Voltage-Activated K+ Currents of Hypoglossal Motoneurons in a Brain Stem Slice Preparation From the Neonatal Rat

Remigijus Lape and Andrea Nistri

Biophysics Sector and Istituto Nazionale Fisica della Materia Unit, International School for Advanced Studies, 34013 Trieste, Italy

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Lape, Remigijus and Andrea Nistri. Voltage-activated K+ currents of hypoglossal motoneurons in a brain stem slice preparation from the neonatal rat. J. Neurophysiol. 81: 140-148, 1999. Whole cell, patch-clamp recordings were performed on motoneurons of the hypoglossus nucleus in a brain stem slice preparation from the neonatal rat brain. The aim was to investigate transient outward currents activated by membrane depolarization under voltage clamp conditions. In a Ca2+-free medium containing tetrodotoxin and Cs+, depolarizing voltage commands from a holding potential of -50 mV induced slow outward currents (Islow) with 34 ± 6 ms (SE) onset time constant at 0 mV and minimal decline during a 1 s pulse depolarization. When the depolarizing command was preceded by a prepulse to -110 mV, the outward current became biphasic as it comprised a faster component (Ifast), which could be investigated in isolation by subtracting the two sets of records. Ifast showed rapid kinetics (9 ± 4 ms 10-90% rise time and 70 ± 20 ms decay time constant at 0 mV) and strong voltage-dependent inactivation (half inactivation was at -92.9 ± 0.2 mV) from which it readily recovered with a biexponential timecourse (4.4 ± 0.6 and 17 ± 2 ms time constants at -110 mV membrane potential). Islow was selectively blocked by TEA (10-30 mM) while Ifast was preferentially depressed by 2-3 mM 4-aminopyridine. Analysis of tail current reversal indicated that both Islow and Ifast were predominantly due to K+ with minor permeability to Na+ (92/1 and 50/1, respectively). These results suggest that membrane depolarization activated distinct K+ conductances that, in view of their largely dissimilar kinetics, are likely to play a differential role in regulating the firing behavior of hypoglossal motoneurons.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Hypoglossal motoneurons are responsible for tongue muscle movements and for maintaining patent airways through the appropriate tone of tongue protrusor muscles. Dysfunction of these cells results in a condition termed sleep apnea, when serious obstruction to breathing develops periodically in infants or adults (Gauda et al. 1987; Wiegand et al. 1991; Willinger 1989). To understand in detail the intrinsic voltage activated conductances of hypoglossal motoneurons subserving their electrical behavior requires, as an experimental model, a brain stem slice preparation in which the electrophysiological properties of single cells can be studied. Using rodent brain slices for this experimental model has demonstrated that these neurons contain several voltage-activated membrane conductances (Haddad et al. 1990; Mosfeldt Laursen and Rekling 1989; Viana et al. 1993a,b) and that they display characteristic firing patterns following membrane depolarization (Sawczuk et al. 1995, 1997; Viana et al. 1995). The latter aspect is of special relevance because it would reveal how the integration of the electrical behavior of the cell at somatic level is translated into output signals to the tongue muscles. Thus depolarizing current pulses induce primary and secondary range firing with different degrees of accommodation of spike activity (Sawczuk et al. 1995, 1997; Viana et al. 1995). These responses presumably rely on the interplay between inward excitatory and outward inhibitory currents. Whereas inward currents, especially those carried by Ca2+ (Umemiya and Berger 1994, 1995), have been amply characterized, data on outward currents studied under voltage clamp are presently lacking. Our aim was, therefore, to provide a first description of some voltage-activated outward currents of rat hypoglossal motoneurons. In addition to a slow K+ current, reminiscent of the classical delayed rectifier, we also observed a fast transient current hitherto unreported in hypoglossal motoneurons.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Slice preparation

Experiments were carried out using brain stem slices obtained from 0 to 9 day-old-rats. Thin slices were prepared following the procedure described by Viana et al. (1994). The brain stem was isolated from neonatal rats and placed into modified, ice-cold Krebs solution (see below). A tissue block containing the lower medulla was then fixed with insect pins onto an agar block inside a Vibratome chamber filled with ice-cold Krebs solution (bubbled with O2-CO2) to obtain 200 µm thick slices. Slices were first transferred to an incubation chamber for 1 h at 32°C under continuous oxygenation and subsequently maintained at room temperature for >= 1 h before use.

