expressed by pigeon vestibular type II hair cells in situ. In high-K(+) external solution with 2 mM Mg(2+), Kir inward current showed openings to at least four amplitude levels. The two most frequent open states (L2 and L3) had a mean slope conductance of 13 and 28 pS, respectively. L1 (7 pS) and L4 (36 pS) together accounted for less than 6% of the conductive state. Closed time distributions were fitted well using four exponential functions, of which the slowest time constant (tau(C4)) was clearly voltage-dependent. Open time distributions were fitted well with two or three exponential functions depending on voltage. The mean open probability (P(O)) decreased with hyperpolarization (0.13 at -50 mV and 0.03 at -120 mV). During pulse-voltage protocols, the Kir current-decay process (inactivation) accelerated and increased in extent with hyperpolarization. This phenomenon was associated with a progressive increase of the relative importance of tau(C4). Kir inactivation almost disappeared when Mg(2+) was omitted from the pipette solution. At the same time, P(O) increased at all membrane voltages and the relative importance of L4 increased to a mean value of 47%. The relative importance of tau(C4) decreased for all open states, while L4 only showed a significantly longer open time constant. The present work provides the first detailed quantitative description of the elementary properties of the Kir inward rectifier in pigeon vestibular type II hair cells and specifically describes the Kir gating properties and the molecule's sensitivity to extracellular Mg(2+) for all subconductance levels. The present results are consistent with the Kir2.1 protein sustaining a strong inwardly rectifying K(+) current in native hair cells, characterized by rapid activation time course and slow partial inactivation. The longest closed state (tau(C4)) appears as the main parameter involved in time- and Mg(2+)-dependent decay. Finally, in contrast to Kir2.1 results described so far for mammalian cells, external Mg(2+) had no effect on channel conductance.

Na+ currents in vestibular type I and type II hair cells of the embryo and adult chicken.

In birds, type I and type II hair cells differentiate before birth. Here we describe that chick hair cells, from the semicircular canals, begin expressing a voltage-dependent Na current (INa) from embryonic day 14 (E14) and continue to express the current up to hatching (E21). During this period, INa was present in most (31/43) type I hair cells irrespective of their position in the crista, in most type II hair cells located far from the planum semilunatum (48/63), but only occasionally in type II hair cells close to the planum semilunatum (2/35). INa activated close to -60 mV, showed fast time- and voltage-dependent activation and inactivation, and was completely, and reversibly, blocked by submicromolar concentrations of tetrodotoxin (Kd = 17 nM). One peculiar property of INa concerns its steady-state inactivation, which is complete at -60 mV (half-inactivating voltage = -96 mV). INa was found in type I and type II hair cells from the adult chicken as well, where it had similar, although possibly not identical, properties and regional distribution. Current-clamp experiments showed that INa could contribute to the voltage response provided that the cell membrane was depolarized from holding potentials more negative than -80 mV. When recruited, INa produced a significant acceleration of the cell membrane depolarization, which occasionally elicited a large rapid depolarization followed by a rapid repolarization (action-potential-like response). Possible physiological roles for INa in the embryo and adult chicken are discussed.

Regional distribution of calcium currents in frog semicircular canal hair cells.

In the present work we studied the regional expression of voltage-dependent Ca channels in hair cells from the frog semicircular canals, employing whole-cell patch-clamp on isolated and in situ hair cells. Although Ca channels are thought to play a major role in afferent transmission, up to now no data were available regarding their distribution in vestibular organs. The problem appears of interest, especially in the light of recent results showing the presence of multiple Ca current components in semicircular canal hair cells. Our data suggest the presence, in all regions of the crista ampullaris, of two classes of cells, one displaying an inactivating Ca current (R1) and one lacking it. In the former cells, Ca current amplitude decreased from the central to the peripheral zone (the maximal currents being observed in the intermediate zone). Only L-type and R2 current components displayed regional differences in expression, whereas the size and properties of R1, although variable among cells, were not regionalized. However, in cells lacking R1, Ca current amplitudes were similar regardless of cell shape and location. The possible contributions of this Ca current distribution to afferent discharge properties are discussed.

Membrane properties of chick semicircular canal hair cells in situ during embryonic development.

