 |
INTRODUCTION |
Neuronal development and synaptogenesis in the rat spinal cord begins during the last week of gestation, and continues throughout the first few weeks after birth (Gao and Ziskind-Conhaim 1995
; Gao et al. 1998
; Kudo and Yamada 1985
, 1987
; Saito 1979
; Seebach and Ziskind-Conhaim 1994
; Takahashi 1992; Walton and Navarrete 1991
; Wu et al. 1992
; Ziskind-Conhaim 1990
; Ziskind-Conhaim et al. 1993
). Increased membrane excitability, as evident by changes in action potential waveform, is one of the fundamental characterizations of motoneuron development during embryonic and postnatal ages (Fitzgerald 1987
; Huizar et al. 1975
; Kellerth et al. 1971
; Naka 1964
; Walton and Fulton 1986
; Ziskind-Conhaim 1988a
). Motoneurons are excitable soon after they clustered in the ventral horn (Mandler et al. 1990
; Saito 1979
; Ziskind-Conhaim 1988a
), but action potential properties and firing behavior markedly change during the last week of gestation and immediately after birth (Fulton and Walton 1986
; Xie and Ziskind-Conhaim 1995
). The increase in motoneuron excitability is functionally important not only for transforming a developmental increase in synaptic transmission (Gao et al. 1998
) into an increasingly complex locomotion activity but also for triggering the onset of activity-dependent mechanisms underlying the differentiation of muscle fibers and synaptic transmission at the neuromuscular junction (reviewed by Navarrete and Vrbová 1993
).
Maturation of membrane excitability in the vertebrate nervous system is a progressive sequence of events that results in changes in passive and active electrical properties. For example, the development of mammalian motoneurons is associated with a negative shift in action potential threshold, thus increasing the likelihood of initiating action potentials. Concurrently, action potential amplitude increases as its duration decreases, and prominent afterhyperpolarizing potential (AHP) is apparent (Huizar et al. 1975
; Kellerth et al. 1971
; Núñez-Abades et al. 1993
; Viana et al. 1994
; Xie and Ziskind-Conhaim 1995
; Ziskind-Conhaim 1988a
). Such changes represent a general developmental trend, but the pattern and time course of the expression of voltage-gated ion channels underlying these changes vary considerably in different neuronal populations (review by Spitzer 1991
). Studies of mammalian and chick spinal neurons have shown that the ionic basis of the action potential is relatively constant during neuronal development, with Na+-dependent action potentials predominant from the onset of membrane excitability (Krieger and Sears 1988
; MacDermott and Westbrook 1986
; McCobb et al. 1990
; Ziskind-Conhaim 1988a
). However, in neurons of Xenopus embryos and cortical neurons of chick embryos, the appearance of short-duration action potential is associated with changes in the ionic currents underlying action potential generation. At the onset of excitability, the long-duration action potentials are Ca2+ dependent, and short-duration Na+-dependent action potentials develop shortly afterward (Baccaglini and Spitzer 1977
; Blair 1983
; Mori-Okamoto et al. 1983
; Willard 1980
).
Most studies that have characterized developmental changes in motoneuron electrical properties and the currents underlying them, have been conducted using dissociated spinal neurons of Xenopus (Spitzer and Lamborghini 1976
; Willard 1980
), chicks (McCobb et al. 1990
; O'Brien and Fischbach 1986
), and mice (Heyer et al. 1981
; Krieger and Sears 1988
; MacDermott and Westbrook 1986
; Randsom et al. 1977
; Rogawski and Barker 1983
). The experimental benefit of cell accessibility in culture might be compromised by the concern that neuronal differentiation in culture differs from that in vivo. An alternative reliable preparation for studying changes in motoneuron membrane properties during spinal cord development in vivo is the spinal cord slice, in which neurons in various lamina of the spinal cord could be identified visually (Bardoni et al. 1997
; Gao and Ziskind-Conhaim 1998; Takahashi 1978
, 1990
). In our study, thick spinal cord slices of embryonic and postnatal rats were used to examine the properties of a repertoire of voltage-gated ionic currents underlying changes in action potential waveforms.
Motoneurons are generated in the thoracic spinal cord at embryonic day 11-12 (E11-12) (Nornes and Das 1974
) and begin to cluster in thoracic and lumbar spinal cords by E13-14 (Smith and Hollyday 1983
; Ziskind-Conhaim 1988a
). Dorsal-root-evoked synaptic potentials are recorded first at E15 (Kudo and Yamada 1987
; Ziskind-Conhaim 1990
), about the same time that motor axons form initial synaptic contacts with muscle fibers (Dennis et al. 1981
). At that age, muscle fibers are multiply innervated and electrically coupled, resulting in wide-spread muscle contraction in response to single nerve stimulation (Dennis et al. 1981
). Although motoneurons and motor axons are excitable at E15 (Ziskind-Conhaim 1988b
), the frequency of spontaneous action potentials is low during embryonic development (Saito 1979
; Ziskind-Conhaim 1988a
), and only one action potential can be produced in response to a prolonged intracellular injection of depolarizing current (Xie and Ziskind-Conhaim 1995
). The frequency of spontaneous action potentials significantly increases immediately after birth (postnatal day 1-3, P1-3), and prolonged current injection produces repetitive action potential firing at that age. To increase our understanding of the ionic currents underlying the postnatal increase in motoneuron excitability, we elected to study the correlation between changes in action potential waveforms and voltage-gated ionic currents at E15-16 and P1-3.
In previous studies, action potentials in embryonic and postnatal spinal motoneurons were recorded intracellularly using high-resistance electrodes, and the contribution of ionic currents to action potential waveform was inferred by examining the effects of blockers of voltage-gated ion channels on action potential properties (Walton and Fulton 1986
; Ziskind-Conhaim 1988a
). In the present study, we used the whole cell voltage-clamp recording technique, a more direct experimental approach to determine the properties of a repertoire of voltage-gated ionic currents during a developmental period characterized by marked changes in action potential waveforms and repetitive firing behavior.