Recording

Brain stem slices placed in a small recording chamber were viewed with an infrared video camera to identify single hypoglossal motoneurons within the XII nucleus. Parallel experiments using choline acetyltransferase immunocytochemistry indicated that >90% of the cells of this area were positively stained (R. Donato, unpublished result), thus identifying them as motoneurons (see Viana et al. 1990). The conventional whole-cell, patch-clamp recording technique (Hamill et al. 1981) was employed with the use of an EPC-7 patch-clamp amplifier. Patch electrodes pulled from borosilicate glass had 3-5 MOmega DC resistance. Seal resistance was usually higher than 2 GOmega . After seal rupture, series resistance (5-25 MOmega ) was routinely monitored and compensated for (usually by 50%, range 20-90%). For the sample of cells used for measurements of outward current kinetics and steady state properties the average voltage error found at the largest depolarized potential employed (+20 mV) was 7.2 ± 0.5 mV (range 4.3-9.6 mV). Voltage pulse generation and data acquisition were performed with a PC running pClamp 6.1 software. Currents elicited by voltage steps were filtered at 3-10 kHz and sampled at 5-10 kHz.

Solutions

The solution for slice preparation and maintenance was (in mM) 130 NaCl, 3 KCl, 26 NaHCO3, 1.5 Na2HPO4, 1 CaCl2, 5 MgCl2, 10 glucose (osmolarity ~290-310 mOsm). The extracellular solution for electrophysiological recording was (in mM) 140 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 10 glucose (pH 7.4, 290-310 mOsm). The patch pipette solution was (in mM) 110 K-gluconate, 20 KCl, 5 NaCl, 2 MgCl2, 1 CaCl2, 10 HEPES, 10 EGTA, 2 ATP-Mg (pH 7.2, 260-270 mOsm).

Voltage-activated fast sodium currents were blocked by tetrodotoxin (TTX; 1-2 µM) in most experiments. In some experiments QX-314 (5-10 mM) was added to the pipette solution to block sodium channels and the hyperpolarization activated inwardly rectifying cationic current (Ih) (Perkins and Wong 1995). Alternatively, CsCl (4 mM) was added extracellularly to block Ih. In most experiments extracellular CaCl2 was replaced by the same amount of CoCl2 (Ca-free-Co solution) or CdCl2 (200 µM) was added to block Ca2+ currents and Ca2+ dependent K+ currents. When tetraethylammonium (TEA; chloride salt) or 4-aminopyridine (4-AP) was added to the recording solution in concentrations larger than 5 mM, an equivalent concentration of NaCl was removed. The recording chamber was continuously superfused at 2-5 ml/min. Drugs were added by switching to an appropriate extracellular solution maintained for 5-10 min for equilibration.

Analysis

Cell input resistence (Rin) was calculated from 10 or 20 mV hyperpolarizing commands from a holding potential (Vh) of -70 mV or from the linear part of the I/V line (ramp test) near the cell resting potential (Vrest). Sigma Plot and Clampfit softwares were used for exponential fitting of membrane currents and for linear regression analysis of experimental data. Data are presented as means ± SE. All potential values were corrected off-line for the liquid junction potential, which was measured as 10 mV. Current leak subtraction was performed either on-line using P/8 subtraction procedure or off-line using the Clampfit module.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Basic characteristics of hypoglossal motoneurons

Recordings were obtained from 49 neurons with 44 ± 4 pF somatic capacitance and 400 ± 100 MOmega input resistance (Rin).

In standard solution membrane currents elicited by depolarizing voltage commands comprised multiple components. An example is shown in Fig. 1A where depolarizing steps (10 mV increments from -70 mV holding potential; Vh) evoked fast inward currents (apparent threshold = -50 mV) followed by slower outward currents (apparent threshold = -20 mV). The fast inward current was blocked completely by 1-2 µM TTX (Fig. 1B), suggesting that it was a voltage-activated Na+ current. Because this current was usually too fast, even under the most favorable voltage-clamp conditions, to be adequately recorded, no further analysis of it was attempted. Although not shown in Fig. 1, A and B, most cells also displayed a slower and smaller inward current that was blocked by Cd2+ (0.2 mM) or by replacing Ca2+ with Co2+, indicating that it was mostly carried by Ca2+. As the properties of voltage dependent, Ca2+ currents and channels of hypoglossal motoneurons have been systematically investigated by Umemiya and Berger (1994, 1995), they will not be further reported here.


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FIG. 1. Depolarization elicited membrane currents of hypoglossal motoneurons. A: membrane currents elicited by application of depolarizing voltage steps (10 mV increments) from -70 mV Vh (schematized in top) in normal solution (Ctrl). Pulses (at 10 s intervals) were 1 s long, but only the initial 10 ms traces are shown. Note inward current followed by outward current. All traces are leak subtracted. B: membrane currents elicited by the same voltage protocol applied to the same cell bathed in a solution containing 1 µM TTX (------) or in a solution containing 1 µM TTX and nominally zero Ca2+ replaced by Co2+ (2 mM) (···). Note suppression of inward current while outward current persists.