The electrophysiological properties of developing vestibular hair cells have been investigated in a chick crista slice preparation, from embryonic day 10 (E10) to E21 (when hatching would occur). Patch-clamp whole-cell experiments showed that different types of ion channels are sequentially expressed during development. An inward Ca(2+) current and a slow outward rectifying K(+) current (I(K(V))) are acquired first, at or before E10, followed by a rapid transient K(+) current (I(K(A))) at E12, and by a small Ca-dependent K(+) current (I(KCa)) at E14. Hair cell maturation then proceeds with the expression of hyperpolarization-activated currents: a slow I(h) appears first, around E16, followed by the fast inward rectifier I(K1) around E19. From the time of its first appearance, I(K(A)) is preferentially expressed in peripheral (zone 1) hair cells, whereas inward rectifying currents are preferentially expressed in intermediate (zone 2) and central (zone 3) hair cells. Each conductance conferred distinctive properties on hair cell voltage response. Starting from E15, some hair cells, preferentially located at the intermediate region, showed the amphora shape typical of type I hair cells. From E17 (a time when the afferent calyx is completed) these cells expressed I(K, L), the signature current of mature type I hair cells. Close to hatching, hair cell complements and regional organization of ion currents appeared similar to those reported for the mature avian crista. By the progressive acquisition of different types of inward and outward rectifying currents, hair cell repolarization after both positive- and negative-current injections is greatly strengthened and speeded up.

Calcium channels functional roles in the frog semicircular canal.

Different types of voltage-operated calcium channels have been described in hair cells; however, no clear functional role has been assigned to them. As a first functional characterization of vestibular calcium channels, we studied the effect of several calcium channel agonists and antagonists on whole nerve firing rate in an isolated frog semicircular canal preparation. Resting activity was affected by all dihydropyridines tested and by omegaconotoxin GVIA, whereas only nimodipine was able to reduce the mechanically evoked activity. These results indicate that nimodipine-sensitive channels play a major role in afferent transmitter release, and omega-conotoxin GVIA sensitive channels regulate the afferent firing (possibly on the postsynaptic side) but with a less important role.

Artifactual voltage response recorded from hair cells with patch-clamp amplifiers.

Patch-clamp amplifiers (PCAs) are commonly used to characterize voltage- and current-clamp responses in the same cell. However, the cell membrane voltage response can be severely distorted by PCAs working in the current-clamp mode. Here we compare the voltage response of pigeon semicircular canal hair cells in situ, recorded with two different PCAs, and with a classic microelectrode bridge amplifier (BA). We found that the voltage response of hair cells recorded with PCAs differed significantly from that recorded with the BA. The true hair cell membrane voltage response to positive current steps was characterized by a strongly damped oscillation, whose frequency and duration depended on hair cell location in the sensory crista ampullaris.

Electrophysiological properties of vestibular sensory and supporting cells in the labyrinth slice before and during regeneration.

