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1Institute of Physiology, Autonomous University of Puebla, Puebla and 2Institute of Cellular Physiology, National Autonomous University of Mexico, Mexico City, Federal District, Mexico
Submitted 18 February 2005; accepted in final form 14 August 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Soto and Vega 1988; Starr and Sewell 1991). Based on the coefficient of variation of their resting discharge, VANs have been classified into regular and irregular cells. However, there is no clear separation into two neuronal subgroups because the regularity of the discharge varies from the most irregular to regular cells forming a continuum among all cells (Goldberg and Fernández 1971; Honrubia et al. 1989). VAN morphology has been correlated with the regularity of the resting discharge. Calyx-ending neurons, with the largest somas and thick afferent dendrites that innervate type I hair cells mainly located in the central zones of the sensory neuroepithelia, have an irregular resting discharge. The smallest neurons, with thin dendrites that establish bouton synapses with type II hair cells in the peripheral zones of sensory epithelia, have a regular resting discharge. Dimorphic neurons, with medium soma size innervating both type I and type II hair cells, are distributed throughout the sensory epithelia and have intermediate electrical properties (Fernández et al. 1988, 1995; Kevetter and Leonard 2002; Leonard and Kevetter 2002; Lysakowski et al. 1995; Si et al. 2003).
Besides this, vestibular neurons exhibit differences in their dynamic spike response to mechanical (Baird et al. 1988; Curthoys 1982; Goldberg and Fernández 1971; Lysakowski et al. 1995) and electrical stimulation (Brontë-Stewart and Lisberger 1994; Ezure et al. 1983; Goldberg et al. 1987) of the membranous labyrinth. Differences in the firing properties of VANs cannot be explained solely by their synaptic input (Smith and Goldberg 1986) nor by the type of hair cells they innervate because vestibular neurons from animals lacking type I hair cells can also be grouped into irregular and regular types with a similar regional distribution of their terminals within the neuroepithelia (Honrubia et al. 1989; Myers and Lewis 1990). Thus as suggested (Goldberg 2000; Smith and Goldberg 1986), ionic conductance of vestibular neurons may vary depending on the location of the cells within the neuroepithelia that these neurons innervate. However, there is little information about the ionic conductance expressed by vestibular neurons in mammals. Studies in embryonic and neonatal mice have shown that VANs express a tetrodotoxin (TTX)-sensitive Na+ current (Chabbert et al. 1997), a hyperpolarization-activated inward current (Chabbert et al. 2001b), three voltage-dependent K+ currents (Chabbert et al. 2001a), and a voltage-dependent Ca2+ current composed of L-, N-, P/Q-, R-, and T-type channels (Chambard et al. 1999; Desmadryl et al. 1997). Only the T-type current has been shown to have a heterogeneous distribution among afferent neurons (Chambard et al. 1999; Desmadryl et al. 1997), leaving unsolved the question of a putative differential expression of ionic conductance that in turn could be correlated with the differences in the firing pattern observed in VANs.
In mathematical models, interspike-interval statistics, sensitivity to galvanic currents, and the relation between discharge VAN regularity and galvanic sensitivity can be accounted for by interactions between the synaptic noise and the slope of the afterhyperpolarization (AHP) (Smith and Goldberg 1986), which is in part dependent on the Ca2+-activated K+ current (IKCa) characteristics (Cloues and Sather 2003; Sah 1996). At present, there is only one report of big conductance current (BK) channels in the sacular nerve of goldfish, in which it was shown that BK current is selectively expressed in a population of cells innervating the caudal portion of the saccular macula (Davis 1996). In this work, we report for the first time that in the primary afferent neurons of the vestibular system the Ca2+-activated K+ current (IKCa) is composed of four components: 1) big conductance current (BK), sensitive to iberiotoxin (IbTx); 2) small conductance current (SK), sensitive to apamin; 3) intermediate conductance current (IK), sensitive to clotrimazole (CLT) and charibdotoxin (ChTx); and 4) a resistant current (IR) that is not sensitive to any of the drugs used in this work and that is activated by extracellular Ca2+. In addition we found that the current density of total IKCa, the BK, and the IR were correlated with soma size and with the expression of a low-voltage–activated (LVA) Ca2+ current. Current-clamp experiments using the perforated-patch technique indicate that the BK current participates in the adaptation of discharge and in the repolarization of the action potential of cultured VANs.
