Intrinsic membrane properties of rat medial vestibular nucleus neurons and their responses to simulated vestibular input signals / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study the membrane properties of rat medial vestibular nucleus (MVN) neurons and their firing responses to simulated input signals of peripheral vestibular system, and to discuss how the intrinsic membrane properties contribute to physiologic functions in central vestibular system.</p><p><b>METHODS</b>By using infrared differential interference contrast technique, whole-cell recordings were made from rat MVN neurons under direct observation. On the basis of their averaged action potential shapes, the MVN neurons were classified. Linear and non-linear currents were put into the neurons to simulate the input signals of peripheral vestibular system. The differences of intrinsic membrane properties and firing response dynamics were observed between two types.</p><p><b>RESULTS</b>The discharge activities were recorded in MVN neurons, which remained in low Ca2+-high Mg2+ artificial cerebrospinal fluid (ACSF). Neurons are classified as type A (33%) characterized by a single deep after-hyperpolarization (AHP) and A-like rectification, or type B (63%) characterized by double AHP, and another two neurons with all or none of the characters. The passive membrane properties were not significantly different between type A and type B neurons, while part of active membrane properties was significantly different. Both type A and B neurons well responded to simulated current inputs, but disparities existed in response range and firing dynamics.</p><p><b>CONCLUSIONS</b>The discharge activities of MVN neurons were initiated by their intrinsic membrane properties. Most MVN neurons were classified as type A and B, while several showed unrepresentative firing properties. Linear and nonlinear inputs evoked a heterogeneous range of firing responses. The differences of response range and firing dynamics between neurons may determine their different physiological functions.</p>

Properties of cholinergic receptor-mediated ion channels on type I vestibular hair cells of guinea pigs / 生理学报

To confirm the existence of cholinergic receptors on type I vestibular hair cells (VHCs I) of guinea pigs and to study the properties of the cholinergic receptor-mediated ion channels on VHCs I, electrophysiological responses of isolated VHCs I to external ACh were examined by means of whole-cell patch-clamp recordings. The results showed that 7.5% (21/279) VHCs I were found to be sensitive to ACh (10-1000 μmol/L). ACh generated an outward current in a steady, slow, dose-dependent [EC(50) was (63.78±2.31) μmol/L] and voltage-independent manner. In standard extracellular solution, ACh at the concentration of 100 μmol/L triggered a calcium-dependent current of (170±15) pA at holding potential of -50 mV, and the current amplitude could be depressed by extracellularly added calcium-dependent potassium channel antagonist TEA. The time interval for the next complete activation of ACh-sensitive current was no less than 1 min. The ion channels did not shut off even when they were exposed to ACh for an extended period of time (8 min). The results suggest that dose-dependent, calcium-dependent and voltage-independent cholinergic receptors were located on a few of the VHCs I investibular epithelium of guinea pigs. The cholinergic receptors did not show desensitization to ACh. This work reveals the existence of efferent neurotransmitter receptors on VHCs I and helps in understanding the function of vestibular efferent nervous system, and may provide some useful information on guiding the clinical rehabilitative treatment of vertigo.

Co-location of ACh-sensitive BK channels and L-type calcium channels in type II vestibular hair cells of guinea pig / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the mechanisms of the influx of calcium ions during the activation of ACh-sensitive BK channel (big conductance, calcium-dependent potassium channel) in type II vestibular hair cells of guinea pigs.</p><p><b>METHODS</b>Type II vestibular hair cells were isolated by collagenase type IA. Under the whole-cell patch mode, the sensitivity of ACh-sensitive BK current to the calcium channels blockers was investigated, the pharmacological property of L-type calcium channel activator-sensitive current and ACh-sensitive BK current was compared.</p><p><b>RESULTS</b>Following application of ACh, type II vestibular hair cells displayed a sustained outward potassium current, with a reversal potential of (-70.5 +/- 10.6) mV (x +/- s, n = 10). At the holding potential of -50 mV, the current amplitude of ACh-sensitive potassium current activated by 100 micromol/L ACh was (267 +/- 106) pA (n = 11). ACh-sensitive potassium current was potently sensitive to the BK current blocker, IBTX (iberiotoxin, 200 nmol/L). Apamin, the well-known small conductance, calcium-dependent potassium current blocker, failed to inhibit the amplitude of ACh-sensitive potassium current at a dose of 1 micromol/L. ACh-sensitive BK current was sensitive to NiCl2 and potently inhibited by CdCl2. NiCl2 and CdCl2 showed a dose-dependent blocking effect with a half inhibition-maximal response of (135.5 +/- 18.5) micromol/L (n = 7) and (23.4 +/- 2.6) micromol/L (n = 7). The L-type calcium channel activator, (-) -Bay-K 8644 (10 micromol /L), mimicked the role of ACh and activated the IBTX-sensitive outward current.</p><p><b>CONCLUSION</b>ACh-sensitive BK and L-type calcium channels are co-located in type II vestibular hair cells of guinea pigs.</p>