The outward current, clearly detected in TTX medium (Fig. 1B), was partly diminished (for instance by 11% at 0 mV) in Ca-free-Co solution or by extracellular application of Cd2+ (not shown). This outward component was more strongly depressed (40 ± 8% at 0 mV; n = 3 cells) by the K+ channel blocker TEA (20 mM; not shown). These preliminary observations suggested to us that the outward current was presumably due to K+ efflux via several conductances, mainly voltage and partly Ca2+ activated. The present report thus focusses on voltage activated K+ currents.

Slow transient outward current

The delayed transient outward current (Islow) was routinely investigated in Ca-free-Co solution containing TTX (1 µM) and Cs+ (4 mM). The example of Fig. 2A shows a set of current traces generated by steps to potentials between -30 mV and +20 mV, following a 500 ms long conditioning step (prepulse) to -50 mV from -70 mV Vh. The test command to -20 mV was clearly above threshold for eliciting a slowly developing outward current that did not decline during the 1 s pulse. Larger steps generated currents of larger amplitude and faster peaking response. Some decline in current amplitude emerged for responses generated by pulses to positive membrane potentials. Preliminary trials showed that the conditioning step (200-500 ms) to -50 mV did not influence the slow current development, but it removed the contaminating presence of a faster current (described below). For this reason, whenever the slow current was studied in isolation, a 400 ms prepulse to -50 mV was always applied before depolarizing test steps. We also checked that extracellular application of Cs+ did not influence Islow because this monovalent cation can block a variety of K+ channels in other cells (Rudy 1988). Thus, comparing the steady state Islow amplitude (evoked by +20 mV steps) in Cs+ free solution with the one obtained after the addition of Cs+ (4 mM) indicated that there was no significant change (3 ± 8%; n = 4; P = 0.7 with ANOVA test).


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FIG. 2. Hypoglossal motoneurons possess a slow transient outward current (Islow). A: Islow elicited by depolarizing voltage steps from -30 to 20 mV with conditioning step to -50 mV for 400 ms. B: current-voltage relation of Islow (n = 15 cells). C: Islow chord conductance vs. membrane voltage for the same group of cells. D: Islow activation time constant (estimated by fitting the current activation phase with a single exponential) vs. membrane voltage (n = 8 cells).

The average I/V relation (n = 15 cells) for Islow is shown in Fig. 2B. Islow activated at membrane potentials positive to -50 mV. Its amplitude monotonically increased with increasing depolarization within the tested potential range of -50 to +20 mV. The dependence of Islow chord conductance (g; expressed in nS) on membrane potential is shown in Fig. 2C. After measuring the peak of Islow the values for g were calculated according to the equation
<IT>g = I</IT><SUB>slow</SUB>/(<IT>V</IT><SUB>m</SUB><IT>− E</IT><SUB>rev</SUB>) (1)
where Erev is the K+ equilibrium potential (measured as described below) and Vm is the test membrane potential at which Islow was recorded. The values of g grew for potentials positive to -50 mV and showed an average increment of 3.9 ± 0.4 nS/mV. Fitting these data according to the Boltzmann equation yielded an average slope of 19 ± 3 mV; the g maximum value was estimated as 26 ± 1 nS as it occurred at a potential level (+60 mV) outside the range tested in the present experiments.

Figure 2D shows a plot for the voltage dependence of Islow activation in which the current rising phase could be fitted monoexponentially: it is clear that it had a slow onset, which for example at 0 mV had a time constant of 34 ± 6 ms. Once Islow reached its peak, it remained at a plateau or gradually declined for test depolarizations positive to -10 mV. For example, Islow decline had a time constant of 4.2 ± 0.4 s at +20 mV membrane potential (n = 3 cells). Using the time constant of tail currents at the end of depolarizing voltage commands, the deactivation properties of Islow were studied. In this case, the deactivation time constant was on average 40 ± 4 ms (n = 7 cells; range 27-60 ms within -105 and -40 mV). These data, therefore, indicate that Islow had rather slow kinetic characteristics, which made it unsuitable to control motoneuronal excitability within a narrow time frame and prompted a more systematic search for a faster outward current component in these cells that was hitherto undescribed.

