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Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada
Submitted 16 January 2008; accepted in final form 7 April 2008
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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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; Whelan 2003). For example, rodents are capable of swimming and crawling at birth; however, the ability to raise the body off the ground and walk does not develop fully until the second or third postnatal week (Brocard et al. 1999; Jiang et al. 1999a; Westerga and Gramsbergen 1990). One important feature of postnatal neuromuscular development is the matching of motoneuron discharge rates with the twitch properties of the muscles they innervate (Vrbova et al. 1985). This matching likely ensures the force generation in limb muscles necessary for the production of overground locomotion (Martin-Caraballo et al. 2000). As the muscle twitch time decreases during postnatal development (Close 1964) a corresponding increase in the motoneuronal firing rate is required.
During the late embryonic and early postnatal periods, many types of motoneurons demonstrate an increase in maximal firing rate and duration of repetitive firing. This developmental phenomenon has been described in phrenic (Greer and Funk 2005; Martin-Caraballo and Greer 1999), oculomotor (Carrascal et al. 2006), hypoglossal (Viana et al. 1995), and spinal motoneurons (Fulton and Walton 1986; Vinay et al. 2000; Vrbova et al. 1985). This ability of motoneurons to discharge action potentials at an increasing rate has been attributed to various factors including an increase in sodium current density (Gao and Ziskind-Conhaim 1998; Garcia et al. 1998; McCobb et al. 1990), development of repolarizing conductances (McCobb et al. 1990; Viana et al. 1994; Vinay et al. 2000), afterhyperpolarization amplitude increase (Fulton and Walton 1986), the development of calcium channels (McCobb et al. 1989; Miles et al. 2004; Mynlieff and Beam 1992), or changes in modulatory inputs (reviewed in Kernell 2003; Schmidt and Jordan 2000). In addition to an increase in firing frequency, a second defining characteristic of mature spinal motoneurons is their ability to generate plateau potentials (Perrier and Hounsgaard 2000). When the conductances underlying the plateau are activated, they create a second quasi-stable potential approximately 10–20 mV more positive to the resting potential (Hounsgaard and Kiehn 1989; Hounsgaard et al. 1986). Depending on the class of motoneuron, this underlying depolarization can persist for many seconds (Lee and Heckman 1998). For a cell to fire a train of action potentials at such depolarized membrane voltages, sodium channels must resist entering an inactivated state so as to remain available for activation. In the present study we assess the biophysical properties of the sodium current generating the action potential as a potential mechanism contributing to postnatal development of the firing properties of spinal motoneurons.
In the mammalian nervous system, voltage-gated sodium currents can be generated by nine different channel subtypes (Catterall et al. 2003), each displaying some unique biophysical properties (Rush et al. 2007). The sodium channel subtype expression in mammalian spinal cord changes during the early postnatal period as demonstrated by studies examining expression at either the protein (Gordon et al. 1987; Schaller and Caldwell 2000) or mRNA level (Beckh et al. 1989; Felts et al. 1997; Garcia et al. 1998; Schaller and Caldwell 2000). Some of these changes have been directly localized to the spinal motoneurons (Schaller and Caldwell 2000) and have been supported by electrophysiological data from mice expressing a spontaneous mutation in a late-developing sodium channel subtype (med mutant; Garcia et al. 1998). Taken together, these data suggest that a change in the biophysical characteristics of the sodium channels may, in part, be responsible for the change in firing properties of developing spinal motoneurons. However, this type of kinetic data is absent from the literature on motoneurons, in all likelihood due to the inherent difficulties associated with voltage- and space-clamping the sodium current in these cells (e.g., Garcia et al. 1998).