Electrophysiological characteristics of muscarinic cholinergic receptor in rat medial vestibular nucleus neurons by visual patch clamp technique / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To establish the visual patch clamp whole-cell recording technique and study the properties and functional significance of muscarinic receptor-mediated currents in rat medial vestibular nucleus neurons (MVNn).</p><p><b>METHODS</b>Brain slices containing the MVN were prepared from fifteen Wistar rats. By combining infrared differential interference contrast (IR-DIC) technique and CCD-Camera system with visual patch clamp whole-cell recording technique, twenty healthy neurons were located and muscarinic receptor-mediated currents in rat MVNn were observed and analyzed.</p><p><b>RESULTS</b>Visual patch clamp technique can be used to make direct localization and to make sure of active neuron. In MVNn, a comparison of the current-voltage relationships before and during the application of muscarine, which revealed an increase in the slope of the I-V curve and the reversal potential for this response lay at (-88.4 +/- 4.9) mV (x +/- s), indicates that the activation of muscarinic cholinergic receptors leads to a decrease in potassium current. The test in the voltage sensitivity of the muscarine-induced response, which showed that the effect had a linear current-voltage relationship and reversed at (-86.7 +/- 3.5) mV, indicates that the potassium current blocked by muscarine corresponds to the voltage-insensitive leak potassium current.</p><p><b>CONCLUSIONS</b>Visual patch clamp technique, which was considered better than blind patch clamp technique, can improve the success of sealing process. By the analysis of muscarinic receptor-mediated currents, the data provide support that muscarinic cholinergic mechanisms play a prominent role in the modulation of the excitability of MVNn and also offer a new idea for the efficacy of anticholinergic drugs.</p>

Feature of ACh-sensitive potassium current in outer hair cells of guinea pig cochlea / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the feature of the ACh-sensitive potassium current in guinea pig cochlear outer hair cells.</p><p><b>METHODS</b>Cochlear outer hair cells of guinea pigs (n=38) were isolated by collagenase type IV. Under the whole-cell patch mode, the ions nature and the pharmacological properties of the ACh-sensitive potassium current were investigated by applying the inhibitors of calcium-dependent potassium currents and the inhibitors of nicotinic ACh receptor.</p><p><b>RESULTS</b>Following application of ACh, cochlear outer hair cells displayed a rapidly activating outward potassium current with a fast desensitized kinetic and a reversal (x +/- s) potential of (-67.3 +/- 8.2) mV (n=10). At the holding potential of -50 mV, the current amplitude of ACh-sensitive potassium current activated by 100 micronmol/L ACh was (506.6 +/- 186.3) pA (n=9). ACh-sensitive potassium current was sensitive to TEA (tetraethylammonium chloride, 10 mmol/L) and potently inhibited by the small conductance calcium-dependent potassium current (SK) blocker, apamin (1 micromol/L). Iberiotoxin (IBTX), the well-known blocker of big conductance calcium-dependent potassium current (BK), failed to inhibit the amplitude of the ACh-sensitive potassium current at the dose of 200 nmol/L. The dose for half-maximal response (EC50) of the ACh-sensitive potassium current was (33.5 +/- 5.7) micromol/L (n=7). The ACh-sensitive potassium current was sensitive to the GABA (gamma-aminobutyric acid)-A receptor blocker, bicuculline, and strongly inhibited by the selective blocker of the alpha 9-nicotinic ACh receptor, strychnine. Strychnine and bicuculline showed the dose-dependent blocking effect with a half inhibition-maximal response (IC50) of (22.3 +/- 2.6) nmol/L (n=7) and (1.2 +/- 0.4) micromol/L (n=6), respectively.</p><p><b>CONCLUSIONS</b>This work provides direct evidences that the ACh-sensitive SK current was present on guinea pig cochlear outer hair cells. The activation of the ACh-sensitive SK current was most possibly mediated by a alpha 9-nicotinic ACh receptor.</p>

