P. Valli

Activation of the efferent system in the isolated frog labyrinth: Effects on the afferent EPSPs and spike discharge recorded from single fibers of the posterior nerve

Intra-axonal recordings were obtained from single afferent fibres of the posterior nerve in the isolated labyrinth of the frog (Rana esculenta). EPSPs and spike discharge were recorded both at rest and during rotatory stimulation of the canal. Electrical stimulation of either the distal end of the cut posterior nerve or of the central stumps of the anterior-horizontal nerves elicited a frequency-dependent inhibitory effect on the afferent discharge arising from the posterior canal. Denervation experiments revealed that inhibition is mediated by efferent fibres exhibiting a high degree of branching in the proximal part of the eighth nerve. The inhibitory effect was selectively cancelled by (1)D-tubocurarine 10(-6) M; (2) atropine 5 x 10(-5) M; (3) acetylcholine or carbachol 10(-4) M; (4) eserine 10(-5) M. Inhibition is thus most likely to be sustained by the release of acetylcholine from the efferent nerve terminals. Experiments in which the ionic composition of the external medium was modified suggest that the transmitter acts mainly by opening the chloride ion channels of the hair cell membrane. In some units the same stimulation pattern evoked a consistent increase in both EPSP and spike discharge, instead of inhibition. Such facilitation was unaffected by drugs for ionic modifications which block the efferent synapse, but disappeared after denervation. Inhibition and facilitation, therefore, act as two control mechanisms which are able to modify substantially, at the first stage of processing, the sensory information which is sent to the vestibular second order neurons.

The vestibular hair cells: post-transductional signal processing.

Hair cells in mechanosensory systems transduce mechanical stimuli into biological signals to be presented to and analyzed by the brain. Vestibular hair cells transduce stimuli primarily associated with the organisms orientation and motion in space. When examined superficially it may appear that the hair cells act as passive transducers whereby mechanical stimulation of their hair bundle results in transmitter release at their afferent synapses. In fact, hair cell functions are more complicated, and the mechanical signals are heavily processed even before being encoded in afferent nerve activity. Hair cells are different from one another in morphology, biophysics, transmitter and transmitter receptor complements, not only across different organs (as one might expect), but even in the same organ. This review focuses on hair cell morpho-physiological properties, ionic conductances, neurotransmitters/modulators and their receptors, second messengers and effectors. Special features of hair cell neurotransmission, as the synaptic body and the presence of autoreceptors and local circuits, are also discussed, as is the possibility of a differential modulation of hair cell transmitter release in the resting and mechanically-stimulated states.

Post-synaptic potentials recorded from afferent nerve fibres of the posterior semicircular canal in the frog

Glass microelectrode recordings were made from single fibres of the posterior ampullary nerve in the isolated labyrinth of the frog (Rana esculenta). Potentials were recorded both at rest and during rotatory stimulation of the canal. At rest, the tracings revealed an intense background of small, largely summated potentials (0.5-10 mV amplitude, 3-6 msec duration), which underlay the discharge of spikes in all the impaled units. The frequency of the subthreshold events was related to the frequency of the propagated spikes, the latter ranging from 0 to 40/sec. Stimulation modulated the frequency of both spikes and subthreshold potentials, whose summation during excitation led to a positive shift of the fibre membrane potential. The small potentials proved to be dependent on Ca2+ and Mg2+ levels in the bath. Antidromic-stimulation of the posterior ampullary nerve indicated that the observed events do not represent an artifact due to extracellular field interference related to spike activity in the neighbouring fibres. Tetrodotoxin (10(-7)-10(-6) g/ml) applied externally to the preparation or previously perfused through the frog vessels abolishes the propagated spikes but left unaffected the small potentials which, even under drug treatment, were normally modulated by the stimulus. The subthreshold potentials thus appear to be EPSPs generated at the cyto-neural junction between the hair cells and the endings of the ampullary nerve fibres.

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.

Peripheral organization of the vestibular efferent system in the frog: an electrophysiological study

The distribution and the properties of efferent fibers in vestibular nerve were studied in the isolated frog labyrinth. Electrical stimulation of the central stump of any vestibular nerve branchlet elicited compound action potentials in all the other eighth nerve branchlets, indicating the existence of neural links between the various vestibular organs. The same experimental paradigm, when repeated in frogs with chronic section of the eighth nerve roots, demonstrated that these pathways are efferent collaterals extending to all vestibular organs. There are more collaterals linking the 3 semicircular canals than the otolith organs and the otoliths with the canal organs. Efferent connections in the eighth nerve were preserved in full after ablation of the ipsilateral hemi-cerebellum, suggesting that the efferent pathways probably originate in the brainstem. Intracellular recordings from single afferent fibers of both canal and otolith organs revealed that efferent fiber activation could elicit either inhibition or facilitation of the receptor discharge. It was concluded that the frog efferent vestibular system is endowed with non-selective control channels which allow single neurons to influence the receptor activity of different labyrinthine organs.

Perilymphatic potassium changes and potassium homeostasis in isolated semicircular canals of the frog.