Some of these results were previously presented in abstract form (Limón et al. 2003).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Cell somata of rat VANs initiate their myelinization around the eighth day after birth (p8) (Toesca 1997). To maintain accessibility to neuronal plasmalemma with patch pipettes, we decided to use rats at ages between p7 and p10, a time at which we can obtain isolated neurons without a myelin sheath. Some of the experiments were done in acutely dissociated vestibular neurons as indicated. However, because enzymatic dissociation could alter properties of KCa channels (Spreadbury et al. 2004) and produce soma size changes caused by cell swelling (Emgard et al. 2002), we decided to use primary cultured (18–24 h) VANs to allow cells to recover from possible alterations of membrane ionic channels. Also, some of the acutely isolated cells have a thin myelin sheet covering the cell body. Because of the culturing procedure, this myelin sheet disappears, facilitating patch-clamping procedures (Santos-Sacchi 1993). Because the culture procedure also removes the myelin in some adult neurons, a small experimental sample was done in VANs from rats p23 to p26. Soma size of neurofilament (NF)-immunoreactive cultured VANs follows a Gaussian distribution [21.8 ± 5 µm (mean ± SD); Soto et al. 2002] similar to the distribution of NF-immunoreactive VANs in the rat vestibular ganglion in situ (19 ± 4 µm; Demêmes et al. 1992), indicating that morphologically they are representative of the in vivo conditions.
Cell culture
Young Wistar rats (p7–p10) were anesthetized with ether and decapitated. The head was cleaned with 70% alcohol, the inferior maxillary was removed, and the cranium immersed in L-15 medium (Gibco, Grand Island, NY). The upper part of the skull and the brain were removed and both the otic capsule and the vestibular ganglia were identified by using a stereoscopic microscope (Nikon, Tokyo, Japan). The superior vestibular ganglion was dissected and placed in fresh L-15 with added collagenase IA at 1.25 mg/mL (Sigma-Aldrich, St. Louis, MO) and porcine trypsin 1.25 mg/mL (USB, Cleveland, OH) for 30 min at 37°C. Tissue pieces were then washed three times in L-15 medium. After each wash, tissue was centrifuged 5 min at 4,000 rpm, after which the tissue was suspended in incubated feeding medium. The feeding medium contained L-15, 10% fetal bovine serum, 100 IU/mL penicillin (Lakeside, Toluca, Mexico), 2.5 µL/mL Fungizone (Gibco), 15.7 mM NaHCO3 (Merck, Naucalpan, Mexico), and 15.8 mM HEPES (Sigma-Aldrich), and was adjusted with NaOH to pH 7.7 (medium was incubated 30 min in a CO2 tissue-culture incubator for it to reach pH 7.4). The cells were mechanically dissociated from the tissue by gently sucking it back and forth with a flame-narrowed Pasteur pipette. The cell suspension was diluted with incubated feeding medium and seeded into 35-mm Nunclon tissue-culture petri dishes (Nunc Denmark, Roskilde, Denmark) previously treated with 100 µg/mL poly-D-lysine (Sigma-Aldrich). The petri dishes were transferred to a 5% CO2 tissue-culture incubator (Nuaire, Plymouth, MN) and maintained there at 37°C until the electrophysiological recording (2 h for acutely dissociated neurons and 18 to 24 h for cultured cells). Three vestibular ganglia per dish were used in each experiment.
Soma size measurements
Although the electrical capacitance of a membrane depends on the membrane area of the cells, and it is an indirect measurement of soma size, we decided to analyze the correlation between soma diameter and membrane capacitance to ensure that there were no differences in cellular-membrane characteristics between small and large cells, such as differences in the membrane folding (García-Pérez et al. 2004) that could prevent the use of membrane capacitance as an indicator of soma size. For this, cell images were acquired with a CCD camera (TI-24A, NEC, Elk Grove Village, IL) mounted in a triocular microscope (Diaphot, Nikon) and digitized with a frame-grabber board DT2867-LC (Data Translation, Marlboro, MA). The soma diameter was calculated as the average of major and minor longitudinal axes using the Global Lab Image (Data Translation) software tools.
