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Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115
Submitted 26 February 2004; accepted in final form 26 March 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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40 and 55%, respectively. The resurgent current present in Nav1.6–/– null STN neurons was similar in voltage dependence to that in wild-type STN and Purkinje neurons, differing only in having somewhat slower decay kinetics. These results show that sodium channels other than Nav1.6 can make resurgent sodium current much like that from Nav1.6 channels. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Felts et al. 1997; Goldin 2001; Schaller and Caldwell 2003). A current challenge is to explore ways in which expression of particular sodium channels might influence cellular excitability in various neurons.
Nav1.6 channels are present in nodes of Ranvier, unmyelinated axons, cell bodies, dendrites, and presynaptic terminals (Boiko et al. 2001; Caldwell et al. 2000; Krzemien et al. 2000; Schaller and Caldwell 2000, 2003). Mice in which expression of Nav1.6 is eliminated show deficient function of the motor system, including ataxia and progressive paralysis of hind limbs (reviewed by Meisler et al. 2001). Disruption of Nav1.6 expression in mice causes symptoms resembling some human idiopathic dystonias (Hamann et al. 2003). Partial paralysis might be plausibly attributed to disruption of axonal conduction in motor neurons, where Nav1.6 is normally expressed in nodes (Caldwell et al. 2000). Ataxia might be partially accounted for by the widespread expression of Nav1.6 in the cerebellum (Burgess et al. 1995; Schaller and Caldwell 2003). In cerebellar Purkinje neurons of mice that are homozygous for a null allele of Nav1.6, there is a dramatic reduction (80–90%) of an unusual "resurgent" sodium current that flows on repolarization after action-potential-like waveforms (Raman et al. 1997). Loss of Nav1.6 is associated with altered electrophysiological function of Purkinje neurons, including slower and less robust spontaneous firing and reduced burst firing (Khaliq et al. 2003; Raman et al. 1997).
Recent experiments have shown the existence of resurgent sodium current in a number of neuronal types in the motor system in addition to Purkinje neurons, including deep cerebellar nuclei (Raman et al. 2000), subthalamic nucleus neurons (Do and Bean 2003), and globus pallidus neurons (Mercer et al. 2003). Thus it is possible that disrupted function of the motor system in Nav1.6-null mice involves altered function of a number of cell types. To explore this prospect, we examined the consequences of loss of Nav1.6 for sodium current and for the electrophysiological function of subthalamic neurons. We find that without Nav1.6, resurgent current in subthalamic nucleus (STN) neurons is reduced but still present, with only modest changes in kinetic properties or voltage dependence. This adds to other evidence showing that sodium channels in addition to Nav1.6 can make sizeable resurgent sodium current. In addition, loss of Nav1.6 was associated with remarkably little alteration of firing properties of STN neurons, in contrast to the situation in Purkinje neurons.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Heterozygous Scn8amed mice were obtained from Jackson Laboratories (Bar Harbor, ME). The med mutation produces complete loss of Nav1.6 expression unlike the medJ mutation, which is hypomorphic and results in a reduction in channel expression by 90% (Kearney et al. 2002). This had the advantage of totally eliminating Nav1.6 channels but confined our measurements to animals younger than 3 wk, when homozygous med mutants die. To compare only homozygous null animals with wild-type animals, we genotyped mice before use and used homozygous med (Nav1.6–/–) or wild-type (Nav1.6+/+) litter-mates. Genotyping used DNA extracted from mouse tails (DNeasy Tissue Kit, Qiagen, Valencia, CA). PCR amplification used the following primers (5' to 3'): for the wild-type allele, GGA GCA AGG TTC TAG GCA GCT TTA AGT GTG and GTC AAA GCC CCG GAC GTG CAC ACT CAT TCC (Kohrman et al. 1996); for the mutant allele, TCC AAT GCT ATA CCA AAA GTC CC and GGA CGT GCA CAC TCA TTC CC (Jackson Labs). The reaction consisted of 20 s at 94°C, 30 s at 66°, and 35 s at 72°C (12 repetitions), followed by 20 s at 94°C, 30 s at 60°C, and 35 s at 72°C (25 repetitions), and 5 min at 72°C. PCR products were separated on a 2% agarose gel, allowing resolution of a 230-bp product for the wild-type allele and a 194-bp product for the mutant allele.