The whole cell patch-clamp technique in combination with the slice preparation was used to investigate the electrophysiological properties of pigeon semicircular canal sensory and supporting cells. These properties were also characterized in regenerating neuroepithelia of pigeons preinjected with streptomycin to kill the hair cells. Type II hair cells from each of the three semicircular canals showed similar, topographically related patterns of passive and active membrane properties. Hair cells located in the peripheral regions (zone I, near the planum semilunatum) had less negative resting potentials [0-current voltage in current-clamp mode (Vz) = -62.8 +/- 8.7 mV, mean +/- SD; n = 13] and smaller membrane capacitances (Cm = 5.0 +/- 0.9 pF, n = 14) than cells of the intermediate (zone II; Vz = -79.3 +/- 7.5 mV, n = 3; Cm = 5.9 +/- 1.2 pF, n = 4) and central (zone III; Vz = -68.0 +/- 9.6 mV, n = 17; Cm = 7.1 +/- 1.5 pF, n = 18) regions. In peripheral hair cells, ionic currents were dominated by a rapidly activating/inactivating outward K+ current, presumably an A-type K+ current (IKA). Little or no inwardly rectifying current was present in these cells. Conversely, ionic currents of central hair cells were dominated by a slowly activating/inactivating outward K+ current resembling a delayed rectifier K+ current (IKD). Moreover, an inward rectifying current at voltages negative to -80 mV was present in all central cells. This current was composed of two components: a slowly activating, noninactivating component (Ih), described in photoreceptors and saccular hair cells, and a faster-activating, partially inactivating component (IK1) also described in saccular hair cells in some species. Ih and IK1 were sometimes independently expressed by hair cells. Hair cells located in the intermediate region (zone II) had ionic currents more similar to those of central hair cells than peripheral hair cells. Outward currents in intermediate hair cells activated only slightly more quickly than those of the cells of the central region, but much more slowly than those of the peripheral cells. Additionally, intermediate hair cells, like central hair cells, always expressed an inward rectifying current. The regional distribution of outward rectifying potassium conductances resulted in macroscopic currents differing in peak-to-steady state ratio. We quantified this by measuring the peak (Gp) and steady-state (Gs) slope conductance in the linear region of the current-voltage relationship (-40 to 0 mV) for the hair cells located in the different zones. Gp/Gs average values (4.1 +/- 2.1, n = 15) from currents in peripheral hair cells were higher than those from intermediate hair cells (2.3 +/- 0.8, n = 4) and central hair cells(1.9 +/- 0.8, n = 21). The statistically significant differences (P < 0.001) in Gp/Gs ratios could be accounted for by KA channels being preferentially expressed in peripheral hair cells. Hair cell electrophysiological properties in animals pretreated with streptomycin were investigated at approximately 3 wk and approximately 9-10 wk post injection sequence (PIS). At 3 wk PIS, hair cells (all zones combined) had a statistically significantly (P < 0.001) lower Cm (4.6 +/- 1.1 pF, n = 24) and a statistically significantly (P < 0.01) lower Gp(48.4 +/- 20.8 nS, n = 26) than control animals (Cm = 6.2 +/- 1.6 pF, n = 36; Gp = 66 +/- 38.9 nS, n = 40). Regional differences in values of Vz, as well as the distribution of outward and inward rectifying currents, seen in control animals, were still obvious. But, differences in the relative contribution of the expression of the different ionic current components changed. This result could be explained by a relative decrease in IKA compared with IKD during that interval of regeneration, which was particularly evident in peripheral hair cells. (ABSTRACT TRUNCATED)

Ionic currents in regenerating avian vestibular hair cells.

By applying the conventional whole-cell patch-clamp technique in combination with the slice procedure, we have investigated the properties of avian semicircular canal hair cells in situ. Passive and active electrical properties of hair cells from control animals have been compared with those of regenerating hair cells following streptomycin treatment (that killed almost all hair cells). Regenerating type II hair cells showed patterns of responses qualitatively similar to those of normal hair cells. However, parameters reflecting the total number of ionic channels and the surface area of type II hair cells changed during recovery-suggesting that new hair cells came from smaller precursors which (with time) reacquired the same electrophysiological properties as normal hair cells. Finally, we have investigated the ionic properties of a small sample of type 1 hair cells. Ionic currents of regenerating type I hair cells did not show, at least in the temporal window considered (up to 10 weeks from the end of the streptomycin treatment), the typical ionic currents of normal type I hair cells, but expressed instead ionic currents resembling those of type II hair cells. The possibility that regenerating type I hair cells can transdifferentiate from type II hair cells is therefore suggested.

Mechanisms for sensory adaptation in frog vestibular organs.

The effects of external low-Ca, high-Mg solutions were tested both on frog isolated semicircular canals and on single cells isolated from these sensory organs. Our results showed that these media were able to cancel slow adaptation of the ampullar microphonic current in the whole organ and to abolish a Ca-dependent K current (IK(Ca)) in single hair cells, suggesting that IK(Ca) is involved in vestibular sensory adaptation.

Isolation of A-type K+ current in hair cells of the frog crista ampullaris.

Different procedures to isolate the K+ A-type current (IA) from other membrane currents were tested on the complex inactivating outward K+ current generated in hair cells from the peripheral regions of the frog crista ampullaris. Experiments were performed in thin slices of epithelium using the whole-cell configuration of the patch-clamp technique. The conventional conditioning voltage protocol did not allow a satisfactory isolation of IA, due to the presence of other K+ currents showing overlapping steady-state inactivation properties. An attempt to block other K+ currents using calcium-free saline containing 50 mM TEA also failed to provide a satisfactory isolation of IA, due to contamination by a residual sustained current, probably consisting of a slow delayed outward K+ current (IK). Use of the selective A-channel blocker 4-aminopyridine (4-AP) at concentrations < 12 mM was also unsatisfactory because at these concentrations 4-AP produced a voltage-dependent blockade. Conversely, use of 4-AP at concentrations of 15-20 mM allowed a good separation of an uncontaminated IA. These results indicate that IA in hair cells of vestibular epithelium can be isolated most effectively by the 4-AP procedure, provided that sufficiently high concentrations of the A-channel blocker are used.

