Ivo Prigioni

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.

Storage and release of acetylcholine in the isolated superior cervical ganglion of the rat.

The storage and release of acetylcholine and choline were studied in the isolated superior cervical ganglion of the rat by a radioenzymic method. The acetylcholine and choline contents were 202.2 +/- 5.1 and 624.7 +/- 20.2 pmole/ganglion, respectively. The transmitter tissue store was unaffected during 1 h of superfusion in choline--Krebs solution, while a 20% decrease was exhibited after 2 h and then remained approximately stable. Conversely, choline content declined to 50% within 1 h and further to 37% of the original level by 4 h. About 24% of the choline assayed in the intact preparation is located in the connective sheath. Preganglionic nerve stimulation at 10--20/sec or potassium stimulation (40 mM KCl) invariably decreased the transmitter tissue stores by 25--45%; such a depletion is independent of the presence or absence of external choline. By contrast, the presence of choline proved to be a prerequisite for the efficient release of acetylcholine from eserinized ganglia during continuous 10/sex stimulation. A drastic depression in the acetylcholine release is described which is related to the time of preincubation of the ganglia with eserine prior to stimulation. Indeed, a 30 min exposure to eserine, compared with a 5 min period, resulted in a 4-fold decrease in the steady output rate. Under optimal conditions, the initial volley output at 10/sec was 1.3 X 10(-4) of the releasable transmitter pool and 1.9 X 10(-4) during the steady-state output. These results are discussed in the light of the electrophysiological knowledge of the quantal release process at the ganglionic synapse.

Potassium currents of pear-shaped hair cells in relation to their location in frog crista ampullaris.

Voltage-dependent K+ currents in pear-shaped hair cells of the frog crista ampullaris were investigated in thin slice preparations using the whole-cell variant of the patch-clamp technique. Microscopy observation revealed that pear-shaped cells are located in intermediate and peripheral regions of the crista, whereas they are absent in the central region. Voltage-clamp recordings in cells from the peripheral regions revealed that the total outward K+ current could be separated pharmacologically into three distinct components: a A-type K+ current (IA); an inactivating calcium-activated K+ current (IK(Ca)) and a delayed rectifier K+ current (IK). IK and IK(Ca) exhibited similar magnitude and accounted for most of the membrane cell conductance. The same experimental protocol applied to cells from the intermediate regions showed the presence of a large and sustained IK(Ca) which represented 95% of the total outward current. In this region IA was absent. The present results demonstrated that pear-shaped hair cells located in two discrete regions of frog crista ampullaris exhibit a different complement of voltage-dependent conductances, suggesting that they can play a different role in processing the natural stimulus.

IP3 receptor in the hair cells of frog semicircular canal and its possible functional role.

The presence and functional role of inositol trisphosphate receptors (IP3R) was investigated by electrophysiology and immunohistochemistry in hair cells from the frog semicircular canal. Intracellular recordings were performed from single fibres of the posterior canal in the isolated, intact frog labyrinth, at rest and during rotation, in the presence of IP3 receptor inhibitors and drugs known to produce Ca2+ release from the internal stores or to increase IP3 production. Hair cell immunolabelling for IP3 receptor was performed by standard procedures. The drug 2‐aminoethoxydiphenyl borate (2APB), an IP3 receptor inhibitor, produced a marked decrease of mEPSP and spike frequency at low concentration (0.1u2003mm), without affecting mEPSP size or time course. At high concentration (1u2003mm), 2APB is reported to block the sarcoplasmic‐endoplasmic reticulum Ca2+‐ATPase (SERCA pump) and increase [Ca2+]i; at the labyrinthine cytoneural junction, it greatly enhanced the resting and mechanically evoked sensory discharge frequency. The selective agonist of groupu2003I metabotropic glutamate receptors (RS)‐3,5‐dihydroxyphenylglycine (DHPG, 0.6u2003mm), produced a transient increase in resting mEPSP and spike frequency at the cytoneural junction, with no effects on mEPSP shape or amplitude. Pretreatment with cyclopiazonic acid (CPA, 0.1u2003mm), a SERCA pump inhibitor, prevented the facilitatory effect of both 2APB and DHPG, suggesting a link between Ca2+ release from intracellular stores and quantal emission. Consistently, diffuse immunoreactivity for IP3 receptors was observed in posterior canal hair cells. Our results indicate the presence and a possibly relevant functional role of IP3‐sensitive stores in controlling [Ca2+]i and modulating the vestibular discharge.

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.

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.