Electrophysiological recording
The culture dish with attached neurons was mounted on the stage of an inverted phase-contrast microscope (TMS, Nikon). Membrane ionic currents and voltage changes in the cell membrane were studied by standard protocols of whole cell voltage-clamp and current-clamp techniques at room temperature (23–25°C) using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Some of the current-clamp experiments, as indicated in RESULTS, were done at temperatures of 35 to 37°C using the perforated-patch technique with 260 µM amphotericin B (Sigma-Aldrich). Command-pulse generation and data sampling were controlled by pClamp 7.0 software (Axon Instruments) using a 12-bit data-acquisition system (Digidata 1200, Axon Instruments). Signals were low-pass filtered at 5 or 2 kHz and digitized at 20 or 10 kHz depending on the ionic current under study. Patch pipettes were pulled from borosilicate glass capillaries (TW120-3; WPI, Sarasota, FL) using a Flaming–Brown electrode puller (80/PC; Sutter Instruments, San Rafael, CA); they typically had a resistance of 1 to 3 M
when filled with internal solutions. Cells were bathed with different solutions depending on the experimental protocol (Table 1). To isolate the Ca2+ current (ICa), Cs+, tetraethylammonium (TEA), and 4-aminopyridine (4-AP) were used to eliminate outward K+ currents (Table 1, external and internal Cs+ solutions) and TTX was used to block Na+ currents. For the analyses of the Ca2+-activated K+ current (IKCa), Na+ and 4-AP–sensitive K+ currents were blocked. The outward current elicited using internal and external choline solutions consisted of the IKCa and a voltage-dependent, Ca2+-independent outward current resistant to 4-AP. To evaluate the total IKCa component, cells were perfused with a Ca2+-free, 5 mM EGTA external choline solution (Table 1) and the IKCa was obtained by subtraction. The free Ca2+ concentration in the internal solutions using 2 mM EGTA was about 4 nM estimated with the Maxc software (from Chris Patton, Hopkins Marine Station, Stanford University). In current-clamp experiments, internal and external solutions were the same as those used to analyze IKCa except that choline-Cl was replaced by NaCl 18 (internal) and by 140 mM (external) and the CaCl2 in the external solution was set to 1.8 mM (Table 1, external and internal CC). All internal solutions also contained 2 mM ATP-Mg and 0.5 mM GTP-Na. The pH was adjusted for the external solution to 7.4 and for the internal solution to 7.2. Osmolarity was monitored by a vapor pressure osmometer (Wescor, Logan, UT) and was around 300 mOsm for internal solutions and adjusted with dextrose to 310 mOsm for external solutions.
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). Cells that showed instability of ICa or IKCa were eliminated from the analysis. For current-clamp experiments, only cells with a membrane potential at rest above –35 mV were considered.
The residual series resistance after 80% of compensation in whole cell patch-clamp experiments was 2.6 ± 0.3 M (n = 107; range 0.8–5.9 M). Measured liquid junction potentials were 3 mV for solutions used to study ICa and 2 mV for solutions for IKCa. Solutions with Na+ used to analyze membrane voltage responses in current clamp had a liquid junction potential of 1 mV. To plot the current density versus membrane capacitance, the inward Ca2+ current was measured at the peak of the current. The K+ currents were measured at 800 ms after the onset of depolarizing pulses. The maximum current density of total IKCa and of its components was estimated from their respective I–V plots, which were obtained by subtraction of I–V relationships (corrected for residual series resistance and liquid junction potential) before and during perfusion of pharmacological agents or external free Ca2+ solution. This was done instead of constructing I–V curves from isolated currents to avoid possible errors that could emerge because of an uncompensated series resistance when large currents with different kinetics are subtracted from one another (Marcotti et al. 2004).
Action potential analysis
Some of the current-clamp experiments as indicated in RESULTS were done at temperatures of 35 to 37°C using the perforated-patch technique with 260 µM amphotericin B. In these experiments, action-potential variables were measured. For this, the voltage value of the threshold level was used as a reference point. Threshold was defined in two forms: 1) as the point where the time course of a voltage response to a suprathreshold pulse diverts from a single exponential fit to the electrical charging of the membrane [the exponential fit was of the form V(t) = exp(–t/
) + C, where V is the voltage, t is time, and is the time constant of the cell membrane]; 2) as the point where the first-order derivative dV/dt of membrane potential reached a value higher than the noise 5 ms before spike onset (Leger et al. 2005), there were no significant differences using both methods. The amplitude of the action potential was defined as the peak action-potential value minus the threshold voltage. The action-potential duration was measured at 75% of the spike amplitude.