Preliminary descriptions of these results have been presented in abstract form (Gao and Ziskind-Conhaim 1993
, 1994
).
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METHODS |
Spinal cord preparation
Lumbar spinal cords were isolated from Sprague-Dawley rat embryos at days 15-16 of gestation (E15-16, birth is at E21-22) and from 1- to 3-day-old postnatal rats (P1-3). The procedures for lumbar spinal cord isolation and preparation of thin spinal cord slices were identical to those described previously (Gao and Ziskind-Conhaim 1995
). Before electrophysiological recording, the transverse slices (150 µm) were incubated at room temperature (21-23°C) for 1 h.
Slices were transferred to a recording chamber, which was fixed on the stage of an upright microscope (Nikon Labophot). The submerged slices were perfused with extracellular solution gassed with 95% O2-5% CO2 and maintained at room temperature. The extracellular solution contained (in mM) 113 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 25 NaHCO3, 1 NaH2PO4, and 11 glucose (pH 7.3).
Whole cell recordings
Motoneurons were identified as the largest cells in the lateral and medial ventral horn (Gao and Ziskind-Conhaim 1995
; Takahashi 1978
). Motoneurons were visualized at ×400 using Hoffman contrast optics. Recordings were carried out using a whole cell configuration of the patch-clamp recording technique (Hamill et al. 1981
). Patch electrodes were pulled from borosilicate glass capillaries using a Flaming-Brown puller (Sutter Instruments). The pipettes had tip diameters of 2-3 µm. The shank and tip of the electrodes were coated with silicone elastomer (Sylgard) to reduce pipette capacitance. Unless otherwise stated, the pipette solution contained (in mM) 140 KCl, 1 Mg-ATP, 0.1 GTP, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 0.2 ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid (pH 7.2). An Axopatch-1D amplifier (Axon Instruments) was used for current and voltage recordings.
After the formation of a giga seal, the pipette resistance and capacitance were compensated electronically. After the rupture of the membrane and the establishment of a whole cell voltage-clamp configuration, 10-mV hyperpolarizing pulses were applied to obtain capacitative transient currents from which the capacitance of the cell membrane and the pipette series resistance were calculated. Series resistance was estimated by measuring the decay time constant of the capacitive current; it ranged from 5 to 10 M
. Electronic compensation partially reduced this resistance by 50-80%.
Motoneuron capacitance was assessed from the integral of the capacitative current. This was possible because the average membrane resistances of embryonic (947 M
) and postnatal (734 M
) motoneurons were >70-fold higher than the series resistance. Membrane capacitances varied considerably, but they significantly lower in embryonic (29.3 ± 1.0 pF, mean ± SE, n = 65) than in postnatal motoneurons (33.2 ± 1.6 pF, n = 68, P < 0.05). It is likely that the average membrane resistance of postnatal motoneurons was overestimated and the membrane capacitance was underestimated, because ~10% of postnatal motoneurons were too large for adequate voltage clamping, and recordings from these neurons were discontinued (see following section).
Leakage currents were eliminated using either on-line subtraction of a series of currents generated by small hyperpolarizing pulses (P-N4 program, pClamp 5.5, Axon Instruments) or by subtracting a linearly scaled current elicited by a 10-mV depolarizing step. Current signals were digitized at 50 kHz, and filtered at 5 kHz.
Recordings were performed only in motoneurons in which fast inward currents were produced in response to depolarizing test potentials. The adequacy of the whole cell clamp recording was tested by examining the waveforms of the fast inward and slow outward currents generated at various test potentials (Fig. 1). Recordings were discontinued in ~10% of postnatal motoneurons that were clamped inadequately as evident by the delayed activation of inward currents or the generation of several peaks of inward currents in response to a single test potential.

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| FIG. 1.
Fast-rising inward currents followed by slow-rising, noninactivating outward currents in a postnatal day 1 (P1) motoneuron. Whole cell voltage-clamp technique was used to record the currents generated by a series of depolarizing test pulses to membrane potentials of 10 to +20 mV (in 10-mV increments). Holding potential (HP) was 70 mV.
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Data analysis
Peak amplitudes of action potentials generated by brief (30 ms) depolarizing currents were measured from a holding potential (HP) of
70 mV, and their durations were measured at threshold potential. Maximum rate of rise was estimated by dividing threshold-to-peak amplitude by the time to peak. Amplitude of AHP during a brief depolarizing current was measured as the peak of the repolarizing potential that was more negative than
70 mV (Table 1). During repetitive action potential firing in response to a prolonged current injection (150 ms), peak amplitude and duration of the first AHP were measured from the threshold potential (e.g., Fig. 11 and Table 4). Measurements were performed using pClamp software (Axon Instruments).

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| FIG. 11.
Blocking the high and small conductance Ca2+-dependent K+ channels increased action potential firing rate, while blocking IK and IA decreased it. Blocking the high IK(Ca) by charybdotoxin (100 nM) increased action potential duration, and decreased AHP duration and amplitude (also see Table 4). Blocking the small conductance IK(Ca) by apamin (100 nM) decreased AHP duration (see Table 4), which was sufficient to increase the rate of action potential firing. Blocking IK by tetraethylammonium (TEA, 30 mM) dramatically increased action potential duration and decreased AHP amplitude. Suppressing IA with 4-aminopyridine (4-AP, 2 mM) increased action potential duration but did not affect the AHP duration and amplitude (see Table 4). Experiments were carried out in 4 different motoneurons (P2-3), and action potentials were generated from a HP of 70 mV. In each experiment, the 1st action potentials in each firing train were overlapped (insets).