1. Endolymphatic and perilymphatic potassium concentrations were measured with K(+)‐sensitive microelectrodes in isolated semicircular canals of the frog. K+ levels were evaluated both at rest and during sinusoidal stimulation (0.05 Hz) of the sensory organ. 2. Mechanical stimulation of hair cells was associated with sinusoidal changes (about 0.2 mM) in the perilymphatic K+ concentration. 3. Perilymphatic K(+)‐fluctuations were modified neither by impairment of the synaptic transmission at cyto‐neural junctions nor by chronic denervation of the crista ampullaris, thus indicating that K+ ions were actually released by hair cells. 4. Voltage‐clamp experiments of the whole sensory organ showed that K+ flows across the crista ampullaris can vary from 3 X 10(11) molecules of K+ s‐1 at rest up to about 15 X 10(11) molecules of K+ s‐1 during mechanical stimuli. 5. Measurement of intra‐ampullar K+ concentration demonstrated that the amount of K+ transported from the perilymph towards the endolymph can be rapidly altered by modifying its perilymphatic levels. This suggests that vestibular organs are endowed with K+ homeostatic mechanisms able to buffer in a very efficient way the concentration of K+ in both the fluids bathing the crista ampullaris. 6. The possible role of K+ homeostatic mechanisms in hair cell adaptation is discussed.

Functional organization of the peripheral efferent vestibular system in the frog.

The functional organization of the efferent vestibular system (EVS) was studied in the isolated frog labyrinth. To ascertain whether, besides the efferent branching fibres that innervate several end-organs, the EVS is also endowed with efferent non-branching axons which might control a given population of sensory units in each end-organ, the 8th nerve and one of its branchlets were electrically stimulated while recordings of the spontaneous activity arising from the different sensors were made by impaling single afferent axons in all the 8th nerve branchlets. The results demonstrated that the vast majority of the sensory units whose activity was modified by stimulating the whole 8th nerve was also affected by stimulating an 8th nerve branchlet. These findings therefore rule out the possibility that the EVS is endowed with projective fibres and strengthen the view that the EVS is a highly divergent system with collaterals arising from single parent axons that innervate several end-organs. These experiments have also shown that the percentage of sensory units which are actually controlled by the EVS varies amongst the different labyrinthine organs. It is maximal in the sacculus (ca. 90%), somewhat lower in canal organs (ca. 80%) and the utriculus (ca. 70%) and considerably lower in the lagena (ca. 50%). This EVS arrangement therefore might allow information arising from some organs to be modified more extensively than that from others.

The metabotropic glutamate receptors of the vestibular organs

This research sought to test the presence and function of metabotropic excitatory amino acid receptors (mGluR) in the frog semicircular canal (SCC). The mGluR agonist +/- 1-aminocyclopentane-trans-1,3-dicarboxylate (ACPD) produced an increase in afferent firing rates of the ampullar nerve of the intact posterior canal. This increase was not due to a stimulation of cholinergic efferent terminals or the acetylcholine (ACh) receptor, since atropine, in concentrations which blocked the response to exogenous acetylcholine, did not affect the response to ACPD. Likewise, ACPD effects were not due to stimulation of postsynaptic NMDA receptors, since the NMDA antagonist D(-)-2-amino-5-phosphonopentanoate (AP-5) did not affect the response to ACPD, reinforcing the reported selectivity of ACPD for mGluRs. When the SCC was superfused with artificial perilymph known to inhibit hair cell transmitter release (i.e. low Ca-high Mg), ACPD failed to increase afferent firing. This suggests that the receptor activated by ACPD is located on the hair cell. Pharmacological evidence suggested that the mGluRs involved in afferent facilitation belong to Group I (i.e. subtypes 1 and 5). In fact, the Group III agonist AP-4 had no effect, and the ACPD facilitatory effect was blocked by the Group I mGluR antagonists (S)-4-carboxyphenylglycine (CPG) and (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). Additional pharmacological evidence supported the presence of Group I mGluRs. Interestingly, the mGluR antagonists, AIDA and 4CPG, by themselves did not affect the resting firing rates of ampullar afferents. This may suggest that the mGluRs are not involved in resting activity but perhaps only in evoked activity (as suggested in Guth et al. (1991) Hear. Res. 56, 69-78). In addition, the mRNA for the mGluR1 has been detected in hair cells of both SCC, utricle, and saccule. In summary, the evidence points to an mGluR localized to the hair cell (i.e. an autoreceptor) which may be activated to produce a positive feedback augmentation of evoked but not resting transmitter release and thus affect afferent activity.

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.

Ionic Mechanisms Sustaining Activity in Ampullar Receptors of the Frog

Ionic mechanisms sustaining sensory transduction in crista ampullaris sensory cells have been investigated chiefly by replacing the endolymph with solutions with K+ and Ca++ chelators added. The effects of the modified solutions were evaluated by extracellular and intracellular recordings of both and the presynaptic and postsynaptic activity of ampullar receptors. The results strongly suggest that the receptor current in labyrinthine cells is carried exclusively by K+. Moreover evidence are reported indicating that the transducer membrane in ampullar receptors is provided with Ca++ sensitive potassium channels whose opening is depending on free Ca++ released from cupular structures during excitatory deflections.