Data analysis
Recordings were analyzed off-line using Clampfit in the pClamp 7.0 suite and Origin software (Microcal Software, Northampton, MA). Statistical differences of means were determined using a Student's t-test, considering significant those with a P < 0.05. Curve-fitting routines were made by using a nonlinear least-squares method. Pooled data are presented as means ± SE unless otherwise stated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Desmadryl et al. 1997). Thus we analyzed the Ca2+ current to define whether cultured rat VANs have the same relationship between soma size and Ca2+ current expression. Voltage-dependent calcium current ICa
The isolated Ca2+ current (ICa) was studied in 35 neurons. In 26% of the cells (n = 35), ICa was formed exclusively by HVA components, whereas in the remaining cells (74%) both LVA and HVA components were identified (Fig. 2, A and B). The ICa in LVA-lacking [LVA (–)] cells activated near –45 mV has its maximum amplitude around –10 mV, inactivating partially during the 800-ms pulse (n = 9). The maximum peak current density in these cells was –30 ± 6 pA/pF. In the LVA-expressing [LVA (+)] neurons, the Ca2+ current activated around –60 mV and completely inactivated with a single time constant of 31 ± 2 ms for pulses at –40 mV (n = 26). The HVA current in LVA (+) cells was evident around –30 mV and partially inactivated during the 800-ms pulse. The maximum peak current density around –10 mV in LVA (+) neurons was –49 ± 8 pA/pF.
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Calcium-activated potassium current IKCa
The expression of IKCa in VANs was analyzed by recording outward currents in external and internal choline solutions and during perfusion with Ca2+-free external choline solution (Table 1). The control current was composed of ICa, IKCa, and a voltage-dependent, 4-AP–insensitive K+ current. The perfusion with Ca2+-free external choline solution reversibly reduced the outward current, leaving the Ca2+-independent components. The IKCa component of the outward current was obtained by subtraction of the current remaining after perfusion with the Ca2+-free external solution from the control current (Fig. 3A). The IKCa component of the outward currents was observed in 100% of acutely dissociated (n = 6) and cultured neurons (n = 47). The IKCa activated above –40 mV reached its maximum around 10 mV, followed by a decline in the current amplitude for more depolarized values (Fig. 3B). Total IKCa current density was 87 ± 7 pA/pF and it was not significantly different (P > 0.05) in cultured or acutely dissociated neurons.
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IKCa was also observed in the 100% of cells obtained from adult p23 to p26 rats (n = 15). The total IKCa current density in VANs from p23 to p26 rats was 318 ± 75 (range 105 to 968 pA/pF). The difference in IKCa current density between young and adult VANs was considerably significant (P < 0.0001), indicating an increase in IKCa expression during the postnatal development. The IKCa current density of LVA (–) cells (681 ± 99 pA/pF; n = 5) was also significantly higher than that found in LVA (+) cells (137 ± 10 pA/pF; n = 10) (P < 0.001) (Fig. 3D).
The correlation between current density and membrane capacitance shows that IKCa decreased as the cell membrane capacitance increased (r = 0.92), indicating a qualitatively similar relationship between p7 to p10 and p23 to p26 VANs. The range and the mean of membrane capacitance of the p23 to p26 cells were smaller because of a smaller culturing time (5 h for p23–p26 compared with 18 h in p7–p10 neurons).
The outward current that remains during perfusion with Ca2+-free solution showed a significant difference (P < 0.05) between LVA (–) (58 ± 7 pA/pF; n = 8) and LVA (+) (38 ± 4 pA/pF; n = 17) cells. No efforts to characterize that current were made.