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Protocols for isolating specific voltage-gated ionic currents are described in RESULTS. The amplitude of the peak current, time to peak, and time to half-maximum peak current were calculated using pClamp software. Inactivation time constant was measured by fitting a single exponential curve on the initial falling phase of the current. Current density was estimated by dividing the peak amplitude of a specific current by motoneuron membrane capacitance. Data are presented as means ± SE. The statistical significance of the data were evaluated using Student's t-test.
 |
RESULTS |
Developmental changes in action potential properties
Action potential properties and the pattern of repetitive firing were examined in E15-16 and P1-3 motoneurons using whole cell current-clamp recordings. To correlate changes in action potential waveforms with the ionic currents underlying them, soma action potentials were generated from a membrane potential of
70 mV, the holding potential from which voltage-gated inward currents were produced. The following action potential properties were analyzed: action potential threshold, amplitude and duration, action potential maximum rates of rise and repolarization, and amplitude of the AHP. The results are summarized in Table 1.
Motoneurons were excitable at E15, but action potential waveforms were distinctively different from those produced after birth (Fig. 2A). Action potential thresholds shifted toward more negative potentials in postnatal motoneurons (Table 1), thus increasing the probability of generating an action potential.

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| FIG. 2.
Developmental changes in action potential waveforms shown in embryonic day 16 (E16) and P3 motoneurons. A: short-duration (30 ms) depolarizing currents were injected to generate single action potentials. These potentials were overlapped at their threshold potentials (inset) to demonstrate that the action potential amplitude increased while its duration decreased after birth. B: increasing the intensity and duration (0.1 nA, 150 ms) of the depolarizing currents generated an afterdepolarizing potential (ADP, ) at E16, and a sustained action potential firing at P3. Action potentials were produced from a HP of 70 mV (- - -).
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Action potential amplitudes significantly increased after birth (Fig. 2A, inset), when potentials overshooting 0 mV were recorded in all motoneurons. In contrast, overshooting action potentials were generated in only 40% of embryonic motoneurons. Concurrently, action potential duration declined from a mean of 9.3 ms at E15-16 to 3.4 ms at P1-3 (Fig. 2 and Table 1). The shorter duration resulted from four- to sevenfold increases in action potential maximum rates of rise and repolarization (Table 1).
During action potential repolarization in embryonic motoneurons, the membrane potential remained more positive than the potential from which the action potential was generated (
70 mV), but an AHP more negative than
70 mV was apparent in postnatal motoneurons (Fig. 2A). The mean amplitude of the AHP was 7.5 mV (Table 1), and its duration was 8.5 ms. Unlike action potential repolarization in spinal motoneurons of 3- to 10-day-old postnatal rats (Walton and Fulton 1986
), the AHP recorded in our study was not preceded by afterdepolarizing potential (ADP). Moreover, AHP waveform consisted of what appeared to be medium-duration AHP and could not be separated into distinctive fast- and medium-duration AHPs that characterize neurons in the sensorimotor cortex and brain stem of neonatal and mature mammals (Nishimura et al. 1989
; Schwindt et al. 1988
; Viana et al. 1993
).
Prolonged (150-ms) intracellular injection of depolarizing currents produced action potentials followed by large ADPs in embryonic motoneurons (Fig. 2B,
). Similar current injections generated repetitive action potential firing with intermittent AHPs in postnatal motoneurons.
Little is known about the ionic currents underlying the increase in motoneuron excitability during spinal cord development in embryonic and postnatal rats. In a few studies, the ionic mechanism was inferred from changes in action potential waveforms in response to blockers of voltage-gated ion channels (Walton and Fulton 1986
; Ziskind-Conhaim 1988a
). In the present study, a more direct experimental approach was adopted to examine the time course of differentiation and properties of voltage-gated macroscopic Na+, Ca2+, and K+ currents that are correlated with increased motoneuron excitability.
Voltage-gated Na+ currents
To isolate inward sodium currents (INa), outward K+ currents were blocked by replacing KCl in the pipette with isotonic CsCl and adding tetraethylammonium (TEA, 10-20 mM) and 4-aminopyridine (4-AP, 2 mM) to the extracellular solution. INa was separated from inward Ca2+ currents (ICa) by including either CdCl2 (0.5 mM) or CoCl2 (5 mM) in a low-Ca2+ extracellular solution (0.5 mM). Ca2+ was not removed from the extracellular solution, because stable recordings could not be sustained in its absence. Fast-rising inward currents were generated during depolarizing test pulses to potentials between
50 and +50 mV from a HP of
70 mV (Fig. 3). These currents were reversibly blocked by tetrodotoxin (TTX, 1 µM) or by substituting extracellular NaCl with equimolar concentration of Tris[hydroxymethyl] aminomethane hydrochloride (not shown), indicating that they were mediated by activation of voltage-gated Na+ channels. TTX-resistant Na+ currents, similar to those recorded in dorsal root ganglion and hippocampal neurons (Akopian et al. 1996
; Bossu and Feltz 1984
; Hoehn et al. 1993
) were not expressed in embryonic and postnatal motoneurons.

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| FIG. 3.
Development of inward Na+ currents. A: Na+ currents (INa) were generated during a series of depolarizing test pulses to membrane potentials of 30 to +10 mV (in 10-mV increments), applied from a HP of 70 mV. Na+ current (INa) rate of rise increased with increasing membrane depolarizations. Currents evoked at P1 were significantly larger than those produced at E16. B: voltage dependence of INa in embryonic and postnatal motoneurons. Similar bell-shape current-voltage relationships were apparent for Na+ currents at E15-16 and P1-3. Na+ currents were significantly larger after birth. Threshold for INa activation was about 40 mV at E15-16 and about 50 mV at P1-3. INa peaked at 10 mV. Values are means ± SE for n = 22-24.