In the present study we circumvent these technical difficulties by using dissociated postnatal lumbar spinal motoneurons cultured overnight in conditions that did not promote process formation. We then tested the hypothesis that the ensemble sodium current demonstrates age-related biophysical changes consistent with the known mature firing pattern of these cells. Specifically, we assessed the ability of the maturing complement of sodium channels to resist accumulating in an inactivated state during repetitive depolarizing stimulations that mimicked repetitive firing and evaluated the tendency of these channels to undergo closed-state inactivation during prolonged depolarizations similar to the voltage change sensed by these channels during the expression of a plateau potential.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Mixed spinal cord cultures were prepared from the lumbar spinal cords of BALB/c mice (P1–P12, where P0 is the day of birth). Motoneurons were identified in the cultures by prelabeling these cells through an intraperitoneal injection of the fluorescent maker Fluoro-Gold (10–20 µl of a 2% solution; Fluorochrome, Denver, CO) 
24 h prior to sacrifice (Miles et al. 2005). Spinal cords were isolated and transverse slices prepared from the lumbar cord (
L1 to L5 segments) as described previously (Carlin et al. 2000a). Care was taken to ensure that there were no adherent dorsal root ganglia. This resulted in spinal cord slices in which labeling was restricted to ventral motoneurons (Fig. 1 A). After removing the agar from the periphery of the slices, the slices were placed in a calcium- and magnesium-free room temperature (RT; 22°C) Earle's salt solution supplemented with 10 mM HEPES (pH 7.4). The slices were then digested with trypsin (0.25%; Invitrogen, Carlsbad, CA) for 20 min at RT with frequent agitation of the solution. The slices were then rinsed twice with RT Leibovitz's L-15 media (Invitrogen; Anderson et al. 2004) and triturated with a fire-polished Pasteur pipette. The cell-containing solution was then transferred to 35-mm culture dishes (tissue culture treated; Corning #430165) and supplemented with a penicillin/streptomycin solution (1%; Invitrogen) and B-27 nutritional supplement (1%; Invitrogen). The plated cells were then incubated overnight at 35°C in a humidified air environment. These culture conditions were optimized to reduce the formation of processes and at the same time promote healthy cells suitable for assaying voltage-gated sodium channel function. All experimental protocols used in this study followed the guidelines set by the Canadian Council on Animal Care and the University of Manitoba.
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After approximately 18–24 h of incubation the culture media was replaced with the external recording solution designed to isolate sodium currents. The culture dishes were then placed directly on the stage of an inverted Nikon Diaphot 300 inverted microscope with a Nikon UV-2A filter set. To limit potential phototoxic damage to labeled cells, large, healthy-looking, round cells were first identified under brightfield illumination, then quickly exposed to the fluorescent light to determine whether labeling was present (Fig. 1B). It is interesting to note that many large cells were identified in these cultures that lacked the fluorescent label and therefore recordings were not made from these cells. This observation is consistent with our previous observations (Carlin et al. 2006; Huang et al. 2000) as well as the observations of others (Thurbon et al. 1998) of large non-motoneuronal cells in the lumbar cord. Whole cell recordings were made from Fluoro-Gold–positive cells using an Axopatch 1D amplifier, DigiData1200 A/D converter, and Clampex 8 software (Axon Instruments, Union City, CA). A holding potential of –70 mV was used for all experiments. This potential was used because it was well tolerated by the cells and it likely produced a pseudophysiological amount of steady-state sodium channel inactivation, thereby increasing the physiological relevance of these data. Data were captured at 50 kHz and low-pass filtered at 5 kHz. The borosilicate glass pipettes had resistances between 1.5 and 2.8 M
when filled with intracellular solution. Series resistance was compensated as required so that the calculated voltage error did not exceed 6 mV. Using a flowing KCl electrode (Neher 1992), a liquid junction potential of –4 mV was determined, which made the true potentials more negative than those reported. All experiments were performed at RT (22°C). For current traces in figures, depolarizing and hyperpolarizing capacitive transients have been either removed or truncated for clarity.
Solutions and chemicals
The intracellular solution contained (in mM): CsMeSO3 (125), TEA-Cl (30), NaCl (5), MgCl2 (1), CaCl2 (0.5), HEPES (10), EGTA (10) Mg-ATP (3), and GTP (0.3). The solution pH was adjusted to 7.2. The extracellular solution contained (in mM): NaCl (124), TEA-Cl (30), MgCl2 (2), CaCl2 (2), HEPES (10), 4-AP (4), CsCl (3), glucose (10), KCl (3), CdCl2 (0.3), and NiCl2 (0.1). The solution pH was adjusted to 7.4. Earle's salt solution contained (in mM): NaCl (117.24), KCl (3.96), NaHCO3 (26.19), NaH2PO4 (0.522), and HEPES (10). The solution pH was adjusted to 7.4. All chemicals were purchased from Sigma (Sigma–Aldrich Canada, Oakville, Ontario, Canada).