Ionic currents in hair cells dissociated from frog semicircular canals after preconditioning under microgravity conditions

The effects of microgravity on the biophysical properties of frog labyrinthine hair cells have been examined by analyzing calcium and potassium currents in isolated cells by the patch-clamp technique. The entire, anesthetized frog was exposed to vector-free gravity in a random positioning machine (RPM) and the functional modification induced on single hair cells, dissected from the crista ampullaris, were subsequently studied in vitro. The major targets of microgravity exposure were the calcium/potassium current system and the kinetic mechanism of the fast transient potassium current, I(A). The amplitude of I(Ca) was significantly reduced in microgravity-conditioned cells. The delayed current, I(KD) (a complex of I(KV) and I(KCa)), was drastically reduced, mostly in its I(KCa) component. Microgravity also affected I(KD) kinetics by shifting the steady-state inactivation curve toward negative potentials and increasing the sensitivity of inactivation removal to voltage. As concerns the I(A), the I-V and steady-state inactivation curves were indistinguishable under normogravity or microgravity conditions; conversely, I(A) decay systematically displayed a two-exponential time course and longer time constants in microgravity, thus potentially providing a larger K(+) charge; furthermore, I(A) inactivation removal at -70 mV was slowed down. Stimulation in the RPM machine under normogravity conditions resulted in minor effects on I(KD) and, occasionally, incomplete I(A) inactivation at -40 mV. Reduced calcium influx and increased K(+) repolarizing charge, to variable extents depending on the history of membrane potential, constitute a likely cause for the failure in the afferent mEPSP discharge at the cytoneural junction observed in the intact labyrinth after microgravity conditioning.

Potassium currents in the hair cells of vestibular epithelium: position‐dependent expression of two types of A channels

The complement of voltage‐dependent K+ currents was investigated in hair cells of the frog crista ampullaris. The currents were recorded in transversal slices of the peripheral, intermediate and central regions of the crista by applying the patch clamp technique to cells located at different positions in the slices. Voltage‐clamp recordings confirmed that cells located in each region have a distinctive complement of K+ channels. Detailed investigation of the currents in each region revealed that the complement of K+ channels in intermediate and central regions showed no variations among cells, whereas peripheral hair cells differed in the expression of two classes of A‐type currents. These currents showed different kinetics of inactivation as well as steady‐state inactivation properties. We termed these currents fast IA and slow IA based on their inactivation speed. The magnitude of both currents exhibited a significant gradient along the transversal axis of the peripheral regions. Fast IA magnitude was maximal in cells located in the external zone of the crista slice and decreased gradually to become very small in the median zone (centre) of the section, while the gradient of slow IA magnitude was reversed. A‐type currents appear to act as a transient buffer that opposes hair cell depolarization induced by positive current injections. However, fast IA is partially active at the cell resting potential, while slow IA can be recruited only following large hyperpolarizations. Thus, two types of A currents are differentially distributed in vestibular hair cells and have different roles in shaping receptor potential.

Position-dependent expression of inwardly rectifying K+ currents by hair cells of frog semicircular canals.

The identity and the expression of inwardly rectifying ionic currents were studied using the whole-cell variant of the patch-clamp technique in frog semicircular canal hair cells in situ. The currents were examined in club-, cylindrical- and pear-shaped sensory cells located in three discrete regions of the crista. A unique current of I(K1) type was distinguished based on its K+ selectivity, rapid monoexponential activation, dependence of activation on external K+ and blockade by Ba2+ and Cs+. I(K1) was found in virtually all cylindrical hair cells of the central region and in club-shaped cells located in the halves of the peripheral regions closest to the centre of the crista. Pear-shaped cells of the intermediate regions showed no inward rectification. The I(K1) density (pA/pF) varied along the crista depending on cell position, being maximal in cells located in the middle of the central region and decreased towards its ends. In the peripheral regions, the gradient of I(K1) increased towards the centre of the crista. Current clamp experiments showed that sensory cells having larger I(K1) constantly exhibited more negative resting potentials and required more depolarizing current to elicit an active response than cells having small or no I(K1).

Localization of Ca-ATPase in frog crista ampullaris

THE distribution of Ca-ATPase in frog crista ampullaris was mapped ultracytochemically by using a one-step lead citrate reaction. Electron-dense precipitates, as an expression of Ca-ATPase activity, were observed on the surface of stereocilia and on the apical membrane surrounding the cuticular plate of hair cells. Sensory cells of the isthmus region showed more reactivity than those of the peripheral regions of the crista. No reaction products were detectable on the basolateral membranes and in cytoplasmatic organelles. Supporting cells of the crista showed a quite variable Ca-ATPase reaction on microvilli and on basolateral membranes. The presence of an evident reactivity on the stereocilia is consistent with the existence of an apical calcium microdomain involved in the mechano-transduction process and supports the current view that calcium ions enter the stereocilia during natural stimulation. On the other hand, the lack of an observable reactivity on the basolateral membrane of hair cells suggests that in semicircular canals other mechanisms of active transport of calcium ions across the plasma membrane, such as Na—Ca exchange, may be involved in homeostasis of the ion.