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Similar INa waveforms were elicited in embryonic and postnatal motoneurons (Fig. 3A), but the threshold potential for INa activation was more negative in postnatal (
50 mV) than in embryonic motoneurons (
40 mV, Fig. 3B). The 10-mV potential change was similar to the 12 mV negative shift in action potential threshold (Table 1), suggesting that the latter was determined primarily by the change in threshold for INa activation. INa significantly increased after birth, which might reflect an increase in the number of Na+ channels, assuming that single channel conductance and its open probability remained unchanged. Concurrently, the time to peak INa decreased from 0.8 to 0.6 ms, but there was no change in the INa inactivation time constant (~1 ms, Table 2). Similar bell-shaped curves of INa amplitudes as a function of depolarizing test potentials were apparent in both embryonic and postnatal motoneurons, and INa peaked at
10 mV (Fig. 3B).
To determine whether the postnatal increase in INa resulted from a developmental increase in Na+ channel density, current densities were estimated by dividing peak INa amplitudes (generated at test potential of
10 mV) by motoneuron membrane capacitances. Our data demonstrated that peak INa densities increased threefold after birth, from an average of 38.9 pA/pF at E15-16 to 127.3 pA/pF at P1-3 (Table 2).
Voltage-gated Ca2+ currents
To record inward ICa, extracellular Ca2+ concentration was elevated to 10 mM. Inward INa was suppressed by TTX (1 µM), and outward IK was blocked by replacing KCl in the pipette with CsCl and adding TEA and 4-AP to the extracellular solution (see preceding section). Relatively small, slow-rising inward currents were produced by depolarizing test pulses to potentials between
40 and +50 mV from a HP of
70 mV (Fig. 4). Those currents were blocked by either CdCl2 (0.5 mM) or CoCl2 (5 mM), confirming that they were Ca2+-dependent currents.

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| FIG. 4.
Amplitude of inward Ca2+ currents significantly increased after birth. A: Ca2+ currents were generated by depolarizing pulses to membrane potentials of 10 to +10 mV (in 10-mV increments), applied from a HP of 70 mV. Small Ca2+ currents were present at E15, but larger currents were recorded after birth. At both ages, the Ca2+ current (ICa) consisted of fast-inactivating and relatively slow-inactivating components. ICa amplitude was measured as the peak fast-inactivating current ( ). B: voltage dependence of ICa during motoneuron differentiation. Similar current-voltage relationships were apparent at E15-16 and P1-3, but significantly larger ICa were generated in postnatal motoneurons at depolarizing test potentials more positive than 20 mV. ICa began to activate at 30 mV and peaked at 10 mV. Values are means ± SE for n = 9-14.
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ICa consisted of two components: relatively fast-inactivating and slow-inactivating currents (Fig. 4A). ICa amplitude was measured as the peak of the transient fast-inactivating current. Similar bell-shaped curves of ICa amplitude versus depolarizing test potentials were apparent in both embryonic and postnatal motoneurons. Throughout this developmental period, the threshold for ICa activation was about
35 mV, and the current peaked at +10 mV (Fig. 4B). Similar to INa, ICa density significantly increased after birth, and at +10 mV, it was threefold larger than in embryonic motoneurons (Table 2). Comparison of ICa and INa densities demonstrated that in both embryonic and postnatal motoneurons, peak ICa densities were fourfold smaller than INa densities. The mean time to 50% peak ICa was faster after birth, and at test potential to +10 mV, it changed from 1.6 ms in embryonic motoneurons to 0.8 ms in postnatal motoneurons (Table 2). The time constant of inactivation of the transient current did not significantly change after birth, and values of 23.2 and 17.2 ms were measured at E15-16 and P1-3, respectively (Table 2).
At least four functionally and pharmacologically distinct Ca2+ currents have been described in neurons: N-, L-, T-, and P/Q-type currents (Bean 1989
; Dunlap et al. 1995
; Huguenard 1996
; Snutch and Reiner 1992
). The kinetics of these currents differ so that N-, L-, and P/Q-type currents consist of fast- and slow-inactivating components, whereas T-type ICa current inactivates relatively fast (McCobb et al. 1989
). Based on the relatively large, slow-inactivating ICa component that was recorded in our study, we hypothesized that ICa might be composed of N-, L-, and/or P/Q-type Ca2+ currents. To determine the contribution of N-type channel to ICa, ICa was recorded in the presence of
-conotoxin GVIA (1 µM, n = 4), a strong blocker of N-type ICa. The toxin reduced ICa amplitude by ~70% suggesting that ICa was mediated primarily by N-type Ca2+ channels. To examine the contribution of L-type channel to ICa, Ca2+ currents were recorded in the presence of nifedipine (100 µM, n = 7) and verapamil (100 µM, n = 7), blockers of L-type ICa. Nifedipine reduced ICa by 6-10%, whereas verapamil did not affect it, suggesting that the L-type current did not contribute significantly to ICa generation. Unlike our finding, verapamil reduced the amplitude of the sustained ICa by ~50% in embryonic chick motoneurons (McCobb et al. 1989
).
It is conceivable that the 20% of ICa that was not suppressed by blockers of N- and L-type Ca2+ channels consisted of P/Q-type current (Churchill and MacVicar 1998
; Mintz et al. 1992
), but its contribution was not tested using
-agatoxin-IVA, a specific blocker of the P/Q-type channel. It is unlikely that the contribution of T-type current was significant, because as much as 50% of this current can be inactivated at a HP of
70 mV (Li et al. 1998
).
