Gianpiero Zucca

The effect of glutamate on the frog semicircular canal.

L-glutamate (Glu) has at least two sites of action in the frog semicircular canal: the hair cell (presynaptic) and the primary afferent nerve fibres (postsynaptic). Glus action on the hair cell results in an increased release of the natural transmitter which is responsible for a substantial increase in the frequency of firing in primary afferents. The presynaptic action of Glu is antagonized by D-alpha-aminoadipate (D alpha AA). Glu produces a long-lasting depolarization on the afferent nerve fibres which does not by itself elicit any afferent discharge of impulses when the release of the natural transmitter is prevented. Glu-induced nerve depolarization is only partially antagonized by D alpha AA. The difficulty of reconciling some of the observations made of the effects of Glu in semicircular canals with its presumed role as an afferent transmitter in this organ is discussed, but this role is not definitely rejected.

WHY DO BENIGN PAROXYSMAL POSITIONAL VERTIGO EPISODES RECOVER SPONTANEOUSLY

It is well known that most episodes of benign paroxysmal positional vertigo (BPPV), even in untreated, recover spontaneously in 2 to 6 weeks. In the present study, we put forward the hypothesis that this is mainly due to the fact that endolymph, owing to its low calcium content (20 microM) is able to dissolve otoconia. To support this, the fate of frog saccular otoconia immersed in normal endolymph (Ca2+ content 20 microM) and in Ca2+-rich endolymphatic fluids (up to 500 microM) was studied by observing the crystals at regular intervals for 3 weeks. The results demonstrated that normal endolymph can dissolve otoconia very rapidly (in about 20 hours). When the endolymphatic Ca2+ content was increased (50 to 200 microM) otoconia dissolution time was slowed down (about 100 to 130 hours, respectively) and completely stopped when the endolymphatic Ca2+ content was of 500 microM. The present results therefore suggest that the major process involved in the spontaneous recovery of BPPV episodes is the capability of the endolymph to dissolve dislodged otoconia.

The origin of slow potentials in semicircular canals of the frog

Slow potentials generated in the sensory organ of the ampulla were recorded in isolated semicircular canals of the frog by means of fluid electrodes. These potentials, which may be picked up from the intrampullar fluid and those from the ampullar nerve appear to be generated at different stages of the process taking place in crista ampullaris. Slow intra-ampullar potentials apparently reflect receptor potentials of hair cells. They are preserved after degeneration of the nerve fibre endings and are relatively insensitive to DNP poisoning; their amplitude is maximum at high K+ concentrations. Slow nerve potentials appear to be due to electronic spreading of post-synaptic excitatory potentials generated at cytoneural junctions. They disappear after degeneration of the nerve fibres, in low Ca++ and high Mg++ solutions and are extremely sensitive to DNP poisoning. An analysis of the time-course of the slow ampullar and nerve potentials referred to the discharge of impulses in afferent fibres was performed with a view to interpreting the transduction mechanism of semicircular canals.

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.

Effects of Betahistine on Vestibular Receptors of the Frog

Betahistine is widely used in the symptomatic treatment of peripheral and central vestibular disorders. However, its remains unknown whether the drug can act directly on inner ear sensory organs. To this end, the effects of betahistine (10(-7)-10(-2) M) were examined on isolated preparations of frog semicircular canal mounted in a double-celled bath which allowed drug administration both in the endolymphatic and in the perilymphatic fluid. The effects of betahistine were evaluated by recording ampullar receptor potentials and nerve firing rate both at rest and during mechanical stimulation of the isolated preparation. The results demonstrated that endolymphatic administration of betahistine had no effect, whereas its perilymphatic administration could reduce greatly ampullar receptor resting discharge but had little effect on mechanically evoked responses. This observation may explain the anti-vertigo effects of betahistine. Vertigo is normally due to uncontrolled changes in vestibular receptor resting discharge. It is therefore probable that any factor able to reduce the resting firing rate of vestibular receptors and, in consequence, its variations, may have an anti-vertigo action.

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.

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 ω-conotoxin 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 ω-conotoxin GVIA sensitive channels regulate the afferent firing (possibly on the postsynaptic side) but with a less important role.

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.

Effects of betahistine metabolites on frog ampullar receptors.

Previous studies have demonstrated that betahistine, an histamine-like substance used widely as an anti-vertigo drug, can decrease ampullar receptor resting discharge without affecting their mechanically evoked responses. Pharmacokinetic studies have shown that this drug is transformed, mainly at the hepatic level, into aminoethylpyridine (M1), hydroxyethylpyridine (M2), then excreted with the urine as pyridylacetic acid (M3). The goal of the present study was to investigate whether betahistine metabolites are also able to affect vestibular receptor activity. Results demonstrated that, in the range tested (10–10 M), M2 and M3 exerted no effect, whereas M1, at concentrations higher than 10 −6 M, was able to reduce the resting discharge of ampullar receptors without affecting the evoked responses. M1 therefore exerts effects similar to those of betahistine on ampullar receptors. This might be of some clinical interest. On the basis of our data, the hypothesis may be put forward that the anti-vertigo action of betahistine is at first achieved by betahistine itself and then sustained by M1.Previous studies have demonstrated that betahistine, an histamine-like substance used widely as an anti-vertigo drug, can decrease ampullar receptor resting discharge without affecting their mechanically evoked responses. Pharmacokinetic studies have shown that this drug is transformed, mainly at the hepatic level, into aminoethylpyridine (M1), hydroxyethylpyridine (M2), then excreted with the urine as pyridylacetic acid (M3). The goal of the present study was to investigate whether betahistine metabolites are also able to affect vestibular receptor activity. Results demonstrated that, in the range tested (10(-7)-10(-2) M), M2 and M3 exerted no effect, whereas M1, at concentrations higher than 10(-6) M, was able to reduce the resting discharge of ampullar receptors without affecting the evoked responses. M1 therefore exerts effects similar to those of betahistine on ampullar receptors. This might be of some clinical interest. On the basis of our data, the hypothesis may be put forward that the anti-vertigo action of betahistine is at first achieved by betahistine itself and then sustained by M1.