Differential expression of potassium currents by hair cells in thin slices of frog crista ampullaris.

1. Electrical responses in hair cells located in the peripheral regions and in the central region of the frog crista ampullaris were investigated in thin slice preparations by using the whole-cell configuration of the patch-clamp technique. 2. Hair cells from the peripheral regions exhibited mostly a club-like shape and had an average resting potential of -46 mV, whereas cells from the central region had mostly a cylindrical shape and a more negative resting potential (-57 mV). 3. Voltage-clamp recordings revealed that ionic conductances differed in the two epithelial regions. Cells from the peripheral regions exhibited a transient K+ current of A-type (IA) in conjunction with a slow rectifier outward K+ current (IK). Cells from the central region showed little or no IA and generated an IK together with an inward rectifier K+ current (IIR). In both regions, hair cells showed a rapidly activating Ca(2+)-dependent outward K+ current (IK(Ca)) that rapidly inactivated to reach a steady-state level during 150-ms test pulses. 4. IA activated close to -60 mV and was inhibited by 12 mM 4-aminopyridine (4-AP). The time course of this current showed time to peak values of 3-4 ms at 0 mV. Inactivation was fast and almost voltage-independent. The decay time constant was approximately 35 ms at 0 mV. 5. IK was recruited close to -60 mV and activated slowly, reaching peak values in approximately 100 ms at 0 mV. It showed no evidence of inactivation during 150-ms test pulses and it was insensitive to 4-AP. 6. IIR activated at membrane potentials more negative than -90 mV and was blocked by exposure to 6 mM Cs+ or to a K(+)-free medium. This current showed an outward relaxation at potentials more negative than -140 mV, an effect that disappeared after exposure to a Na(+)-free medium. 7. IK(Ca) was recruited close to -40 mV and was inhibited by exposure to a Ca(2+)-free external medium or to 0.5 mM Cd2+. The time to peak of this current was approximately 3 ms at 0 mV and inactivation was very fast and almost independent from the membrane potential. The decay time constant was approximately 4 ms at 0 mV. 8. IK and IA were prominent in hair cells from the peripheral regions, whereas IK accounted for most of the membrane conductance in cells from the central region. The contribution of IK(Ca) was comparable in cells from both epithelial regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Non-NMDA receptors mediate glutamate-induced depolarization in frog crista ampullaris.

The effect of glutamate on frog crista ampullaris was investigated in order to assess the potential role of this agent as an afferent transmitter in inner ear organs. Intracellular recordings from single afferent axons in the isolated labyrinth showed that, after blocking synaptic transmission with high concentrations of Mg2+, micro-injections of glutamate elicit a dose-dependent postsynaptic depolarization. The amplitude of depolarization was reduced dose-dependently by the competitive non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. When Na+ concentration in the bath was progressively reduced, depolarization decreased gradually and disappeared almost completely in Na(+)-free Ringer. On the contrary, complete substitution of Ca2+ ions in the bath was without apparent effects. These results indicate that the postsynaptic depolarization induced by glutamate in frog semicircular canals involves the activation of non-NMDA amino acid receptors.

Calcium currents in solitary hair cells isolated from frog crista ampullaris.

Some properties of Ca2+ currents in hair cells isolated from frog semicircular canals by enzymatic or mechanical treatment were studied by using the whole-cell configuration of the patch-clamp technique. After blocking the large outward K+ currents by substituting Cs+ for K+ and adding tetraethylammonium to the pipette filling solution, voltage- and time-dependent inward currents were clearly detectable in the presence of 4 mM Ca2+ in the extracellular solution. Ca2+ current was recruited at test potentials more positive than -60 mV, showed a rapid activation, and exhibited no inactivation during 150-ms depolarizing pulses. The maximal amplitude was attained at about -20 mV, with an average value of about 80 pA. When Ca2+ in the extracellular solution was replaced with Ba2+, the magnitude of inward currents increased about twofold. Ba2+ currents were blocked more effectively by Cd2+ than by Ni2+, were suppressed by 0.5 microM omega-conotoxin, and were virtually unaffected by amiloride. The dihydropyridine Bay K 8644 caused a marked voltage-dependent increase in inward currents. The present data suggest that hair cells from frog crista ampullaris are endowed with a homogeneous population of Ca2+ channels having several properties similar to those described for neuronal L channels. Since these channels are recruited in a range of potentials close to the resting level, it is suggested that they subserve the control of both resting and evoked transmitter release from the basal pole of the hair cells.