Voltage-gated K+ currents
To record voltage-gated Ca2+-independent K+ currents, inward INa and ICa were blocked by including TTX (1 µM) and CdCl2 (0.5 mM) or CoCl2 (5 mM) in a low-Ca2+ extracellular solution (0.5 mM). Outward K+ currents were produced from a HP of
80 mV by depolarizing test pulses to potentials between
40 and +50 mV. The outward currents consisted of a transient inactivating and a relatively sustained, noninactivating components (Fig. 5A).

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| FIG. 5.
Large transient A-type K+ currents (IA) were elicited in embryonic motoneurons, and their amplitude did not increase after birth. A: total K+ currents (control, left) were generated in an E16 motoneuron by a series of depolarizing pulses to membrane potential of 30 to +50 mV (in 20-mV increments) applied from a HP of 80 mV. To isolate the transient K+ currents from the noninactivating delayed rectifier-type currents (IK), the latter were produced at the same depolarizing pulses that were preceded by a short (35 ms) prepulse to 40 mV (middle). At each depolarizing test potential, IA was obtained by subtracting IK from total K+ current (right). B: similar IA voltage dependence was apparent in E15-16 and P1-3 motoneurons. Threshold for IA activation was 30 mV. Values are means ± SE for n = 12-14.
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TRANSIENT K+ CURRENTS.
Isolation of transient K+ current was based on previous reports illustrating that this current is partially or completely inactivated at potentials more positive than
50 mV (McCobb et al. 1990
; Nerbonne and Gurney 1989
; Ribera and Spitzer 1990
). Therefore, K+ currents were first generated from a HP of
80 mV and then from a brief prepulse to
40 mV. Activation of K+ channels from a potential of
40 mV produced only noninactivating currents (Fig. 5A). Subtraction of the noninactivating currents from total K+ currents revealed distinct transient currents that were almost completely inactivated during the 60-ms depolarizing pulses. These transient currents were partially blocked by 4-AP (60-90%, 2 mM), suggesting that they were similar to A-type K+ currents (IA).
Plotting peak IA amplitudes as a function of depolarizing test pulses illustrated similar I-V relationships in embryonic and postnatal motoneurons (Fig. 5B). Relatively large transient K+ currents were generated in embryonic motoneurons (>1 nA at 0 mV), and their amplitudes did not further increase after birth. It is unlikely that the channel kinetics changed after birth, because the mean time to peak IA (2.7-2.9 ms at E15-16 and P1-3, Table 3) and the decay time to 50% of peak current (9.8-13.9 ms) remained relatively unchanged.
NONINACTIVATING K+ CURRENTS.
Noninactivating K+ currents were produced by depolarizing test pulses from a HP of
40 mV (Fig. 6A). These currents were substantially reduced by TEA (60-75%, 30 mM), suggesting that they were similar to the delayed rectifier-type K+ current (IK) (Del Negro and Chandler 1997
; McCobb et al. 1990
). From a HP of
40 mV, the threshold potential for activation of IK channel was
10 mV, and it did not change after birth (Fig. 6B). Unlike IA, IK amplitude increased twofold postnatally, and during a depolarizing pulse to 10 mV, for example, mean IK amplitude increased from ~0.5 nA in embryonic motoneurons to ~1.0 nA in postnatal motoneurons (n = 14-17, Fig. 6B). Similar increases were apparent in peak IK density (Table 3).

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| FIG. 6.
Amplitude of noninactivating IK increased after birth. A: IK were generated in E15 and P2 motoneurons during depolarizing pulses to membrane potentials of 10 to +50 mV (in 10-mV increments), applied from a HP of 40 mV. IK rising phase consisted of fast and slow components, and its amplitude was measured at the end of the depolarizing pulse (30 ms). Larger IK were elicited in the P2 motoneuron. B: current-voltage relationships in E15-16 and P1-3 motoneurons demonstrated that significantly larger IK were evoked after birth at depolarizations more positive than 0 mV. Threshold for IK was 10 mV. Values are means ± SE for n = 14-15.
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VOLTAGE-GATED CA2+-DEPENDENT K+ CURRENT.
Extracellular Ca2+ is required to trigger Ca2+-dependent IK (IK(Ca)), but Ca2+ concentration in the extracellular solution was reduced (0.5 mM) to decrease the amplitude of inward ICa, which peaked at about the same time as the outward IK(Ca) (Tables 2 and 3). Under these experimental conditions, the amplitude of inward ICa was <80 pA, and it was not subtracted from the outward current. Therefore, it is possible that IK(Ca) amplitude was underestimated by <80 pA. IK(Ca) was isolated from total K+ current by first generating a series of K+ currents from a HP of
80 mV (Fig. 7A, control), followed by the generation of Ca2+-independent K+ currents in the presence of CdCl2 (0.5 mM, n = 14). IK(Ca) was measured by subtracting Ca2+-independent K+ current from the total current (Fig. 7A).

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| FIG. 7.
Amplitude of the Ca2+-dependent K+ currents (IK(Ca)) significantly increased after birth. A: total K+ currents were generated in a P3 motoneuron by depolarizing pulses to membrane potentials of 30 to +30 mV (in 20-mV increments), applied from a HP of 80 mV (control, left). Ca2+-independent K+ currents were elicited in the presence of Cd2+ (0.5 mM, middle), and the latter were subtracted from total currents to obtain IK(Ca) (right). Calibration bars: 2 nA for control currents and those recorded in the presence of Cd2+ and 1 nA for IK(Ca). B: voltage dependence of IK(Ca) changed during development. At E15-16, IK(Ca) amplitude slightly increased at depolarizing pulses to potentials between 20 and +10 mV and remained relatively constant at depolarizing pulses to potentials more positive than +10 mV. In contrast, almost linear increase in IK(Ca) was apparent in P1-3 motoneurons. At both ages, the threshold for IK(Ca) activation was 30 mV. Values are means ± SE for n = 9-10.