Tissue preparation for electrophysiology
In most experiments, the experimenter was blind to genotype and phenotype until after data analysis. Because the ataxia of the mutant animals is generally evident at the ages used (P13-18), another researcher (Dr. Gui-lan Yao) selected animals that had been previously genotyped, anesthetized the animals with isoflurane, and decapitated them. The experimenter received the head, from which the phenotype could not be determined, for further dissection. One or two slices of 300 µm were cut in a cold sucrose solution [(in mM) 87 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 Na2HPO4, 7 MgCl2, 25 glucose, and 75 sucrose, equilibrated with 95% O2-5% CO2]. Slices were incubated in holding solution (sucrose solution with 0.5 mM CaCl2 added) at 35°C for 30 min then kept at room temperature. For dissociation, slices were exposed to 3 mg/ml protease XXIII for the last 7–8 min at 35°C and then transferred to holding solution with 1 mg/ml BSA and 1 mg/ml trypsin inhibitor during the first 15–30 min at room temperature. The STN was removed into trituration solution [(in mM) 70 Na2SO4, 1.5 K2SO4, 7 MgCl2, 10 HEPES, 25 glucose, and 75 sucrose, pH 7.4 with NaOH] and passed through fire-polished Pasteur pipettes to release individual neurons. The STN is well-defined by white matter tracts and contains a highly homogeneous cell population, making cell identification straightforward (Afsharpour 1985; Do and Bean 2003; Song et al. 2000).
For experiments on Purkinje neurons, sections of cerebellar vermis were minced in oxygenated, cold dissociation solution [(in mM) 82 Na2SO4, 30 K2SO4, 5 MgCl2, 10 HEPES, and 10 glucose, 0.001% phenol red, pH 7.4 with NaOH] and transferred to the same solution with 3 mg/ml protease XXIII added. After 7 min at 35°C, tissue was transferred to dissociation solution with 1 mg/ml BSA and 1 mg/ml trypsin inhibitor for 10–15 min at room temperature, then maintained in cold dissociation solution. Tissue was withdrawn as needed and triturated with fire-polished Pasteur pipettes. Purkinje neurons were recognized by their large size and characteristic stump of apical dendrite.
Electrophysiology
Recordings were made using an Axopatch 200B amplifier (Axon Instruments, Union City, CA) in voltage-clamp or fast current-clamp mode. Borosilicate patch pipettes were wrapped to near the tip with stretched parafilm to reduce capacitance. For voltage-clamp recordings, cell capacitance was nulled and series resistance, usually <10 M
, was compensated 60–95%. For current-clamp recordings, series resistance was compensated 100%.
Voltage-clamp experiments used solutions designed to isolate sodium currents. The internal solution was based on N-methyl-D-glucamine (NMDG) phosphate: (in mM) 70 NMDG2PO4, 13.5 NaCl, 1.8 MgCl2, 9 EGTA, 9 HEPES, 14 phosphocreatine (Tris salt), 4 MgATP, and 0.3 GTP (Tris salt), pH 7.2 with H3PO4. The external solution was designed to block calcium currents and reduce potassium currents to facilitate accurate TTX-subtractions: (in mM) 150 NaCl, 3.5 KCl, 1 BaCl2, 0.2 CdCl2, 1 MgCl2, 10 HEPES, 10 glucose, 10 TEACl, and 3 CsCl, pH 7.4 with 4.4 mM NaOH. Sodium channel currents were defined by subtraction of currents remaining in 300 nM TTX. Resurgent current was measured as time-dependent current flowing when the voltage was repolarized to voltages between –80 and –20 mV after a 10-ms step to +30 mV, and persistent current was measured as the steady-state current flowing at the end of the steps (100-ms long) after resurgent current had decayed. Persistent current measured in this way is quantitatively very similar to that measured using slow ramps of voltage; in a series of experiments on STN neurons of rats, the average persistent current flowing during a step repolarization to –40 mV was –32 ± 5 pA (n = 6), while that flowing at –40 mV during a 20-mV/s depolarizing ramp from –90 mV was –33 ± 6 pA (n = 3).