Calcium-activated potassium current components SK, BK, IK, and IR
To analyze IKCa components in cultured vestibular-afferent neurons from p7 to p10 rats, the pharmacological agents used were specific blockers for BK (100 nM IbTx) (Galvez et al. 1990; Giangiacomo et al. 1992), SK (100 nM apamin) (Hugues et al. 1982), and IK (1 µM CLT) (Grissmer et al. 1993; Jensen et al. 2001; Kaczorowski and Garcia 1999). In some experiments, ChTx was also used to isolate the IK current. Because ChTx blocks both BK and IK channels (Jensen et al. 2001; Kaczorowski and Garcia 1999), BK channels were first blocked with IbTx and then cells were subsequently perfused with an external solution with ChTx + IbTx added. IK was obtained by the subtraction of the current during IbTx perfusion minus the current elicited with IbTx + ChTx. We found a Ca2+-activated K+ current that was resistant to classical IKCa blockers (100 nM IbTx, 100 nM ChTx, 100 nM apamin, and 1 µM CLT). Because no specific blockers for this current exist, we obtained the resistant current (IR) by removal of external Ca2+ after IbTx + apamin + ChTx/CLT application.
The cells used to study IKCa components extend within all the range of capacitances. Values (mean ± SD) of the initial total outward current (Ca2+-independent K+ current + IKCa) of the cells used to analyze each IKCa component before drug application were 115.4 ± 83.8 pA/pF for SK (n = 35), 117.2 ± 86.8 pA/pF for IK (n = 15), 114.1 ± 87.7 pA/pF for BK (n = 30), and 148.7 ± 130.3 for IR (n = 15). There were no statistical differences between these data (P > 0.05), indicating that the cell sample used to make the analyses of IKCa components was not biased.
The perfusion of 100 nM apamin reduced the outward current in 86% of the cells (n = 30/35) (Fig. 4A). Subtraction of the current during apamin perfusion from control current gave the SK current. The SK component activates for voltages positive to –30 mV and reaches its maximum amplitude between 5 and 10 mV (data not shown). The SK current had a slow activation and did not decay during the 800-ms pulse, indicating a minimal, if any, inactivation. The maximum current density of SK was classified according to LVA expression. The SK current density was not different between LVA (–) and LVA (+) cells (8.3 ± 3.8 vs. 13.6 ± 3.5 pA/pF; P > 0.05). No significant correlation between SK expression and cell membrane capacitance was found (r = 0.21), indicating that the SK current is expressed independently of VAN size (Fig. 5, A and B).
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Perfusion of 100 nM ChTx (after BK blockade with IbTx) reduced the outward current in 86% of the cells (n = 6/7; Fig. 4C). The perfusion of 1 µM CLT (n = 8) allowed us to isolate an outward current whose characteristics were indistinguishable from the ChTx-sensitive current. Therefore for IK analysis, data obtained from ChTx- and CLT-sensitive currents were pooled. The IK did not inactivate during 800-ms voltage pulses. The IK activated above –40 mV, reaching its maximum amplitude at 10 mV (data not shown). The mean IK current density was higher in LVA (–) than in LVA (+) cells, although because of its large variability this difference was not statistically significant (26 ± 9 vs. 11 ± 3.8 pA/pF; P > 0.05). The analysis of correlation between IK current density and membrane capacitance gave a weak correlation (r = 0.58) (Fig. 5, A and B).
In 80% of the cells (n = 12/15), the application of IKCa blockers (100 nM IbTx, 100 nM ChTx, 100 nM apamin, and 1 µM CLT) did not remove a Ca2+-dependent outward current (Fig. 4D). This resistant current (IR) was evident by removal of external Ca2+ after IbTx + apamin + ChTx/CLT application (Fig. 4E). The IR current density in LVA (–) cells was significantly higher than that in LVA (+) cells (86 ± 3 vs. 6.2 ± 1.8 pA/pF; P < 0.01). The plot of IR current density versus membrane capacitance shows a significant correlation (r = 0.82) and confirms the existence of two separated cell groups (Fig. 5, A and B). The first was composed of LVA (–) cells with higher IR current density dispersed in a wide range from 36 to 165 pA/pF. The second was composed of LVA (+) cells that did not express a significant IR component, with a range of IR current density from 0 to 14 pA/pF. Note that the IR current density is always underestimated because it is contaminated with ICa. This is because removal of Ca2+ from the external solutions used to obtain IR simultaneously abolishes the Ca2+ current. The range of ICa current density at 800 ms (time at which IR was measured) was between –4 and –43 pA/pF, so the IR could be underestimated within this range. Because HVA-Ca2+ current density was not different between LVA (–) and LVA (+) subgroups, the difference observed in IR current density between LVA (–) and LVA (+) cells is produced exclusively by differences in IR expression.