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The threshold for IK(Ca) was about
20 mV in embryonic motoneurons, and about
30 mV in postnatal motoneurons (Fig. 7B). Plotting peak IK(Ca) amplitude as a function of depolarizing test pulses demonstrated that in embryonic motoneurons IK(Ca) reached a plateau (0.3 nA) at a potential of ~0 mV. Depolarizations to potentials more positive than 10 mV only slightly increased IK(Ca) amplitude (0.4 nA at +40 mV, Fig. 7B). In contrast, IK(Ca) amplitude increased almost linearly with depolarizing pulses in postnatal motoneurons, resulting in significantly larger current density after birth (Table 3). The larger postnatal IK(Ca) might be partially attributed to the developmental increase in inward ICa. In embryonic and postnatal motoneurons, ICa and IK(Ca) were significantly smaller than the rest of the ionic currents studied.
Unlike the large increase in IK(Ca) amplitude, there was no appreciable developmental change in its time to peak (Table 3). For example, during a depolarizing pulse to potential of +10 mV, the times to peak IK(Ca) were 3.0 and 3.3 ms at E15-16 and P1-3, respectively.
Ionic mechanism underlying action potential generation
Developmental changes in action potential waveforms and the ionic currents underlying them are summarized in Fig. 8. The action potentials and the fast-rising INa peaked at about the same time. The relatively slow-rising ICa peaked during action potential repolarization, implying that it did not contribute to the action potential rising phase. The sustained IK(Ca) and the transient IA peaked during action potential repolarization but before the onset of AHP. Peak AHP was correlated with maximal IK(Ca), large IK, and reduced IA.

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| FIG. 8.
Summary of the changes in action potential waveform and properties of voltage-gated inward and outward ionic currents in embryonic and postnatal motoneurons. Traces are from different motoneurons. Action potentials were generated from a HP of 70 mV (- - -) using suprathreshold stimuli (0.1 nA, 30-ms duration). Ionic currents were elicited by depolarizing test pulses to 10 mV. Fast-rising phase of action potentials was aligned with the stimulus artifacts of the ionic currents.
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The contribution of inward and outward ionic currents to the action potential waveform and repetitive firing behavior was determined by examining the actions of specific blockers of voltage-gated ion channels on action potential properties.
INWARD CURRENTS.
Blocking Na+ channels with TTX (1 µM) reversibly suppressed action potential generation (n = 5). This finding suggested that during early stages of development of motoneuron excitability, action potential rising phase was dependent primarily on Na+ influx. Although Ca2+ channels were expressed early in motoneuron development, Ca2+ influx could not functionally substitute for Na+ influx in triggering an action potential. It is likely that the charge carried by the relatively slow-rising, small Ca2+ influx (Fig. 8) was effectively counterbalanced by the large K+ efflux. Indeed, only when both Na+ and K+ channels were blocked did depolarizing pulses generate slow-rising, prolonged (>20 ms) action potentials that were blocked by CoCl2 (not shown, but see Ziskind-Conhaim 1988a
).
To evaluate the role of ICa in modulating action potential waveforms, Ca2+ channels were blocked by either CdCl2 (0.5 mM) or CoCl2 (5 mM). At E16, blocking ICa decreased the action potential duration from a mean of 6.4 to 4.1 ms (n = 5, Fig. 9A, inset), suggesting that Ca2+ influx prolonged action potential duration. Blocking ICa greatly reduced the ADP recorded in embryonic motoneurons (Fig. 9A,
), indicating that the ADP was mediated by ICa. After birth, the shorter duration action potential (3.4 ms, n = 7) was not affected by Ca2+ channel blockers (3.2 ms, n = 7, Fig. 9B), probably because of the development of large K+ currents (Fig. 8) that played a more prominent role than ICa in regulating action potential duration. The most striking effect of blocking Ca2+ channels in postnatal motoneurons was the twofold decrease in AHP duration, which was associated with an almost twofold increase in action potential firing rate (Fig. 9B). It is likely that this effect was related to the suppression of IK(Ca) (see next section).

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| FIG. 9.
Effects of blocking ICa on action potential waveforms in E16 and P1 motoneurons. A: at E16, a prolonged (150 ms) current injection generated only 1 action potential that overshot the 0 potential and was followed by ADP ( ). Action potential duration was reduced and the ADP was eliminated in the presence of Co2+ (5 mM). Threshold potential was slightly more positive in the presence of Co2+ presumably because of the surface charges generated by the high concentration of Co2+. Action potentials were overlapped at their threshold potentials to illustrate that blocking ICa reduced action potential duration (inset). B: sustained action potential firing was produced in a P1 motoneuron. Blocking ICa by Cd2+ (0.5 mM) did not affect action potential duration (inset), but it decreased AHP duration and increased the rate of action potential repetitive firing.
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K+ OUTWARD CURRENTS.
To examine the effect of blocking K+ currents on action potential generation, CsCl was substituted for KCl in the pipette solution (Fig. 10). Suppressing K+ efflux significantly increased action potential duration in both embryonic and postnatal motoneurons. At E15-16, mean action potential duration increased fivefold in the presence of CsCl, changing from 9.3 ms (Table 1) to 45.4 ms (n = 4). After birth the effect of CsCl was markedly larger, and an increase of more than eightfold was apparent: from 3.4 to 29.3 ms (n = 7; Table 1). Blocking K+ currents eliminated the AHP in postnatal motoneurons and prevented the generation of sustained action potential firing, confirming the importance of K+ currents in regulating firing behavior.

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| FIG. 10.