Characterization of a Voltage-dependent Calcium Current in the Human Neuroblastoma Cell Line SH-SY5Y During Differentiation.

In the presence of retinoic acid, cultured human neuroblastoma SH-SY5Y cells grow processes indicative of neuronal differentiation. A voltage-gated Ca current is already present in undifferentiated cells. A gradual increase of the Ca current density occurs during cell differentiation. According to kinetic and pharmacological properties, Ca currents in differentiated cells are indistinguishable from those elicitable in undifferentiated cells and resemble features of the high-voltage activated currents present in mammalian neuronal cells. omega-conotoxin strongly depresses high-voltage activated currents, both in undifferentiated and in differentiated SH-SY5Y cells. Interestingly, the Ca agonist Bay K 8644 is effective, albeit with great variability from cell to cell, only in differentiated cells and only when barium is the current carrier through the Ca channels. A diversity of high-voltage activated Ca channels of distinct pharmacology has been recently observed in other kinds of neurons. This requires a redefinition of the role that voltage-dependent Ca channel subtypes can play in mammalian neurons.

[K+ and Ca++ currents in hair cells isolated from the semicircular canals of the frog]. / Correnti di K+ e di Ca++ in cellule ciliate isolate dai canali semicircolari della rana.

Hair cells of the inner ear are endowed with different types of ionic channels. To characterize voltage- and ion-dependent channels in vestibular hair cells, experiments were performed in enzymatically isolated hair cells of frog semicircular canals by using the whole-cell configuration of the patch-clamp technique. A large outward current, identified as a K+ current, was recorded when 132 mM KCl were present in the pipette filling solution. It could be dissected pharmacologically into three different components. The first component, which was transient and selectively blocked by 10 mM external 4AP, is most likely an IA-type current. The second one, sensitive to 20 mM external TEA, might be a delayed rectifier K+ current, while the third component insensitive to TEA and showing faster activation time course has been interpreted as a K+ current of IKCa-type. After blocking the outward current by substituting Cs+ for K+ and adding 20 mM TEA to the internal solution, a sustained inward current, identified as a Ca++ current, could be recorded. This current did not inactivate, and was blocked by Cd++ more effectively than Ni++, thus suggesting the presence of Ca++ channels similar to the neuronal "L" channels. Since both K+ and Ca++ channels were recruited at potentials near the resting level, it is suggested that they are involved in the modulation of the resting as well as the evoked transmitter release from the basal pole of the hair cells.

Pre- and postsynaptic excitatory action of glutamate agonists on frog vestibular receptors.

In order to investigate the localization and the type(s) of excitatory amino acid receptors in the frog vestibular system, the exogenous amino acid agonists Quisqualic acid, Kainic acid and N-methyl-D-aspartic acid were tested on the sensory organ of semicircular canals. Intracellular recordings of the resting discharge from single afferents showed that these agonists exerted a complex excitatory action consisting in a rapid and brief increase in frequency of both EPSPs and spikes, followed by a slower and longer lasting membrane depolarization. The progressive impairment of natural transmitter release achieved by adding Mg2+ or Co2+ in the bath caused a dose-dependent decrease of the agonist-induced afferent discharge, without substantially affecting axonal depolarization. These results suggest that the exogenous amino acid agonists act both pre- and postsynaptically on the vestibular organs. Quisqualic acid and kainic acid were much more potent than N-methyl-D-aspartic acid in inducing excitatory effects, suggesting that the amino acid receptors located on both hair cells and afferent endings are mainly of the non-NMDA type. The present findings, while not excluding that an excitatory amino acid may be the afferent transmitter, highlight its possible function as a presynaptic modulator of the afferent transmission in the frog vestibular system.