Data analysis
Current traces were analyzed using Clampfit v.9 software (Axon Instruments). Statistical analysis and curve fitting were performed with Microsoft Excel, Microcal Origin 6.0, and SigmaPlot 9.0 (Systat) software programs. Activation curves were constructed using the calculated sodium reversal potential of 83 mV. Both the activation and steady-state inactivation conductance data were fitted with a single Boltzmann equation of the form: G/Gmax = Gmin + (Gmax – Gmin)/{1 + exp[(V1/2 – x)/k]}, where Gmax is the maximal conductance value, gmin is the minimal conductance value, k is the slope factor, and V1/2 is the voltage of the half-maximal conductance value. Recovery and closed-state inactivation data were fitted with double-exponential functions of the form: y = {A1 x [1 – exp(–x/
1)]} + {A2 x [1 – exp(–x/2)]}, where 1 and 2 are the fast and slow time constants. These curves were compared by taking the mean of the raw normalized data with the exclusion of the first point over the time period indicated in the text.
Parametric statistics were used to assess differences between age groups if the data were normally distributed (Kolmogorov–Smirnov test for normality) and groups were of equal variance. If these criteria were not met, groups were compared using the Kruskal–Wallis one-way ANOVA on ranks. Cell capacitance was determined from the mean of 10–18 current transients integrated over 1 ms divided by the 10-mV step that elicited the transients. Current density was obtained by dividing the cell capacitance by the peak current elicited by a voltage step to –10 mV from a 100-ms x –120-mV hyperpolarization.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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As a first step in exploring developmental changes in sodium channel functioning we examined the voltage dependence of activation and inactivation of channel gating because these parameters can have dramatic effects on channel availability and gating at a given membrane potential. The voltage dependences of activation and fast inactivation of the sodium channels were measured using standard voltage protocols (Fig. 2, A and B). Both the activation and inactivation conductance–voltage relationships were fitted well with a single Boltzmann function. The average V1/2 values and slope (k) values of both the activation and inactivation fits were not significantly different across the measured developmental period (Table 1). Given the remarkable consistency of these parameters across the age groups, an average of the full data set was generated and is illustrated in Fig. 2C. This analysis resulted in an average V1/2 of activation of –21.3 mV (k = 4.6) and V1/2 of inactivation of –51.5 mV (k = 7.0) that describes the voltage dependence of sodium channel gating in spinal motoneurons during this postnatal period. The highly consistent data obtained from motoneurons over this developmental period indicate that a change in the voltage dependence of activation or fast inactivation does not contribute to the described developmental increase in excitability of these cells.
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For a cell to fire multiple action potentials a sufficient number of sodium channels must remain available before each spike. To assess channel availability during repetitive stimulations, such as would occur during a period of repetitive action potential firing, we assessed the sodium current amplitude during various frequencies of stimulation. To do this, cells were depolarized for 20 ms from –70 to –10 mV x 20 pulses at 1, 10, 20, and 30 Hz (Fig. 3 A). It was found that the sodium current amplitude was reduced in a frequency-dependent manner, with the degree of inhibition by the 20th pulse increasing as the frequency of stimulation increased. This was consistent across the age groups (Fig. 3B). Also across all age groups and at frequencies >1 Hz, the current exhibited an initial rapid reduction in amplitude followed by a more gradual reduction. The rate of the initial reduction was almost identical across the age groups, consistent with the comparable fast-inactivation process seen across age groups (Table 1). The latter slower component likely represents channels being driven into a slow-inactivated state (Blair and Bean 2003; Rush et al. 1998). As depicted in the insets in Fig. 3B the current from the P10–P12 age group consistently had the slowest transitions into this slow-inactivated state across all frequencies.
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Recovery from inactivation
Having excluded differences in the voltage dependence of channel gating (Table 1), the ability to maintain current during repetitive stimulations is likely a consequence of either an ability of the channels to quickly recover from an inactivated state (Bean et al. 1983) or an ability to resist entering an inactivated state. These two possibilities were investigated further to understand the factors permitting the more mature cells to better follow repetitive stimulations without losing current.
The ability to recover from the inactivated state, or "reprime" (Bean et al. 1983), was assayed using a double-pulse protocol with an increasing interval (Fig. 4 A). The data for the recovery process in these cells were best fitted with two exponentials (see legend of Fig. 4 for values), suggesting that even with a short 20-ms stimulation a small percentage (20%) of channels transition into a slow-inactivated state. As can be seen from the averaged data in Fig. 4B, significant differences between age groups could be seen both in the rate at which the channels recovered and in the completeness of recovery over the 300 ms assayed. Given longer intervals, such as the 1,000-ms interval used in the 1-Hz stimulation protocol (Fig. 3B), channels in all age groups tended to become equally available (95%). Currents elicited from cells in both the youngest (P1–P3) and oldest (P10–P12) age groups tended to have a greater proportion of channels recovering in the fast phase of recovery and trended to transition into a slow-inactivated state to a lesser degree than the two middle age groups.