Blocking outward K+ currents prolonged action potential duration and suppressed the sustained action potential firing in a P2 motoneuron. A: short-duration (30 ms) depolarizing current generated a single action potential, whereas long-duration (150 ms) current injection elicited a sustained burst of action potentials in a P2 motoneuron. B: in a different P2 motoneuron, K+ currents were blocked by replacing KCl in the pipette solution with CsCl. Inhibition of outward K+ currents substantially prolonged action potential duration and blocked the AHP and action potential repetitive firing.
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The specific contribution of IK(Ca), IK, and IA to action potential waveforms and repetitive firing was studied by selectively blocking those currents. Ca2+-activated K+ channels can be separated into two families of high (BKCa) and small (SKCa) conductance channels (reviewed by Sah 1996
), which can be selectively blocked by charybdotoxin and apamin, respectively. Charybdotoxin (100 nM, n = 6) significantly increased action potential duration, shortened AHP duration and decreased its amplitude (Table 4). Similar to the effect of blocking ICa, charybdotoxin markedly increased action potential firing rate (Fig. 11). Apamin (100 nM, n = 9) significantly reduced AHP duration but only slightly affected action potential duration and AHP amplitude. Apamin-induced shorter AHP duration was sufficient to significantly increase the rate of action potential firing. Blocking IK with TEA (30 mM, n = 7) had the most profound effect on prolonging action potential duration and decreasing AHP amplitude (Fig. 11, Table 4), and these changes resulted in a substantially reduced frequency of repetitive action potentials. Blocking the transient IA with 4-AP (2 mM, n = 5) significantly increased action potential duration and effectively reduced the frequency of repetitive firing in most motoneurons (4/5) (Fig. 11, Table 4). These findings suggested that blocking IK(Ca) had opposite effect on firing behavior than blocking IK and IA, and the effects were primarily modulated by reducing AHP duration and increasing action potential duration, respectively.
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DISCUSSION |
Developmental changes in action potential waveforms and properties of voltage-gated ionic currents were examined in rat spinal motoneurons during the last week in utero and the first few days after birth. This period is characterized by marked changes in motoneuron properties (Fulton and Walton 1986
; Gao and Ziskind-Conhaim 1995
; Mandler et al. 1990
; Takahashi 1990
; Takahashi et al. 1992
; Walton and Navarrete 1991
; Ziskind-Conhaim 1988a
), the formation of sensorimotor pathways (Gao et al. 1998
; Kudo and Yamada 1987
; Nishimaru et al. 1996
; Saito 1979
; Seebach and Ziskind-Conhaim 1994
; Wu et al. 1992
; Ziskind-Conhaim 1990
), and the establishment of peripheral synaptic connections with the target muscle fibers (Dennis et al. 1981
; reviewed by Grinnell 1995
). To determine the ionic mechanism underlying developmental changes in action potential waveforms, we examined the properties and time course of expression of voltage-gated macroscopic Na+, Ca2+ and K+ currents during early stages of differentiation of motoneuron excitability in spinal cord developing in vivo. Most previous studies have characterized the properties of voltage-gated ionic currents in rodent spinal neurons developing in culture (Heyer et al. 1981
; Krieger and Sears 1988
; MacDermott and Westbrook 1986
; Rogawski and Barker 1983
).
Voltage and current measurements were performed using the whole cell technique either in current- or in voltage-clamp mode. A possible complication of the whole cell technique is intracellular dialysis from the electrode, resulting in diffusion of important intracellular constituents into the pipette solution (Pusch and Neher 1988
). However, continuous recordings for periods >60 min showed no evidence for time-dependent changes in action potential waveform and pattern of repetitive firing, implying that intracellular dialysis affected neither the ionic basis of action potential generation nor motoneuron membrane properties. A previous study comparing membrane properties using whole cell and high-resistance electrode recordings illustrated that intracellular dialysis did not affect membrane resting potential, input resistance, or time constant in adult dentate granule cells (Staley et al. 1992
).
Development of motoneuron excitability
The changes in action potential properties and repetitive firing behavior reported in this study are similar to those documented in developing motoneurons using high-resistance intracellular electrodes, but some differences in the absolute values were apparent (Xie and Ziskind-Conhaim 1995
; Ziskind-Conhaim 1988a
). For example, estimated membrane resistances were 8- to 10-fold larger and action potential amplitudes were >20 mV larger when recorded with whole cell electrodes compared with high-resistance electrodes (Xie and Ziskind-Conhaim 1995
, Table 1). These differences, at least in part, could be attributed to the nonspecific leak conductance associated with high-resistance electrodes (Staley et al. 1992
). Another probable factor contributing to these differences is the potential from which action potentials were generated. In this study, action potentials were elicited from a membrane potential of
70 mV, whereas in our previous studies they were generated from variable resting membrane potentials, ranging from
58 mV at E15 to
65 mV at P1-3 (Xie and Ziskind-Conhaim 1995
).
In embryonic motoneurons, prolonged current injections generated relatively slow-rising Na+-dependent action potentials and ADPs, whereas similar currents produced sustained firing in postnatal motoneurons. It is conceivable that the persistent depolarization during action potential repolarization increased the probability of prolonging Na+ channel inactivation, therefore decreasing the likelihood of generating more than one action potential. The inability to fire repetitively might be functionally advantageous in limiting the contraction forces generated by developing muscle fibers. During embryonic and early postnatal development, muscle fibers are multiply innervated and electrically coupled (Dennis et al. 1981
), and single-nerve stimulation produces wide-spread muscle contraction. The inability to generate sustained action potential firing is correlated functionally with the expression of predominantly slow myosin heavy chains in the developing muscles (Hughes et al. 1993
).