Dependence of Ca2+ on the acetylcholine-sensitive current in guinea pig type II vestibular hair cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the dependence of Ca2+ on the acetylcholine (ACh)-sensitive potassium current in guinea pig type II vestibular hair cells.</p><p><b>METHODS</b>Under the whole-cell patch mode, the current amplitude of the ACh-sensitive potassium current was recorded in response to the concentration change of the extracellular or intracellular Ca2+.</p><p><b>RESULTS</b>Following application of ACh, type II vestibular hair cells displayed the sustained potassium current, which was inhibited by tetraethylammonium chloride (TEA), but not inhibited by 4-aminopyrine (4-AP). The activation of the ACh-sensitive potassium current was strongly affected by the concentration of the extracellular Ca2+. The current amplitude of the ACh-sensitive potassium increased following the increase of Ca2+ concentration from 0 mmol/L to 4 mmol/L At the concentration of 4 mmol/L Ca2+, the current amplitude of the ACh-sensitive potassium current reached the maximal response. Lowering the Ca2 concentration in the external solution from 4 mmol/L to 0. 5 mmol/L, the current amplitude of the ACh-sensitive potassium current was inhibited to (36.5 +/- 6.5)%. However, no difference was found in the presence and in the absence of the intracellular heparin, which was a well-known blocker of the inositol trisphosphate-dependent calcium release channels. In addition, the calcium channel blocker, Cd2+, inhibited the ACh-sensitive potassium current.</p><p><b>CONCLUSIONS</b>The activation of the ACh-sensitive potassium current in guinea pig type II vestibular hair cells was dependent on the extracellular Ca2+ influx through the calcium channel. The application of ACh would stimulate membrane Ca2+ channels; the influx of Ca2+ will then activate the calcium-dependent potassium current in guinea pig type II hair cells to mediate the hyperpolarization effect.</p>

Cation ions modulate the ACh-sensitive current in type II vestibular hair cells of guinea pigs / 生理学报

Molecular biological studies and electrophysiological data have demonstrated that acetylcholine (ACh) is the principal cochlear and vestibular efferent neurotransmitter among mammalians. However, the functional roles of ACh in type II vestibular hair cells among mammalians are still unclear, with the exception of the well-known alpha9-containing nicotinic ACh receptor (alpha9-nAChR) in cochlear hair cells and frog saccular hair cells. In this study, the properties of the ACh-sensitive current were investigated by whole-cell patch clamp technique in isolated type II vestibular hair cells of guinea pigs. The direct effect of extracellular ACh was to induce a hyperpolarization effect in type II vestibular hair cells. Type II vestibular hair cells displayed a sustained outward current in response to the perfusion of ACh. It took about 60 s for the ACh-sensitive current to get a complete re-activation. The reversal potential of the ACh-sensitive current was (-66 +/- 8) mV, which indicated that potassium ion was the main carrier of this current. The blocking effect by the submillimolar concentration of tetraethylammonium (TEA) further indicated that extracellular ACh stimulated the calcium-dependent potassium current. Following replacement of the compartment of NaCl in the normal external solution with TrisCl, LiCl or saccharose respectively, the amplitude of the ACh-sensitive current was not affected. Blocking of the release of intracellular Ca(2+) stores by intracellular application of heparin failed to inhibit the ACh-sensitive current. Therefore, extracellular Na(+)and the inositol 1,4,5-trisphosphate (IP(3))-dependent intracellular Ca(2+)release were not involved in the activation of the ACh-sensitive current. However, the ACh-sensitive current was strongly affected by the concentration of the extracellular K(+), extracellular Ca(2+) and intracellular Mg(2+). The amplitude of the ACh- sensitive current was strongly inhibited by high concentration of extracellular K(+). In the Ca(2+)-free external solution, ACh only activated a very small current; however, the ACh-sensitive current demonstrated a Ca(2+)-dependent inhibition effect in high concentration of Ca(2+)solution. In addition, the ACh-sensitive current was inhibited by increasing of the concentration of intracellular Mg(2+). In conclusion, the present results demonstrate that ACh plays an important role in the vestibular efferent system. The fact that Na(+) is not involved in the ACh-sensitive current will not favor the well-known profile of alpha9-nAChR, which is reported to display a small but important permeability to Na(+). It is also suggested that, in vivo, the amplitude of the ACh-induced hyperpolarization may strongly depend on the concentration of extracellular Ca(2+)and intracellular Mg(2+).