Effect of IKCa blockers on the voltage response to current pulses
To analyze the influence of IKCa components on the discharge of the cultured vestibular neurons, the effect of IKCa channel blockers on the electrical response of VANs was studied. For this, the perforated patch-clamp technique was used to avoid dialysis of internal constituents of VANs. Recordings were done at temperatures between 35 and 37°C to approximate to more physiological conditions. Voltage responses were recorded using external and internal CC solutions (Table 1), and with holding membrane potential of –60 mV (close to the zero current potential of VANs). In these conditions, 60% of VANs fired one or two action potentials (APs) during 200-ms and 2-s pulses of suprathreshold current injection (n = 21). The remaining 40% of the cells showed a slowly adapting response, firing no more than eight action potentials with long-duration pulses (2 s). The threshold of the first AP in control conditions was –36 ± 1 mV (n = 21). AP amplitude from the threshold to the maximum peak was 53 ± 3 mV. The AP duration (measured at 75%) was 4 ± 0.4 ms (note that external CC solution contains 10 mM 4-AP).
The perfusion of 100 nM apamin or 1 µM CLT did not significantly modify any of these variables (Fig. 6, A and B). However, in 38% of the cells, either apamin or CLT perfusion produced a slight (5–10 mV) oscillatory behavior in the voltage response after the first action potential. This oscillatory response continuously decreased and vanished within 150 ms.
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770% (r = 0.78, n = 7) (Fig. 6C). In those cells with repetitive discharge, the perfusion of 100 nM IbTx removed the adaptation of the response during 200-ms and 2-s current pulses (Fig. 6D). These results indicate that the BK current contributes to repolarization of the action potential and to discharge adaptation, and this contribution is correlated with the membrane capacitance of VANs.
Voltage-clamp experiments indicate that the IR is the principal IKCa component expressed by small LVA (–) cells and its contribution is correlated with the membrane capacitance. Because of the lack of a specific channel blocker for this current, there is no possibility of making a pharmacological analysis of its contribution to the VANs' discharge.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Rat VANs in primary culture expressed a Ca2+ current formed by LVA and HVA components. Whereas the HVA-Ca2+ current was present in 100% of VANs, the expression of LVA-Ca2+ was heterogeneous among cells and preferentially polarized to medium and large neurons. Correlation between T-type Ca2+-current expression and diameter has been reported in vestibular-ganglion neurons of mice (Chambard et al. 1999; Desmadryl et al. 1997) and in the dorsal root and nodosus-ganglion neurons of rat (Fedulova et al. 1985; Lambert et al. 1997). The percentage of cells expressing the LVA-Ca2+ current in this study (74%) was greater than that reported in newborn mice (20%). The differences are because in mice only the largest cells express the LVA-Ca2+ current, whereas in the rat, medium and large cells express this current. This may be an interspecies difference or caused by distinct culturing times of isolated neurons (2–8 h as compared with 18–24 h in the present study). Another possible source for the differences could arise from the distinct age of the animals used (p4–p8 compared with p7–p10 in the present study). In mice, the separation of VANs into two groups on the basis of T-type current starts around E17, and at p4 it is possible to observe two completely separated groups (Chambard et al. 1999). As shown in this work, in rats this separation is still present in the nearly mature (p23 to p26) neurons. The expression of the LVA-Ca2+ current in medium to large cells suggests that the T-type current could contribute to discharge differences between small and large cells in situ. The T-type current is preferentially expressed in dendrites of central neurons and participates in the threshold for action-potential generation, in synaptic integration, and in the configuration of spike discharge (Gauck et al. 2001; Perez-Reyes 2003; Pouille et al. 2000). Therefore as proposed for mice (Desmadryl et al. 1997), the presence of an LVA-Ca2+ current in large cells may decrease the threshold for spike generation, which could explain the increased sensitivity of thick axons that innervate central zones of the neuroepithelium compared with thinner axons that innervate peripheral zones.