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) or fast phase of recovery (Bean et al. 1983). Here the P10–P12 age group showed significantly greater recovery than that of cells of the P7–P9 age group. As the interval length was increased (100 and 150 ms), these significant differences in channel availability extended to all the younger age groups. As seen in Fig. 4C, a "U-shaped" pattern emerged over the developmental period spanned in this study, similar to the pattern in the repetitive stimulation data. A similar pattern was also seen in the data assessing the population of channels in the slow phase of recovery: those channels that tended to remain in a slow-inactivated state (Fig. 4D). Here again the oldest age group (P10–P12) had significantly greater channel availability compared with that of the younger age groups (P < 0.05, one-way ANOVA on ranks). Again the oldest and youngest age groups were similar and a U-shaped pattern was seen. The similarity between the pattern of the age-dependent changes in frequency-dependent inhibition (Fig. 2C) and both the fast and slow components of the recovery from inactivation (Fig. 4, C and D) suggested that these two parameters were related. This relationship is further explored in the following text.
Correlation analysis
Thus far the data have been arranged into age groups to examine the changes that occur during postnatal development. At this point we sought to examine the underlying factors that allowed cells to maintain available channels during repetitive stimulations. For this we grouped cells from all age groups together because some cells in all the age groups showed a remarkable ability to follow high frequencies even though on average the cells in the oldest age group were the most proficient at this task. At all frequencies studied, a strong and highly significant correlation existed between the degree of frequency-dependent inhibition (taken to be the amplitude of the 20th pulse divided by the amplitude of the first pulse) and channel availability over the initial 50-ms interval of recovery (Fig. 5 A). The data for the 100-ms (0.62 < R < 0.93; P < 0.0001; n = 58 cells) and 150-ms (0.86 < R < 0.91; P < 0.0001; 58 cells) intervals showed similarly strong and highly significant correlations at all frequencies (Pearson product-moment correlation; data not shown).
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, 2001; Herzog et al. 2003; Spampanato et al. 2001). Closed-state inactivation
Because sodium channels do not need to actually open to transition into an inactivated state (Taddese and Bean 2002), we assessed the potential contribution of closed-state inactivation to the observed changes during repetitive stimulations. For this assessment the membrane potential was depolarized 20 mV more positive to the holding potential for varying periods of time before a test pulse was used to assess channel availability. The initial depolarization to –50 mV did not cause channel activation (Fig. 6 A). Current amplitudes were normalized to the amplitude of the first evoked current.
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Correlation analysis
Similar to the variability in the repriming data, the ability to resist closed-state inactivation in some cells was not representative of the average of their age group. Therefore we looked at individual cells to determine whether there was a correlation between the ability to retain current during repetitive stimulations and the closed-state inactivation process (Fig. 7). At 10 Hz the correlation just reached significance (P = 0.049), whereas at 20 and 30 Hz these two parameters showed more significant relationships (P = 0.001 and 0.002, respectively). This strong correlation suggests that a component of the cell's ability to maintain available channels is also related to the fast component of closed-state inactivation because this fast process determined the relative channel availability for depolarizations 
2 s. That is, the slow inactivation process was essentially equal across the groups (i.e., similar slope values). The age-dependent pattern seen in Fig. 6C and the significant correlation between closed-state inactivation and the ability of channels to resist entering an inactivated state during repetitive stimulations suggest that inactivation from the closed state also plays an important role in the developmental change of motoneuronal firing patterns.