Fast transient outward current

We suspected that any hypothetical fast outward current (Ifast) might have been largely masked by the use of relatively depolarized Vh values because this appears to be the case for the fast outward current of other central neurons (Rogawski 1985; Rudy 1988). To demonstrate its existence in hypoglossal cells, we used a subtraction procedure as detailed in Fig. 3, A and B. In fact, we first obtained a family of outward currents consisting of an initial, rapid peak followed by a slower component (Fig. 3A) when the 1 s depolarizing test steps were applied immediately after a hyperpolarizing prepulse (to -110 mV). An analogous protocol with only one difference was subsequently repeated on the same cell, namely, instead of a hyperpolarizing prepulse, a depolarizing one (to -50 mV; Fig. 3B) was used. In the latter case the outward current typically consisted of Islow only (see also Figs. 1 and 2). After point-to-point subtraction of the current traces obtained with depolarized prepulses from those obtained with hyperpolarizing prepulses, a transient and rapid outward current (Ifast) could be demonstrated in isolation (Fig. 3C). Ifast grew to a peak and inactivated in <200 ms (Fig. 3C). We also investigated whether the presence of 4 mM Cs+ in the bathing solution might have affected Ifast development. Comparing the peak amplitude of Ifast (induced by steps to +20 mV) before and after adding Cs+ showed that there was no significant change (7 ± 9%; n = 3; P = 0.8), suggesting that this cation did not interfere with Ifast recording.


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FIG. 3. Hypoglossal motoneurons possess a fast transient outward current (Ifast). A and B: two sets of outward current traces evoked by a series of depolarizing voltage steps from -60 to 20 mV preceded by either a hyperpolarizing voltage step to -110 mV (A) or a depolarizing voltage step to -50 mV (B) for 400 ms. C: Ifast isolated by subtracting current traces in B from those in A. D: current-voltage relation of Ifast (n = 8 cells). E: Ifast activation time (calculated as 10-90% rise time) vs. membrane potential (n = 5 cells).

The peak current-voltage (I/V) relation for Ifast is shown in Fig. 3D. The threshold for activation of Ifast was approximately -60 mV with a monotonic increase in Ifast amplitude as the test depolarization increased. The Ifast rise time (calculated as 10-90% time to peak) decreased with increasing depolarization (Fig. 3E), suggesting voltage dependence of the current activation process. The rise time was 24 ± 7 ms at -50 mV and 8 ± 3 ms at +20 mV (n = 5 cells).

The activation and inactivation properties of Ifast g were next examined. For activation the peak conductance (gpeak) was simply determined by dividing the Ifast peak amplitude by the driving force (Eq. 1) and then normalized (gnorm) to the maximum conductance, gmax. The latter value was obtained from chord conductance-voltage plots, which indicated g saturation at +20 mV. Hence
<IT>g</IT><SUB>norm</SUB><IT>= g</IT><SUB>peak</SUB>/<IT>g</IT><SUB>max</SUB> (2)
For nine cells the normalized conductance (gnorm) values were thus plotted against membrane potentials to give the activation curve (Fig. 4A; bullet ). These experimental points were fitted with the Boltzmann equation, which indicated half activation of Ifast at -27.6 ± 0.9 mV with a 16 ± 1 mV slope. The conductance was thus activated at potentials positive to -70 mV and was virtually fully activated at +20 mV.


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FIG. 4. Voltage dependence of activation and inactivation of Ifast. A: activation (bullet ) and inactivation (open circle ) curves for Ifast. Experimental points were obtained by dividing Ifast peak by its driving force (Vm-Erev) and normalizing them by the maximal conductance gmax taken as 1. Lines represent Boltzmann equation fits; the potentials for half inactivation and half activation were -92.9 ± 0.2 mV and -27.6 ± 0.9 mV, respectively, while the slope factors were 10.8 ± 0.2 mV and 16 ± 1 mV for inactivation and activation, respectively (n = 9 cells). B: a set of outward currents elicited by a test voltage step to 30 mV after conditioning prepulses from -140 mV to -50 mV for 200 ms. C1: same set of current traces on a faster time scale. C2: individual Ifast responses were isolated by subtracting the current trace obtained after the depolarizing prepulse to -50 mV from the current traces shown in C1.

The protocol to study steady-state inactivation of Ifast is depicted in Fig. 4B. The motoneuron membrane potential was conditioned (for 200 ms) to different potentials (in the range from -140 mV to -10 mV) and then depolarized to a fixed test potential of +30 mV, which corresponded to full activation of Ifast (see Fig. 4A). The faster timebase records of Fig. 4C1 show that, with progressively more depolarized prepulses, Ifast disappeared. For full isolation of Ifast the current trace after a prepulse to -50 mV (when Ifast was absent) was subtracted from all the other traces. Ifast could thus be measured separately (Fig. 4C2) and the underlying gpeak calculated and normalized to gmax (Eqs. 1 and 2). The inactivation curve was generated by plotting gnorm versus the conditioning step potential (Fig. 4A; open circle ) and fitted with the Boltzmann equation. Half inactivation was estimated at -92.9 ± 0.2 mV with a -10.8 ± 0.2 mV slope (n = 9 cells). The conductance inactivation was complete at -50 mV and was completely removed at -140 mV. When activated Ifast inactivated completely or to a small residual current (Fig. 3C).