For the HVA-Ca2+ currents, although we did not perform a pharmacological dissection, its characteristics were similar to those reported in mice (Desmadryl et al. 1997), where it has been found that the HVA-Ca2+ current is composed of L-, P/Q-, N-, and R-type currents (Chambard et al. 1999; Desmadryl et al. 1997). The whole HVA-Ca2+ current was found in 100% of the rat VANs, and no correlation between its current density and soma size (membrane capacitance) was observed. However, we cannot discard the possibility that some of the ionic channels that make up the HVA-Ca2+ current have a polarized distribution or are coupled differently with some of the KCa channels reported in this work. Therefore further studies focused on the characterization of channels underlying the HVA-Ca2+ current and its functional coupling with KCa channels are needed to determine its particular role in the electrical firing of VANs.
Calcium-activated potassium current expression
The removal of Ca2+ from the external solution decreased the outward current, indicating the presence of an outward current that is activated by the influx of extracellular Ca2+.
The IKCa current was found in 100% of the studied cells. Current-density analysis showed that small LVA (–) neurons had up to four times higher current density than that of larger LVA (+) cells. Our results indicate that in both cell groups four different currents compose the IKCa—SK, IK, BK, and IR—although in different proportions. Of these, only BK and IR have a clear correlation with soma size (membrane capacitance) and with the expression of the LVA-Ca2+ current.
The BK current was preferentially expressed in LVA (+) cells, whereas the IR was strongly expressed in LVA (–) cells. In 60% of the cells, BK was activated at voltages below the AP threshold, indicating its physiological coupling with LVA-Ca2+ channels, similar to data reported in central-vestibular neurons (Smith et al. 2002). If the correlation between soma diameter and BK expression and the functional coupling with T-type currents are present in VANs in situ, it might have important functional consequences. The T-type current decreases the AP threshold, but the major contribution of BK may tend to repolarize the cells, thus producing failures in the AP generation and irregularity of discharge. Actually, the participation of BK in the adaptation of the response to current pulses, revealed by IbTx effects, indicates that BK inhibition converts the firing pattern from phasic to tonic. Furthermore, the BK participates in the repolarization of the action potential and contributes to the spike duration, supporting the hypothesis that the expression of BK significantly contributes to determination of the discharge properties of VANs in situ.
Our current-clamp experiments with the perforated-patch technique, which should maintain endogenous intracellular buffers, reveal that SK and IK have an insignificant participation in the discharge of VANs caused by constant-current pulses, despite the fact that electrical firing was recorded using external solutions with 4-AP, which enhances the excitability of sensory neurons (Sculptoreanu et al. 2004; Soto et al. 2002; Stansfeld et al. 1986). However, the possibility that SK and IK currents participate in the more dynamic and complex conditions in situ cannot be discarded.
SK as well as IK activity depends largely on the concentration of intracellular Ca2+. However, the I–V relationships of SK and IK did not follow the expected decrease for voltages >0 mV (at which the inward Ca2+ current decreases). This can be attributable to contamination with voltage-dependent channels (such as BK currents), although this is unlikely because ChTx and CLT inhibit similar currents and to date there are no reports about nonspecific effects of CLT and apamin on voltage-dependent channels at the concentrations used in this work. Another possibility is that Ca2+ may accumulate by an incomplete clearance by mechanisms of extrusion or sequestration of Ca2+ between voltage pulses. In dorsal root ganglion neurons (Thayer and Miller 1990) and in chromaffin cells of the rat (Herrington et al. 1996) large Ca2+ loads (1–2 µM) produce a rapid initial Ca2+ uptake by mitochondria, but intracellular [Ca2+] decay to resting levels can last >2 min. The modulation of IKCa currents by Ca2+ released from intracellular stores will also be taking place in our system and that particular issue should be addressed in future studies.
The identity of the KCa channels underlying the IR current will require further studies. However, the electrophysiological properties of the IR current are similar to drug-resistant currents from oocytes expressing BK channels coupled with the 
4 subunit (Meera et al. 2000) and astrocytes expressing the 4 subunit (Gebremedhin et al. 2003). The 4 subunit has been reported to confer resistance to IbTx to the BK channels (Gebremedhin et al. 2003; Meera et al. 2000). Unfortunately, the lack of selective blockers of the IR current deters an adequate analysis of its contribution to the firing discharge of VANs. The IR has current densities 160 pA/pF, which are considerably greater than those of other currents reported in this study. Thus the differences found in outward current among cells seems to be caused by the expression of IR.