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With clear age-related changes in both the closed-to-inactivated-state and the inactivated-to-closed-state transitions we assessed whether there were also age-related differences in the open-to-closed-state transitions. The current decay during a voltage step could be fitted with a single-exponential function and was plotted relative to membrane potential (Fig. 8). When comparing the rate of inactivation from the open state of the channel, significant differences between the age groups could not be detected at any voltage examined. There was greater scatter between the means at the more hyperpolarized voltages but, even at a membrane potential of –30 mV, this was not significant (ANOVA on ranks, P = 0.37). Furthermore, there was not a significant correlation between the rate of open-state inactivation and the ability to follow repetitive stimulations (10–30 Hz) when assessed on a cell-by-cell basis (R < 0.11 and P > 0.46; data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Garcia et al. 1998; McCobb et al. 1990) and hyperpolarizing conductances (McCobb et al. 1990; Vinay et al. 2000), to enable the mature motoneurons to discharge at high frequencies for extended durations. The increased ability of the most mature cells to maintain channels in an available state appears to be derived mainly from the ability of the channels to recover quickly from an inactivated state: first, because of the highly significant correlation that was seen between these two parameters and, second, because the developmental pattern of changes in this biophysical parameter paralleled those seen with frequency-dependent inhibition. It is important to note that the developmental change in the ability to follow repetitive stimulations was seen in the absence of an age-related change in the voltage dependence of channel gating because a hyperpolarizing shift in this parameter, relative to the holding voltage, could also result in a greater availability of channels.
The tight association between the ability to maintain available channels during repetitive stimulations and the ability to recover quickly from inactivation is not unexpected. This relationship has been demonstrated in previous studies (Bean et al. 1983; Ilyin et al. 2005) and in fact the retardation of recovery is thought to be an important component of the mechanism of action of many sodium-channel–inhibiting drugs (such as lidocaine, phenytoin, carbamazepine). But the ability to reprime quickly does not necessarily endow channels with the ability to maintain current during repetitive stimulations. For instance, when Nav1.2 and Nav1.6 channels were compared after expression in sensory neurons, the Nav1.2 isoform was found to display faster repriming kinetics than did the Nav1.6 isoform, even though it was the Nav1.6-expressing cells that were better able to maintain current during repetitive stimulations (Rush et al. 2005). Additionally, when Zhou and Goldin (2004) coexpressed these same two isoforms with the β1 subunit in Xenopus oocytes they also observed that the Nav1.6 isoform maintained significantly more current during repetitive stimulations than did the Nav1.2 isoform. However, under these experimental conditions the Nav1.6-mediated current was actually potentiated during repetitive stimulations, whereas the Nav1.2-mediated current showed the typical decline. Clearly this difference could not be due to isoform differences in repriming kinetics and, in fact, was attributed to differences in the activation process that occurred during the repetitive stimulations (Zhou and Goldin 2004).
In the present study the ability of spinal motoneurons to maintain channels in an available state seems to receive a contribution from differences in other state transitions in addition to the transition from the inactivated-to-closed state (i.e., repriming). As demonstrated in Fig. 3B, the channels in the most mature age group consistently had a slower transition rate into a slow-inactivated state compared with the other age groups (see Fig. 3B, insets). This tendency to either resist transitioning into a slow-inactivated state or recover quickly from this state was also evident during the repriming experiments (Fig. 4B). In these latter experiments the current in the mature cells showed a greater degree of recovery over the last 100 ms of the protocol. Interestingly, the transitions into a slow-inactivated state from the closed state were very similar across the age groups (similar slope values; Fig. 5B) and therefore may represent transitions into a different slow-inactivated state. Finally, a decreased tendency to undergo the closed-to-inactivated-state transition correlated well with the ability to maintain available channels during repetitive stimulations. That is, with fewer channels participating in the fast phase of closed-state inactivation, the more channels were available for a subsequent opening. These findings suggest that differences in these inactivation transitions also contribute to the ability to follow repetitive stimulations without losing appreciable current.
The occurrence of closed-state inactivation during the repetitive stimulation protocols at first may seem unusual, but keep in mind that these cells were assessed from a holding potential of –70 mV. From the inactivation conductance–voltage graph (Fig. 2) this voltage corresponds to roughly 10% of channels being inactivated. Therefore, while holding the cell at this potential, one would expect to see transitions to an inactivated state and an increase in the rate of these same transitions as the membrane potential was pulsed to more depolarized potentials where channel openings occurred (Nuss et al. 2000).
A surprising finding in this study was that the current in the youngest cells was also very proficient at following repetitive stimulations and, with respect to other parameters, behaved in a manner similar to that of the most mature cells. A possible explanation for this will be discussed in the following section but this finding suggests that, at least for the very early postnatal cells, the biophysical properties of the voltage-gated sodium current do not limit the maximal firing rates. Other mechanisms such as the low sodium current density (Gao and Ziskind-Conhaim 1998; Garcia et al. 1998; McCobb et al. 1990), or the underdeveloped potassium (McCobb et al. 1990; Vinay et al. 2000), or other conductances (McCobb et al. 1989; Miles et al. 2004; Mynlieff and Beam 1992) likely play a larger role in limiting the maximal firing rate at this stage of development.