It seemed useful to characterize how quickly Ifast inactivation could develop, because this property may influence the firing characteristics of the cell. This result was obtained by measuring the time constants of single exponential decay of Ifast (Fig. 5A). In these examples the values were 86, 75, and 61 ms at -40, -20, and 0 mV membrane potentials, respectively. These observations allowed us to plot the average time constants of inactivation versus membrane potential (Fig. 5B; n = 7 cells). Inactivation was found to be dependent on voltage because the decay time constant was twice as fast at +20 mV than at -40 mV.


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FIG. 5. Inactivation kinetics of Ifast. A: Ifast traces isolated as described in Fig. 3C. The decay phase was fitted monoexponentially with time constants of 86, 75, and 61 ms for currents elicited by depolarizing steps to -40, -20, and 0 mV, respectively. B: decay time constant versus membrane voltage (n = 7 cells). C: current traces elicited by two pulse voltage protocol to measure time course of Ifast recovery from inactivation. D: data obtained with a protocol similar to the one shown in C. Experimental points represent the normalized amplitude of Ifast (ordinate) evoked by the second pulse at different intervals (ms; abscissa) after first pulse for sojourns to -110 mV (open circle ) or -90 mV (bullet ) (n = 3 cells). The time course for recovery was well fitted by two exponentials with time constants of 4.4 ± 0.6 and 17 ± 2 ms for -110 mV and 5.1 ± 0.2 and 25.8 ± 0.9 ms for -90 mV.

Using a two pulse protocol as shown in Fig. 5C, recovery of Ifast from inactivation was studied. A conditioning step (1 s) from -110 mV Vh to +10 mV was first employed to inactivate Ifast completely and then, after a sojourn at -110 mV for 10-1,000 ms, a test pulse to +10 mV was subsequently applied. Note that after progressively longer sojourns at -110 mV, the amplitude of Ifast gradually returned to control value. The Ifast peak (normalized with respect to the initial control amplitude) was plotted versus time (Fig. 5D; open circle ). Figure 5D also shows that on 3 cells similar data were obtained when the initial Vh and sojourns values were -110 mV or -90 mV (open circle , bullet , respectively). In both cases the time course of recovery from inactivation was well fitted by two exponentials (Fig. 5D). The time course for recovery was well fitted by two exponentials with time constants of 4.4 ± 0.6 and 17 ± 2 ms at -110 mV and 5.1 ± 0.2 and 25.8 ± 0.9 ms at -90 mV, indicating a slight voltage dependence for this process. In conclusion, recovery from inactivation was less dependent on membrane potential than the process of inactivation itself.

Deactivation properties of Ifast were obtained by measuring the time constant of decay of tail currents (e.g., Fig. 6) at potentials between -105 and -65 mV because at less negative level the inactivation process became predominant. In this case, deactivation was relatively independent from membrane voltage and averaged 19 ± 3 ms (range 4-38 ms; n = 8 cells).


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FIG. 6. Ionic properties of outward currents. A: tail currents of Ifast (bottom) elicited by voltage steps to different potentials from -95 to -60 mV after a depolarizing step to 0 mV for 15 ms (top). The membrane was hyperpolarized to -110 mV for 400 ms prior to the test pulse to remove inactivation of the transient outward current. B: average tail current amplitude as a function of membrane voltage in the presence of 3 (n = 11), 6 (n = 3), and 12 mM (n = 3) extracellular K+. C: reversal potential as a function of the extracellular K+ concentration. black-square, Ifast; square , Islow. ···, theoretical K+ equilibrium potential calculated from the Nernst equation.

Ionic properties of outward currents

The ionic selectivity of Ifast and Islow was studied with tail current analysis. Voltage steps to 0 mV from -70 mV Vh with or without a prepulse (for 200-500 ms) to -110 mV were applied to evoke these currents. When the current reached its peak (200 ms for Islow and 15 ms for Ifast), the membrane potential was stepped to different voltages and tail currents were recorded. An example of Ifast tail currents recorded at membrane potentials from -95 mV to -60 mV is shown in Fig. 6A. In this cell the tail current reversed at -80 mV. The mean reversal potential of Ifast tail currents was -75 ± 2 mV (n = 11 cells), whereas the corresponding value for Islow tail currents was -81 ± 1 mV (n = 11 cells).