Our findings demonstrated that the ionic dependence of the action potential did not change during early ages of motoneuron development. From the onset of motoneuron excitability, action potentials were largely Na+ dependent, and the shorter duration action potential and the appearance of AHP were correlated with increased K+ currents densities. Similar findings have been reported in other developing neurons including grasshopper interneurons (Goodman and Spitzer 1981
), chick ciliary ganglion neurons (Bader et al. 1983
), rat sympathetic ganglion cells (Nerbonne and Gurney 1989
), and mouse and chick spinal neurons (Krieger and Sears 1988
; McCobb et al. 1990
). In contrast, developmental changes in action potential properties resulted from changes in the ionic currents in spinal neurons of Xenopus embryos and cortical neurons from chick embryos. In those neurons, long-duration Ca2+-dependent action potentials were predominant at the onset of membrane excitability, and short-duration Na+-dependent potentials developed within a short time afterward (Baccaglini and Spitzer 1977
; Blair 1983
; Mori-Okamoto et al. 1983
; Spitzer and Lamborghini 1976
).
Inward currents
Both Na+ and Ca2+ channels were expressed at early stages of motoneuron development, but only Na+ influx was necessary and sufficient for action potential generation. Developmental increases in action potential maximum rate of rise and peak amplitude were attributed to an increase in INa density. This is a developmental trend that has been documented in most differentiating cultured neurons (Bader et al. 1983
; MacDermott and Westbrook 1986
; McCobb et al. 1990
; Willard 1980
). In some cases, such as in sympathetic neurons, INa densities as well as the kinetics of INa activation and inactivation did not change during the period studied (Nerbonne and Gurney 1989
).
Throughout embryonic and early postnatal development, Ca2+ currents were relatively small compared with Na+ and K+ currents. It is unlikely that dominant Ca2+ currents were expressed in motoneurons of rodents younger than E15, because such currents could not be detected in spinal neurons of E12 mice (Krieger and Sears 1988
) and were apparent in motoneurons of E14 rats only when extracellular Ca2+ was elevated (Ziskind-Conhaim 1988a
). Restricting Ca2+ influx might be related to complex cellular mechanisms regulating the fine balance of transient increase in cytoplasmic Ca2+ that promotes neuronal differentiation and synaptogenesis (Fields and Nelson 1993
; Gosh et al. 1993
; Spitzer 1994
) and prevents Ca2+-induced neurotoxicity and cell death (Choi 1992
).
At the onset of membrane excitability, ICa seemed to play an important role in regulating action potential duration and repolarization. Our findings that the ADP was Ca2+ dependent confirmed previous observations in spinal and hypoglossal motoneurons of neonatal rats (Umemiya and Berger 1994
; Walton and Fulton 1986
). Although ICa increased postnatally, it no longer affected action potential duration, probably because of the development of relatively larger counteracting outward K+ currents. It is likely that the contribution of Ca2+ inflow to action potential waveform in postnatal motoneurons was related to the increase in IK(Ca) and the production of predominant AHP (Nishimura et al. 1989
; Viana et al. 1993
; Walton and Fulton 1986
). The functional contribution of ICa to various phases of the action potential might be mediated by the activation of distinct populations of voltage-gated Ca2+ channels (Umemiya and Berger 1994
).
Outward currents
Each of the three types of K+ currents examined in this study had a different role in regulating action potential duration, AHP waveform, and the pattern of motoneuron firing. Transient IA was the largest K+ current expressed in embryonic motoneurons, but neither its amplitude nor its time to peak changed after birth. High ratio between IA and IK during early neuronal development also was reported in gyrus granule cells of postnatal rats (Beck et al. 1992
) and neurons from quail mesencephalic neural crest (Bader et al. 1985
). However, in embryonic chick and Xenopus spinal neurons, the expression of IA lags behind IK (Desarmenien et al. 1993
; McCobb et al. 1990
). Time to peak IA was similar to the time to peak ICa, therefore it is reasonable to assume that its early expression contributed to the relatively short-duration action potential (<10 ms) in embryonic motoneurons. Although IA influenced action potential duration after birth, it was probably the postnatal expression of the large noninactivating IK that resulted in the shortening of action potential duration. The roles of IA and IK seemed to be reversed in other immature neurons such as sympathetic neurons, in which the shorter duration action potential was attributed to the increase in IA (Nerbonne and Gurney 1989
).
Repetitive firing behavior is one of the properties that distinctly characterizes various neuronal populations, and it has been suggested that the firing pattern is determined primarily by the balance between persistent inward and outward currents (Schwindt and Crill 1982
). In rat spinal motoneurons, repetitive firing began at birth and was correlated temporally with large increases in the expression of IK and IK(Ca). IK and IA effectively increased the rate of repetitive firing by shortening action potential duration. IK(Ca) significantly increased after birth, and both high and small conductance Ca2+-dependent K+ channels modulated the AHP duration and reduced the frequency of action potential repetitive firing. Blocking these channels increased the firing rate, similar to the elevated frequency that was apparent during the suppression of inward ICa. The opposite roles of K+ currents in regulating the pattern of action potential firing are likely to provide a fine-tuning mechanism for balancing motoneuron output. Opposite modulatory actions of different types of K+ channels on neuronal discharge have been reported in other populations of developing neurons. Unlike our findings, in rat mesencephalic trigeminal neurons, reducing IA increased the probability of generating a sustained firing, whereas blocking IK eliminated the repetitive firing in most neurons (Del Negro and Chandler 1997
). The different roles of K+ channels in regulating action potential firing behavior might be attributed to their relative amplitudes and subtle differences in their kinetic properties in various neuronal populations.
In summary, the ionic dependence of action potentials generated in spinal motoneurons of immature rats was studied from the onset of their excitability to the age of establishment of repetitive action potential firing. Our finding suggested that changes in the relative roles of a repertoire of ion channels that already were expressed in embryonic motoneurons accounted for the developmental changes in action potential waveforms and the establishment of distinctive pattern of repetitive firing behavior.