Paola Perin

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.

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 effect of proteolytic enzymes on the α9-nicotinic receptor-mediated response in isolated frog vestibular hair cells

In frog vestibular organs, efferent neurons exclusively innervate type II hair cells. Acetylcholine, the predominant efferent transmitter, acting on acetylcholine receptors of these hair cells ultimately inhibits and/or facilitates vestibular afferent firing. A coupling between alpha9-nicotinic acetylcholine receptors (alpha9nAChR) and apamin-sensitive, small-conductance, calcium-dependent potassium channels (SK) is thought to drive the inhibition by hyperpolarizing hair cells thereby decreasing their release of transmitter onto afferents. The presence of alpha9nAChR in these cells was demonstrated using pharmacological, immunocytochemical, and molecular biological techniques. However, fewer than 10% of saccular hair cells dissociated using protease VIII, protease XXIV, or papain responded to acetylcholine during perforated-patch clamp recordings. When present, these responses were invariably transient, small in amplitude, and difficult to characterize. In contrast, the majority of saccular hair cells ( approximately 90%) dissociated using trypsin consistently responded to acetylcholine with an increase in outward current and concomitant hyperpolarization. In agreement with alpha9nAChR pharmacology obtained in other hair cells, the acetylcholine response in saccular hair cells was reversibly antagonized by strychnine, curare, tetraethylammonium, and apamin. Brief perfusions with either protease or papain permanently abolished the alpha9-nicotinic response in isolated saccular hair cells. These enzymes when inactivated became completely ineffective at abolishing the alpha9-nicotinic response, suggesting an enzymatic interaction with the alpha9nAChR and/or downstream effector. The mechanism by which these enzymes render saccular hair cells unresponsive to acetylcholine remains unknown, but it most likely involves proteolysis of alpha9nAChR, SK, or both.

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.

Prevalence and Determinants of Tinnitus in the Italian Adult Population

Background: Limited, outdated, and poor quality data are available on the prevalence of tinnitus, particularly in Italy. Methods: A face-to-face survey was conducted in 2014 on 2,952 individuals, who represented the Italian population aged 18 or more (50.6 million). Any tinnitus was defined as the presence of ringing or buzzing in the ears lasting for at least 5 min in the previous 12 months. Results: Any tinnitus was reported by 6.2% of Italian adults, chronic tinnitus (i.e. for more than 3 months) by 4.8%, and severe tinnitus (i.e. which constitutes a big or very big problem) by 1.2%. The corresponding estimates for the population aged ≥45 years were 8.7, 7.4 and 2.0%, respectively. Multivariable analysis on population aged ≥45 years revealed that old age (odds ratio (OR) = 4.49 for ≥75 vs. 45-54 years) and obesity (OR = 2.14 compared to normal weight) were directly related to any tinnitus, and high monthly family income (OR = 0.50) and moderate alcohol consumption (OR = 0.59 for <7 drinks/week vs. non-drinking) were inversely related. Conclusions: This is the first study on tinnitus prevalence among the general Italian adult population. It indicates that in Italy tinnitus affects more than 3 million adults and is felt as a major problem by more than 600,000 Italians, mostly aged 45 years or more.

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.

Spontaneous low-frequency voltage oscillations in frog saccular hair cells

Spontaneous membrane voltage oscillations were found in 27 of 130 isolated frog saccular hair cells. Voltage oscillations had a mean peak‐to‐peak amplitude of 23 mV and a mean oscillatory frequency of 4.6 Hz. When compared with non‐oscillatory cells, oscillatory cells had significantly greater hyperpolarization‐activated and lower depolarization‐activated current densities. Two components, the hyperpolarization‐activated cation current, Ih, and the K+‐selective inward‐rectifier current, IK1, contributed to the hyperpolarization‐activated current, as assessed by the use of the IK1‐selective inhibitor Ba2+ and the Ih‐selective inhibitor ZD‐7288. Five depolarization‐activated currents were present in these cells (transient IBK, sustained IBK, IDRK, IA, and ICa), and all were found to have significantly lower densities in oscillatory cells than in non‐oscillatory cells (revealed by using TEA to block IBK, 4‐AP to block IDRK, and prepulses at different voltages to isolate IA). Bath application of either Ba2+ or ZD‐7288 suppressed spontaneous voltage oscillations, indicating that Ih and IK1 are required for generating this activity. On the contrary, TEA or Cd2+ did not inhibit this activity, suggesting that IBK and ICa do not contribute. A mathematical model has been developed to test the interpretation derived from the pharmacological and biophysical data. This model indicates that spontaneous voltage oscillations can be generated when the electrophysiological features of oscillatory cells are used. The oscillatory behaviour is principally driven by the activity of IK1 and Ih, with IA playing a modulatory role. In addition, the model indicates that the high densities of depolarization‐activated currents expressed by non‐oscillatory cells help to stabilize the resting membrane potential, thus preventing the spontaneous oscillations.

Voltage-gated Ca2+ channel CaV1.3 subunit expressed in the hair cell epithelium of the sacculus of the trout Oncorhynchus mykiss: Cloning and comparison across vertebrate classes

Full-length sequence (>6.5 kb) has been determined for the Ca(V)1.3 pore-forming subunit of the voltage-gated Ca(2+) channel from the saccular hair cells of the rainbow trout (Oncorhynchus mykiss). Primary structure was obtained from overlapping PCR and cloned fragments, amplified by primers based on teleost, avian, and mammalian sources. Trout saccular Ca(V)1.3 was localized to hair cells, as evidenced by its isolation from an epithelial layer in which the hair cell is the only intact cell type. The predicted amino acid sequence of the trout hair cell Ca(V)1.3 is approximately 70% identical to the sequences of avian and mammalian Ca(V)1.3 subunits and shows L-type characteristics. The trout hair cell Ca(V)1.3 expresses a 26-aa insert in the I-II cytoplasmic loop (exon 9a) and a 10-aa insert in the IVS2-IVS3 cytoplasmic loop (exon 30a), neither of which is appreciably represented in trout brain. The exon 9a insert also occurs in hair cell organs of chick and rat, and appears as an exon in human genomic Ca(V)1.3 sequence (but not in the Ca(V)1.3 coding sequence expressed in human brain or pancreas). The exon 30a insert, although expressed in hair cells of chick as well as trout, does not appear in comparable rat or human tissues. Further, the IIIS2 region shows a splice choice (exon 22a) that is associated with the hair cell organs of trout, chick, and rat, but is not found in human genomic sequence. The elucidation of the primary structure of the voltage-gated Ca(2+) channel Ca(V)1.3 subunit from hair cells of the teleost, representing the lowest of the vertebrate classes, suggests a generality of sensory mechanism for Ca(V)1.3 across hair cell systems. In particular, the exon 9a insert of this channel appears to be the molecular feature most consistently associated with hair cells from fish to mammal, consonant with the hypothesis that the latter region may be a signature for the hair cell.