Plateau potentials
Mature spinal motoneurons generate plateau potentials (Perrier and Hounsgaard 2000), quasi-stable potentials approximately 10–20 mV more positive to the resting potential (Hounsgaard and Kiehn 1989; Hounsgaard et al. 1986), capable of eliciting repetitive action potential discharges. In the present study we sought to identify a possible means whereby sodium channels could remain available to discharge multiple action potentials under depolarized conditions that normally promote transitions into an inactivated and therefore unavailable state. To accomplish this we tested the ability of motoneuronal sodium channels to resist transitioning from the closed state to the inactivated state using a voltage protocol that mimicked a plateau potential (Fig. 6A). In these experiments the most mature cells were better able to resist transitioning into the inactivated state than the P7–P9 age group (but it is unclear as to why the P4–P6 age group was so proficient at this particular task). Regardless, should this trend in closed-state inactivation continue, this biophysical parameter would likely become important for the expression of the mature firing properties of spinal motoneurons. This late-developing resistance to inactivation may contribute to the difficulty of demonstrating plateau potentials in spinal motoneurons of rodents until well into the second postnatal week (Jiang et al. 1999c). Furthermore, postnatal development of the resistance to closed-state inactivation would likely contribute to previous results, suggesting that nifedipine-sensitive plateau potentials play in increasing role in lumbar ventral root output during postnatal development (Jiang et al. 1999b).
Developmental changes in channel subtypes
Previous studies have demonstrated that the complement of voltage-gated sodium channels expressed in the spinal cord in general, and specifically in motoneurons, changes during postnatal development. Channel expression assessed at both protein (Gordon et al. 1987; Schaller and Caldwell 2000) and mRNA levels (Beckh et al. 1989; Black et al. 1994; Felts et al. 1997; Garcia et al. 1998; Schaller and Caldwell 2000) in the rodent spinal cord have demonstrated that around birth the sodium channel subtypes Nav1.1, 1.2, 1.3, and 1.6 are all expressed to varying degrees. There is some discrepancy as to the prominent channel subtype between studies, although there is consensus that over the first two postnatal weeks there is a relative change in the expression of these channels. With respect to the ventral horn cells in particular, at birth these cells express detectable levels of Nav1.1, Nav1.2, and Nav1.3 mRNA transcripts (Felts et al. 1997). During the following two weeks of development the levels of Nav1.1 mRNA are seen to increase, the levels of Nav1.3 decrease, and the levels of Nav1.2 are seen to remain somewhat stable (Felts et al. 1997). The expression of Nav1.6 mRNA (Felts et al. 1997; Schaller and Caldwell 2000) and protein (Schaller and Caldwell 2000) in motoneurons becomes detectable between P2 and P7 and increases until approximately the third postnatal week. These labeling studies are in good agreement with electrophysiological data obtained from the Nav1.6 mutant mouse (med; Garcia et al. 1998). In this study the authors demonstrated that in the absence of the Scn8a gene encoding the Nav1.6 channel, the normal postnatal increase in current density of spinal motoneurons failed to occur despite normal current density at P0. These data support the suggestion that the Nav1.6 channel isoform becomes the major current contributor in these cells by the second postnatal week.
Heterologous expression and function of channel subtypes
The biophysical properties of most of the mammalian sodium channel 
subunits (with or without accompanying β subunits) have been studied in relative isolation after their expression in various heterologous systems. These studies have demonstrated the fundamental properties of the four main sodium channels expressed in spinal motoneurons (Cummins et al. 2001; O'Leary 1998; Smith et al. 1998; Zhou and Goldin 2004). Of those channels that would be expressed during the first days after birth, Nav1.1 and Nav1.2 channels appear to have very similar properties. When expressed under similar conditions (Xenopus oocytes with the same β subunits), the voltage dependences of activation and inactivation were similar, with Nav1.2 having slightly more hyperpolarized values. Both of these channels are capable of quickly recovering from inactivation (fast tau <2.2 ms) and with >80% recovering during the fast time constant (Smith et al. 1998). Again, when compared under similar expression conditions (HEK-293 cells), the voltage dependences of activation and inactivation of Nav1.3 channels were found to be within 4 mV of the Nav1.2 channels (Cummins et al. 2001; O'Leary 1998). The Nav1.3 isoform was found to recover from inactivation threefold slower than the Nav1.2 isoform at –80 mV. On the other hand, at –60 mV the Nav1.3 channels were found to undergo closed-state inactivation more than twofold slower.