Current-clamp recordings were done in brain slice and used more physiological solutions. The internal solution was (in mM) 117 KCH3SO4, 13.5 NaCl, 1.8 MgCl2, 0.09 EGTA, 9 HEPES, 14 phosphocreatine (Tris salt), 4 MgATP, and 0.3 GTP (Tris salt), pH 7.2 with KOH. The external solution for recording from slices was artificial cerebrospinal fluid (ACSF) consisting of (in mM) 125 NaCl, 25 NaHCO3, 3.5 KCl, 1.25 Na2HPO4, 1.2 CaCl2, 1 MgCl2, and 25 glucose, equilibrated with 95% O2-5% CO2. To block fast synaptic transmission, this solution also contained 2–3 mM kynurenate and 100 µM picrotoxin.
Junction potentials (KCH3SO4 solution: –8 mV; 70 mM NMDG2PO4 solution: –4 mV), measured using a flowing KCl bridge (Neher 1992), have been corrected.
Data analysis
Signals were filtered at 5–10 kHz and sampled at 50 kHz or filtered at 5 kHz and sampled at 10 kHz. Some traces were also digitally filtered for clarity. Data were acquired and analyzed with pClamp 8.0 (Axon Instruments), Igor 3.14 (Wavemetrics, Lake Oswego, OR), DataAccess (Bruxton, Seattle, WA), Excel (Microsoft, Seattle, WA), and WinStat (A-Prompt, Whitehall, PA). Descriptive statistics are presented as means ± SD The Mann-Whitney U test was used to assess statistical significance.
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RESULTS |
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, 2001). Persistent current was measured as the steady-state current flowing at the end of the 100-ms steps after resurgent current had decayed.
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), STN neurons from wild-type mice showed clear resurgent current (18 of 18 cells). Qualitatively, sodium currents in STN neurons from Nav1.6–/– mice were indistinguishable from those in wild-type littermates: transient currents were large, and resurgent current was detectable in 10 of 11 cells (e.g., Fig. 1B, top). Quantitatively, however, transient, resurgent, and persistent sodium currents were all smaller in Nav1.6–/– mice compared with wild-type littermates (Fig. 2). The different components of current were reduced to different extents. Transient sodium current, normalized to cell capacitance, was –1,717 ± 956 pA/pF (n = 11) in wild-type mice and –1,019 ± 342 pA/pF (n = 11) in med mice, a reduction of 41% (P = 0.02). Resurgent current was –27 ± 14 pA/pF in wild-type mice (n = 11) and –10 ± 7 pA/pF in med mice (n = 11), a reduction of 63% (P = 0.001). Persistent current was –11 ± 8 pA/pF in wild-type mice (n = 11) and –5 ± 2 pA/pF (n = 11, P = 0.0003) in med mice, a reduction of 55%.
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). However, the previous experiments were done with a mouse line (medTg) where a transgene-induced null allele of Nav1.6 is maintained in strain C57BL/6J. This is different from the med line used here, where the null allele of Nav1.6 was caused by insertion of an L1 element and is maintained in a CH3 background (Sprunger et al. 1999). The previous experiments with Purkinje neurons also used somewhat different internal and external recording solutions, for example using an external solution with reduced sodium. Thus although the results suggest that resurgent current depends less on Nav1.6 in STN neurons than in Purkinje neurons, it is also possible that differences between the mouse strains or recording conditions complicate the comparison. To evaluate this possibility, we performed parallel experiments on cerebellar Purkinje neurons (Fig. 1B, bottom) from the med mice and their wild-type littermates using the same solutions as for the STN recordings. Consistent with the earlier results in the medTg mice (Raman et al. 1997), resurgent current was detected in all wild-type Purkinje neurons assayed (14 of 14 cells) but was undetectable (5 of 16 cells) or very small (11 of 16 cells) in med Purkinje neurons. Transient and persistent sodium currents were also significantly smaller in Purkinje cells from the med mutants. Examining current density, there was a reduction of 89% for resurgent current (P = 0.0001), 43% for transient current (P = 0.002), and 76% for persistent current (P = 0.0004, Figs. 2 and 3). These findings are very similar quantitatively to those of Raman et al. (1997), who found that resurgent, transient, and persistent currents in Purkinje neurons of homozygous medTg mice were reduced by 82, 40, and 70% of the levels in wild-type littermates.