In mathematical models of VAN, it has been proposed that the AHP slope accounts for differences in sensibility and regularity of discharge (Smith and Goldberg 1986). According to this, the irregular discharge of large neurons innervating type I hair cells may be caused by a smaller slope of postspike voltage trajectory compared with small regular neurons innervating type II hair cells. Although the AP duration did not have a significant linear correlation with total outward current, there is a tendency of small cells to have shorter APs, as observed in VANs in situ innervating the peripheral zones of chick vestibular-neuroepithelium (Yamashita and Ohmori 1990).
It is worth noting that the electrophysiological properties of VANs in situ are changing during the first weeks after the birth until reaching a stable phenotype around p23 (Curthoys 1979). Therefore because most of our results were obtained from rats between p7 and p10, our conclusions are mainly circumscribed by this time window in the development of the vestibular system. However, at this age irregular neurons almost show a mature phenotype except for the presence of firing, bursting, and silent neurons at rest (Curthoys 1979). The percentage of regular neurons between p7 and p10 is in the range of 10 to 15%, which is nearly half of those in the adult animal (32%). Regular neurons at p7 to p10 have an electrical discharge at rest of about 20 spikes/s, which is around one third of the rate in the adult rat (Curthoys 1979, 1982). The inability of our cultured VANs to fire tonically during sustained pulses in current clamp suggests that they are in an intermediate stage of maturity or the spatial relationships of ionic channels is not the same as that in the vestibular system in situ. That total IKCa increases in adult p23 to p26 rats reinforces the idea of the immaturity of VANs at p7 to p10 and is in agreement with studies of vestibular-nucleus neurons, which show that electrical properties of vestibular-nucleus neurons change during the embryonic ages (Peusner and Giaume 1997) and beyond birth (Dutia and Johnston 1998). Despite the increase in IKCa current density, the correlation between total IKCa and membrane capacitance in p23 to p26 adult VANs indicates that the differences in the expression of IKCa are maintained in the mature vestibular system. However, the individual contribution of each IKCa subtype to total IKCa in p23 to p26 neurons was not defined. It is also possible that some of the kinetic and electrophysiological properties of IKCa change during the development as previously reported in central neurons (Kang et al. 1996).
The IKCa has been shown to significantly contribute to the shaping of the action potential waveform and to the firing-discharge pattern of neurons (Dutia and Johnston 1998; Faber and Sah 2002; Johnston et al. 1994; Sah and Faber 2002; Smith et al. 2002). In lamprey motoneurons, blockade of the SK current increased the variation coefficient of the spike discharge (El Manira et al. 1994). The heightened contribution of the IKCa in small LVA (–) cells could determine a faster AHP slope, thus significantly influencing discharge regularity of VANs (Smith and Goldberg 1986).
The functional role of KCa channels is not restricted to the modulation of the electrical firing but also may participate at the central synapse controlling neurotransmitter release (Hu et al. 2001; Robitaille et al. 1993). The particular role of KCa channels at central terminals should be analyzed in experimental models keeping intact the synapses between primary afferents and second-order neurons. It is worth noting that our results are limited to analysis of the KCa currents expressed in specific developmental stages and that there is no evidence indicating whether the complement of these currents is also expressed in afferent dendrites or central terminals of the mature VAN. Our results indicate that cultured VANs express an IKCa carried by several channel subtypes. As shown in other systems IKCa may participate in determining the threshold, latency, repolarization rate, and afterhyperpolarization of the action potential. Its differential expression among VANs may have important functional implications in the coding of vestibular sensory information. Future studies to analyze the expression and distribution of IKCa subtypes within the primary sensory neuron in the mature vestibular system, including recordings in the vestibular nerve in situ, would contribute to our understanding of the role of the intrinsic properties of VANs in the coding of the vestibular information.
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Address for reprint requests and other correspondence: A. Limón, Universidad Autónoma de Puebla, Apartado Postal 406, Puebla, Pue, C.P. 72000, Mexico (E-mail: alimonru{at}uci.edu)
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