Average Ifast tail currents plotted versus membrane voltage in 3 (n = 11 cells), 6 (n = 3), or 12 (n = 3) mM external K+ concentrations are presented in Fig. 6B. A compensatory reduction in NaCl was effected whenever K+ was raised. The results indicate that the tail current reversal potential, Erev, moved to more positive values when the external K+ was increased and that there was an approximately parallel, rightward shift of these plots. The Erev dependence on the external K+ concentration is shown in Fig. 6C for both Islow (square ) and Ifast (black-square). The theoretical Erev for K+ at three K+ concentrations was calculated with the Nernst equation and is shown as a dashed line in Fig. 6C. From these data, it is apparent that Erev for both outward currents moved together with changes in external K+ concentration, suggesting that Islow and Ifast were predominantly K+ currents. Nevertheless, as the observed values differed from the calculated one, at least three factors could have accounted for the deviation: 1) K+ accumulation in the extracellular space, 2) a degree of membrane permeability to other ions such as Na+, or 3) imperfect voltage clamp-conditions. The first possibility seemed unlikely because the tail current amplitude of Ifast or Islow at -50 mV (to maximize its size) did not change during 2 Hz test pulses, a condition which should have enhanced any K+ accumulation. The second possibility was explored by 50% replacement of external NaCl with the presumably impermeant N-methyl-glucamine HCl: in this case, the tail Erev of Ifast shifted in the negative direction by 5 ± 1 mV (n = 3 cells). This result suggests a measurable contribution by Na+ to the transient outward currents. Using the Goldman-Hodgkin-Katz equation (Hille 1992), we calculated the permeability ratio of K+ to Na+ (PK/PNa). For the Na+ and K+ concentrations of the patch pipette and extracellular solutions the permeability ratio values of 1/0.01 (92/1) and 1/0.02 (50/1) were obtained for Islow and Ifast, respectively. These data suggest that the contribution by Na+ permeability to the transient K+ currents was relatively small and that the deviation of the outward current Erev from the one calculated for a pure K+ mediated response was perhaps also due to the difficulty to obtain isopotential conditions for a large cell with dendritic arborization in a slice preparation.

Pharmacological dissection of Ifast and Islow

Two well known K+ channel antagonists, TEA and 4-AP, were tested. Application of 10-30 mM TEA (n = 6 cells) readily depressed Islow, as indicated by the example in Fig. 7A 1 and 2, in which the whole set of outward current traces (elicited by the same protocol shown in Fig. 2A) recorded in control solution (Ca-free-Co, TTX, Cs+) was attenuated by 20 mM TEA. Fig. 7A3 shows the I/V curve related to the same data, indicating that there was a consistent depression (by 70 ± 3%) of Islow at various test potentials. This effect was reversible after 15-20 min washout. Ifast was comparatevely resistant to 10-30 mM TEA: in fact, Ifast amplitude at +20 mV slightly increased by 1.1 ± 0.2 times (n = 4 cells).


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FIG. 7. Pharmacological properties of Islow and Ifast. A: current traces obtained by depolarizing voltage steps (same protocol as in Fig. 2.) in control solution (Ctrl, containing TTX, Ca-free-Co, Cs+) (A1) and in the presence of TEA (20 mM) (A2). A3: I/V relation of Islow in control (open circle ) solution or in the presence of 20 mM of TEA (bullet ) for the same cell shown in A1 and 2. B: 4-AP preferentially blocks Ifast. Current traces (elicited by voltage step to 20 mV) were recorded in a solution containing TTX, TEA (20 mM), Ca-free-Co, and different concentrations of 4-AP (0, 1, 2, and 3 mM). All records are from the same cell.

Ifast was preferentially blocked by 4-AP. An example is shown in Fig. 7B in which Ifast elicited by a depolarizing step to 20 mV after a conditioning step to -110 mV was recorded in Ca-free-Co solution containing TTX (1 µM), TEA (20 mM), and Cs+ (4 mM). Increasing concentrations of 4-AP (1, 2, and 3 mM) were cumulatively added to the external solution. The peak amplitude of Ifast was not decreased by 1 mM 4-AP, although the current decay became faster (33 ms time constant in control and 21 ms time constant in presence of 1 mM 4-AP). At 2 or 3 mM concentration, 4-AP diminished Ifast peak by 6 and 19%, respectively, together with a progressive reduction in current decay (13 and 10 ms time constants, respectively). Nevertheless, the late component of Ifast remained relatively unaffected. Even very high concentrations of 4-AP (up to 10 mM) were unable to block Ifast completely, whereas at concentrations higher than 6 mM 4-AP also depressed (by 28% in 7 mM 4-AP solution) Islow. At 5 mM concentration 4-AP depressed Ifast and Islow by 37 and 6%, respectively.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The present report provides the first quantitative description of two voltage-activated, Ca2+-independent K+ currents (Islow and Ifast), under voltage-clamp conditions, in neonatal hypoglossal motoneurons.