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40%), but reduces resurgent current more in Purkinje neurons (89%) than in STN neurons (63%). A straightforward interpretation is that Nav1.6 sodium channels make up most of the resurgent current in both cell types but that other sodium channels can also form resurgent current and do so more effectively in STN neurons than in Purkinje neurons. Of course, it is possible that in the mutants there is compensatory upregulation of the sodium currents formed by nonNav1.6 channels, in which case Nav1.6 channels in wild-type animals might make up more than the 40% "missing" transient current and more than the 89 and 63% "missing" resurgent current in Purkinje and STN neurons, respectively. Whatever the amount of compensation, two reasonably firm conclusions can be drawn with regard to current in STN neurons: Nav1.6 channels account for the majority of resurgent current in wild-type animals, but non-Nav1.6 channels can also produce a sizeable resurgent current. Comparison of resurgent current in Purkinje and STN neurons
The voltage dependence of resurgent current was similar in STN and Purkinje neurons from wild-type and med animals, reaching a peak at –40 mV (Fig. 4). However, in both STN and Purkinje neurons from Nav1.6-null mice, the kinetics of both activation and decay of resurgent current were somewhat slower than in wild-type animals. The substantial magnitude of the resurgent current in Nav1.6–/– STN neurons allowed us to compare the resurgent current carried by Nav1.6 channels (which clearly makes up the great majority of the resurgent current in wild-type Purkinje neurons) with that carried by non-Nav1.6 channels, as recorded in Nav1.6–/– STN neurons. After repolarization from +30 to –40 mV, the resurgent current in Nav1.6–/– STN neurons reached a peak in 5.9 ± 2.2 ms (n = 7), later than that in wild-type Purkinje neurons (3.3 ± 0.4 ms, n = 6, P = 0.045). Resurgent current at –40 mV decayed with a time constant of 29 ± 7 ms in Nav1.6–/– STN neurons, slower than in wild-type Purkinje neurons, with a time constant of 16 ± 2 ms (P = 0.003). Thus both activation and deactivation are slower for non-Nav1.6 resurgent current. It is possible that the later time to peak for resurgent current in Nav1.6–/– STN neurons can be accounted for partly by the slower decay even if the intrinsic activation kinetics are not changed, so the primary kinetic difference in Nav1.6–/– neurons may be a decay that is slower by about twofold.
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Resurgent sodium current appears to arise from an alternative form of inactivation that competes with classical fast inactivation (Raman and Bean 2001). Entry into this second inactivated state can be modeled as an open-channel block, and recovery from it as an voltage-dependent unblock that leaves the channel transiently open, allowing resurgent current to flow. Entry into the inactivated state associated with resurgent current appears to be faster than the competing normal inactivation process, but the normal inactivated state appears to be more absorbing at equilibrium. This predicts a falling phase of transient current that has two components of decay, a fast component reflecting entry into the two inactivated states (initially occurring in parallel) and a slow component of decay arising from the relatively slow redistribution of inactivated channels from the less absorbing to the more absorbing inactivated state; this redistribution is accompanied by transient opening of the channels as the blocking particle leaves, giving rise to the slow component of decay. However, interpretation of the decay kinetics is not straightforward because in addition to two components of decay expected from properties of channels showing resurgence, there could also be multiple components of decay arising from the contribution of multiple channel types with different speeds of inactivation, and even individual channel types can have conventional inactivation with decay described by two time constants (Spampanato et al. 2001).
Figure 5 illustrates the kinetics of transient current in wild-type and med STN and Purkinje neurons. Transient sodium current was evoked by a step to –30 mV from a holding potential of –90 mV. In all cases, the time course of decay could be reasonably well fit by the sum of two exponentials. There was no clear difference in the kinetics of transient current between wild-type and med STN neurons; in both cases, the fast time constant was 0.5–0.6 ms (P = 0.6), the slow time constant 3 ms (P = 0.5), and the slow component accounted for 12–18% of the whole (P = 0.5). On the other hand, the currents in Purkinje neurons did show a consistent difference between the two genotypes in that the slower time constant was significantly shorter (2.6 ± 0.7 ms, n = 7) in med Purkinje neurons than in wild-type Purkinje neurons (6.6 ± 3.7, n = 10, P = 0.003). The changes in Purkinje neurons are similar to those previously seen for medTg versus wild-type mice (Raman et al. 1997). The time course of decay in med Purkinje neurons is very similar to that of both wild-type STN and med STN neurons.