The fourth channel isoform expressed in these cells during this developmental period is Nav1.6. When Nav1.6 channels were coexpressed with the β1 subunit in Xenopus oocytes, these channels were so proficient at remaining in an available state that repetitive stimulations elicited a potentiation of this current (Zhou and Goldin 2004). In the same study the Nav1.2 isoform showed a modest decrease in the current. Moreover, when Nav1.6 was compared with Nav1.2 after expression in dorsal root ganglion cells, the Nav1.6 isoform was significantly better at resisting inactivation during repetitive stimulation protocols (Rush et al. 2005).
The most parsimonious explanation for the main finding of this study—the fact that the older cells can retain more current during repetitive stimulations—is a postnatal increase in the expression of Nav1.6, a channel subtype that has been demonstrated to be able to retain available channels during this type of stimulation. This finding is supported by studies demonstrating that deletion of the Scn8 gene encoding the Nav1.6 channel results in a decreased maximal firing rate of cells that normally express this isoform (Enomoto et al. 2007; Levin et al. 2006; Van Wart and Matthews 2006). Moreover, the absence of this channel isoform in trigeminal neurons of the Nav1.6 null mice resulted in a reduced ability to maintain repetitive discharges compared with that of wild-type littermates (Enomoto et al. 2007). These authors recognized that this was consistent with the channels in the Nav1.6-containing wild-type cells being less susceptible to inactivation and being able to recover from inactivation quicker.
Changes in β-subunit expression
The expression of the various accessory β-subunit isoforms also changes during early development. Coexpression of these β subunits has been shown to have a strong influence on various biophysical parameters of the expressed -subunit–mediated currents (Isom et al. 1992). During the embryonic period high levels of the β3 mRNA transcript are detected in the rat CNS. After P1 these levels continually decline (Shah et al. 2001). On the other hand, levels of β1 transcripts are first detected at approximately P3 and increase until approximately P14, whereas β2 mRNA appears at approximately the same time and the levels increase until reaching adulthood (Shah et al. 2001). All three -subunit isoforms that predominate during the first few days of postnatal development, Nav1.1, Nav1.2, and Nav1.3, can all form complexes with β3. With respect to Nav1.2 (Morgan et al. 2000) and Nav1.3 (Shah et al. 2001) the addition of this subunit increases the degree of channel availability during recovery from inactivation. Under these conditions young cells may be expected to retain a large fraction of available channels during repetitive stimulations as seen in the present study.
The preceding discussion on the postnatal changes in both the sodium channel and β isoforms, and their expected functional effects on the sodium current in spinal motoneurons, provides a possible explanation for the "U-shaped" developmental pattern observed in this study. During the earliest postnatal period the cells would express a combination of Nav1.1, Nav1.2, and Nav1.3 channels in the presence of β3 subunits. This would permit all of these channels to recover from inactivation relatively fast and likely contribute to the ability of the current to resist inactivation during the repetitive stimulations protocol. With further development, while the level of Nav1.1 increases, that of the relatively fast repriming Nav1.3 and β3 combination decreases because there is a change from β3 to β1 and β2. Moreover, during this period the fast repriming Nav1.2 channels contribute a smaller fraction to the total current as the overall current density in these cells increases. These changes likely result in an overall decrease in the ability of the cell to retain current during repetitive stimulations. With the increase of the Nav1.6 isoform during the second postnatal week, the ensemble current would again acquire the ability to retain available channels during repetitive stimulations because this -subunit isoform appears to be the most proficient at this task.
Limitations
An important factor in this present work was the ability to maintain voltage control over the sodium current. This is a difficult task in spinal motoneurons due to the large current density in these cells and the fact that these channels are clustered at the initial segment and in the axon, structures spatially remote from the somatic pipette during whole cell recording. In the present work we sought to circumvent these voltage- and space-clamp problems by removing the motoneuronal processes through mechanical isolation of the soma and then culturing these cells in conditions that did not promote process growth (absence of growth factors, uncoated plastic substrate). The cells were exposed to these culture conditions for a relatively short period of time with the goal of replicating the in vivo channel expression as previously described (Garcia et al. 1998). As discussed in the following text, these experimental procedures are both a strength and a possible limitation of the present work.