Voltage activated currents of hypoglossal motoneurons

Motoneurons integrate excitatory synaptic inputs to convert them into trains of the action potential that determine the characteristics of muscle activity. The pattern and frequency of action potential firing are due to the interplay of various membrane intrinsic conductances. In hypoglossal motoneurons (like in most nerve cells) the TTX-sensitive, fast Na+-inward current (shown in Fig. 1) is presumably responsible for the initial rising phase of the action potential. As it is notoriously difficult to study this Na+ current under voltage-clamp conditions (see for example, Takahashi 1990), it was not further investigated in the present report. Membrane depolarization of hypoglossal motoneurons is also known to activate four types of Ca2+ channels (Umemiya and Berger 1995) that underlie distinct Ca2+ currents systematically investigated by Umemiya and Berger (1994).

In addition to the transient inward currents generated by depolarization, multiple K+ conductances have been suggested to shape action potential repolarization and repetitive firing behavior (Viana et al. 1993b).

Molecular biology studies have indicated that rat brain stem nuclei contain mRNA for various K+ channel subunits, particularly Kv3.3 (and, to a lesser degree, Kv3.1), which in expression studies generates a sustained, slowly inactivating current (Weiser et al. 1994), and Kv4.2 and Kv4.3 both responsible for fast activating and inactivating currents (Serodio and Rudy 1998). It appears likely that hypoglossal motoneurons possess a repertoire of K+ currents that so far have not been systematically analyzed under voltage-clamp conditions.

In our experiments the total outward current could be separated into two broad components: one composed of voltage-activated K+ currents and a second of Ca2+-dependent K+ currents, which are typically responsible for the medium and slow afterhyperpolarization (AHP) (Viana et al. 1993b). In our experiments, Ca2+-dependent K+ currents were very small by comparison with Ca2+-independent K+ currents (Fig. 1B) presumably because of the presence of the strong Ca2+ chelator EGTA, which is known to suppress the Ca2+ dependent AHPs (Viana et al. 1993b).

In our study, voltage-activated K+ currents were investigated in the presence of TTX to block Na+ currents and in Ca-free-Co solution to eliminate Ca2+ currents and any residual Ca2+-dependent K+ currents. Cs+ was always present in the external solution to block the hyperpolarization activated current Ih (Bayliss et al. 1994) and did not appear to interfere with the voltage activated outward currents. Under these conditions a slow outward current (Islow) was generated by 1 s voltage steps from -70 mV Vh (see Fig. 1B), peaking in tens of milliseconds and remaining at a plateau with a slow decline. Hyperpolarizing prepulses allowed us to unmask a much faster current (Ifast). Islow and Ifast could therefore be distinguished on the basis of the substantial differences in their voltage dependence of activation and inactivation.

Islow properties

Based on its activation kinetics and voltage dependence, Islow resembled the classical delayed rectifier first reported for the squid giant axon by Hodgkin and Huxley (1952). Islow activated at membrane potentials positive to -50 mV and showed voltage-dependent kinetics of activation. For example, the activation time constant value decreased nearly 10-fold from -20 mV to +20 mV. At membrane potentials positive to -10 mV, Islow inactivated slowly with a time course of seconds. During repolarization at the end of voltage step, Islow deactivated with monoexponential time constant. In addition, Islow was suppressed by TEA and partly depressed by very high doses of 4-AP.

Ifast properties

Ifast was unaffected by TEA application, a treatment which actually allowed us to observe it in isolation with its characteristically faster kinetics. Ifast resembled the fast transient outward current IA, first observed in gastropod neurons (Connor and Stevens 1971a). Ifast was almost completely inactivated near resting potential because depolarizing voltage steps from -70 mV Vh did not evoke Ifast. Half-maximal inactivation was at -92.9 ± 0.2 mV. By membrane hyperpolarization the inactivation of Ifast was rapidly removed with recovery to 50% of control amplitude in about 10 ms (see Fig. 5D). Subsequent depolarization to values positive to -60 mV activated Ifast (with a time course depending on membrane voltage), which peaked and fully inactivated within 200 ms. (At 0 mV the inactivation time constant was 70 ± 20 ms.) Ifast deactivation after the end of the command pulse was comparatively faster (19 ± 3 ms) and showed little voltage sensitivity. In contrast to Islow, Ifast was selectively attenuated after application of 4-AP (up to 5 mM), which, in mM concentrations, is a well established blocker of Ifast in other nerve cells (Rudy 1988). The characterization of Ifast in hypoglossal motoneurons is thus based on distinct electrophysiological properties and pharmacological sensitivity.