With the use of these experimental conditions the cells had their initial segment removed and subsequently reexpressed their sodium channels. We believe that the recorded currents are from newly expressed channels for two main reasons: 1) cells patch-clamped immediately after trituration did not demonstrate a sodium current (data not shown); and 2) given the same incubation time, cells of the various age groups demonstrated similar current densities (Table 1). It is not surprising that the cells did not demonstrate a sodium current after mechanically removing the processes because a similar result was seen when the same process-removal effect was accomplished by pulling the intact soma away from the rest of the cell in a transverse slice of spinal cord tissue (Safronov et al. 2000). In this case, the sodium current density in the soma-only situation was reduced dramatically compared with the current recorded from the intact cell. The ability to reexpress the sodium channels in isolated somata has also been demonstrated. The cell bodies of the squid giant axon are normally unexcitable, but if dissociated from the axon and cultured these cell bodies will begin to develop an inward sodium current that begins on the day of isolation and increases in density during the culture period (Brismar and Gilly 1987). Moreover, the vast majority of these newly synthesized channels are biophysically indistinguishable from the "normal" axonal channels (Gilly and Brismar 1989). To definitively determine whether the complement of newly expressed sodium channels in the present study are the same as would be expressed in the in age-appropriate in vivo cells will require evaluation of either the protein or the mRNA; however, that the sodium current in these cells demonstrated age-related changes in inactivation properties and that these inactivation changes are consistent with the known maturation of firing properties in these cells strongly suggest that the expression of the age-appropriate channels occurred in these cells. Nonetheless, we cannot discount the possibility that an age-related change in some intracellular modulatory factor also plays a role in the observed changes.
In the present study we isolated and examined one small component of the much more complex intact spinal motoneuron. We view this culture preparation as a model of an intact in vivo motoneuron and, as with any reduced preparation, there is a trade-off between gaining control in one aspect (in this case voltage control) and the loss of other aspects of the original condition. Our goal was to create an experimental condition that allowed a very accurate study of the sodium current in motoneurons and in doing so we may have given up the ability to assess other aspects of motoneuron functioning. Although we did not attempt to assess action potential firing, in all likelihood the somata examined in the present study would not discharge as motoneurons would in a more intact preparation (e.g., slice, whole cord, or in vivo animal). The motoneurons examined in the present experiments lacked some of the fundamental components of spinal motoneurons such as the dense sodium channel clustering on the initial segment (Alessandri-Haber et al. 1999) and not only the dendritic membrane itself (which can have an effect on firing patterns; Mainen and Sejnowski 1996) but also the dendritic conductances important for plateau potential generation (Carlin et al. 2000b; Lee and Heckman 1998). Furthermore, the overall sodium current was many fold lower under the present experimental conditions compared with that recorded from more intact spinal motoneurons (ex. Miles et al. 2005). As such, the degree to which the presently described changes in sodium channel functioning manifest themselves in the behavior of intact motoneurons during development has yet to be fully defined.
Functional consequences
In the present study we have demonstrated that the most mature spinal motoneurons possessed the greatest ability to follow repetitive stimulations due to their ability to maintain channels in a state capable of opening and passing current. This ability stems from age-related changes in various channel transitions but results mainly from an increased ability of the channels to recover quickly from inactivation. The age-related decrease in the tendency of these channels to transition into a slow-inactivated state or undergo closed-state inactivation also contributes to maintaining available channels during repetitive stimulations. The functional consequence of these kinetic changes in the native cells would be amplified by the well-described developmental increase in the sodium current density (Gao and Ziskind-Conhaim 1998; Garcia et al. 1998; McCobb et al. 1990). Therefore not only would these cells have the benefit of a larger pool from which to draw available channels, but these channels would also tend to remain in an available state. It is likely that these two factors greatly impact the ability of these cells to fire repetitively for extended periods of time.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Present address of K. P. Carlin: Purdue Pharma LP, Discovery Research, Cranbury, NJ 08512.
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Address for reprint requests and other correspondence: L. M. Jordan, The University of Manitoba, Department of Physiology, Spinal Cord Research Centre, 730 William Avenue, BMSB 425, Winnipeg, Manitoba R3E 3J7, Canada (E-mail: larry{at}scrc.umanitoba.ca)
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, 30 Hz (one-way ANOVA on ranks with Dunn's multiple pairwise comparison). Note that the P10–P12 group was significantly different from all other groups at 10, 20, and 30 Hz.