It should be noted that Erev for Ifast (and also Islow) was near the calculated value for K+ and was shifted positively (as predicted by the Nernst equation) by raising extracellular K+ concentration. These observations suggest that both currents were mainly mediated by an increased membrane permeability to K+. Nevertheless, there was a small but systematic difference between the current Erev and EK+. This was partly due to a slight, yet consistently measurable permeability to Na+ inherent in both currents and perhaps partly to the difficulty in obtaining complete isopotential conditions in neurons with dendritic arborization.

Functional implications of Ifast and Islow for motoneuronal firing properties

Analogous to spinal (Barrett et al. 1980) and facial (Nishimura et al. 1989) motoneurons, hypoglossal motoneurons were shown to possess at least two transient K+ currents, the functional role of which should be considered. It has been previously demonstrated that TEA- and 4-AP-sensitive K+ conductances shape the action potential of hypoglossal motoneurons (Viana et al. 1993b) because TEA (1-10 mM) or 4-AP (0.1-0.5 mM) prolong action potential duration. When hypoglossal motoneurons are depolarized by long current pulses, their firing frequency decreases in a multimodal fashion with linearly developing initial adaptation (Sawczuk et al. 1995, 1997). In the study by Viana et al. (1993b), firing accommodation could not be tested in 4-AP solution owing to the development of intense synaptic activity while it seemed to be enhanced by TEA, although a quantitative analysis was not provided.

Our experiments suggest that only a very small part of Ifast could by blocked by 1 mM 4-AP. Thus it seems plausible that spike lengthening by such a small dose of 4-AP does not involve depression of Ifast. Furthermore, as the resting potential of neonatal motoneurons is about -70 mV and the spike duration about 1 ms (Viana et al. 1995), it is likely that at rest level Ifast is largely inactivated and that any available fraction of Ifast possesses activation and deactivation kinetics (~10 and 20 ms, respectively) that are much slower than the spike duration. For these characteristics we suggest that Ifast, rather than controlling the duration of a single action potential (which perhaps relies on a distinct Ca2+-dependent K+ current not investigated in the present study), has a role in determining the initial adaptation of firing (lasting about 40 ms) which is only moderately dependent on Ca2+-activated K+ currents (Sawczuk et al. 1997). The AHP after the first action potential, typically lasting about 100 ms and with <10 mV undershoot from rest (Viana et al. 1995), would, however, be long enough to remove part of Ifast inactivation. Thus, the second action potential could be delayed by a more substantial Ifast activation. A similar mechanism can be suggested to operate for the third and fourth spike observed within the initial 50 ms of a train (Sawczuk et al. 1995), particularly because Ifast deactivation required about 20 ms. Ifast would then be able to control firing frequency through a mechanism similar to the one proposed by Connor and Stevens (1971b) for IA.

Only a small part of Islow could by activated during the first spike because of its slow kinetics. Nevertheless, because Islow deactivated with a time constant (40 ms) longer than first interspike interval (Sawczuk et al. 1995, 1997), a certain fraction of activated Islow would be present at the beginning of the second spike in a train. Likewise, further accumulation of Islow would occur during the spike train with summating properties after each successive spike.

Unfortunately, direct experimental test of Islow and Ifast participation in spike frequency adaptation is presently made difficult by the lack of highly selective blockers. For instance, although Ifast is sensitive to 4-AP, even a 5 mM concentration of this substance could not block it completely. The same applies to TEA acting on Islow. Along the same line it might be difficult to rely on holding potential changes to remove Ifast inactivation selectively, as this approach would also affect the kinetic properties of voltage dependent inward currents. Thus it seems useful to employ computer stimulation studies (see for instance, Powers 1993), which in the future might provide a better understanding of the functional role of these currents in hypoglossal motoneurons, but they will require strictly quantitative data. The availability of kinetic parameters pertaining to these currents is expected to be useful for this purpose.

    ACKNOWLEDGEMENTS

  We thank R. Donato for making available the CAT immunocytochemistry data, and Dr. Gytis Baranauskas for performing pilot studies on hypoglossal motoneurons.

  This work was supported by grants from Telethon Foundation project 823, Istituto Nazionale Fisica della Materia, the Consiglio Nazionale della Ricerche and the Ministero dell' Università e della Ricerca Scientifica e Tecnologica.

    FOOTNOTES

  Address for reprint requests: A. Nistri, Biophysics Sector and Istituto Nazionale Fisica della Materia Unit, International School for Advanced Studies, Via Beirut 4, 34013 Trieste, Italy.

  Received 9 June 1998; accepted in final form 25 September 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

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