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The firing properties of Purkinje neurons lacking Nav1.6 are disrupted, with a reduction in pacemaking frequency (Khaliq et al. 2003) and reduced (though not eliminated) tendency to fire bursts of spikes in response to brief injections of currents (Raman et al. 1997). To test how the loss of Nav1.6 affects the physiology of med STN neurons, we performed a series of current-clamp experiments in the brain-slice preparation. As expected from previous recordings from rat STN neurons in brain slice (Beurrier et al. 1999; Bevan and Wilson 1999; Do and Bean 2003), mouse STN neurons showed rhythmic spontaneous firing in the absence of current injection. The STN neurons of med as well as wild-type mice showed spontaneous activity. On average, there was no clear difference in the frequency of firing in med compared with wild-type mice, although individual neurons varied widely in their frequency of firing. The frequency of firing was 12 ± 9 Hz (n = 10) in wild-type neurons and 12 ± 5 Hz (n = 16, P > 0.5) in med neurons. We also examined firing of STN neurons when driven by current injection (Fig. 6). In these experiments, too, there was surprisingly little difference between med and wild-type neurons. There was no clear difference in the frequency of firing in response to injections of increasing amounts of current (Fig. 6B). The only noticeable and reproducible difference in firing behavior was in the tendency to fire action potentials after the cessation of current injection for large stimuli. Most wild-type neurons fired a burst of action potentials during repolarization after a strong stimulus (asterisks, Fig. 6A). Of 15 wild-type neurons studied, 11 fired at least one spike after interruption of the simulating current pulse, and 9 fired at least two spikes, and 5 fired five or more spikes. Of 11 med neurons studied, 5 fired at least one spike, 2 fired at least two spikes, and none fired more than four spikes. Thus the reduction of sodium current in med STN neurons does impact the firing properties of these cells, but in a relatively subtle manner.
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DISCUSSION |
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)—confirmed by our own measurements in a different null mutant—it adds to accumulating evidence that sodium channels other than Nav1.6 can form resurgent current. Although resurgent sodium current in Nav1.6-null Purkinje neurons was very small when initially assayed (Raman et al. 1997) with 50 mM external sodium, used to improve voltage control, it was clearly present in some neurons, and its presence is more obvious and unambiguous when studied with physiological solutions (present results; T. M. Grieco and I. M. Raman, personal communication). Additionally, even in individual Purkinje neurons from Nav1.6-null mice where resurgent current is undetectable, resurgent current can be induced if the remaining sodium channels, most likely Nav1.1 channels (Schaller and Caldwell 2003; Vega-Saenz de Miera 1997), are treated with beta-pompilido toxin, which slows inactivation (Grieco and Raman 2004). The simplest interpretation of these results is that the open-channel-blocking mechanism underlying resurgent current can operate with all sodium channel types and is normally more prominent with Nav1.6 channels, probably because in those channels this mechanism competes more effectively with conventional inactivation (Grieco and Raman 2004), thought to be due to block of channels by the domain III-IV linker (Catterall 2000). Conversely, resurgent current was not detected in some cell types that clearly express Nav1.6, including CA3 pyramidal neurons and motor neurons (Garcia et al. 1998; Pan and Beam 1999; Raman and Bean 1997).
While showing that channels other than Nav1.6 can form resurgent current, our results add to previous results in suggesting that Nav1.6 channels are more effective than other sodium channel types in forming resurgent current. In our results with STN neurons, loss of Nav1.6 had a substantially larger effect in reducing resurgent current (by 63%) than transient current (by 40%). Similarly, loss of Nav1.6 reduced persistent current (by 55%) somewhat more than transient current in STN neurons. This is similar to previous results for a disproportionate reduction of persistent current in both Purkinje neurons (Raman et al. 1997) and prefrontal cortex pyramidal neurons (Maurice et al. 2001) from Nav1.6-null mice, and the results are also consistent with data from heterologous expression studies showing that Nav1.6 channels are more effective than Nav1.1 and Nav1.2 channels in producing persistent current (Smith et al. 1998). In the heterologous expression studies, Nav1.6 channels did not produce clear resurgent current, presumably because the hypothetical blocking particle is lacking.
It is notable that persistent current is substantially larger in relation to transient current in wild-type Purkinje neurons (ratio of persistent current to transient current 2.7%, Raman et al. 1997; 1.7%, this study) than in wild-type STN neurons (0.6%) or Nav1.6-null STN neurons (0.6%). The simplest possibility is that Nav1.6 channels in Purkinje neurons have conventional inactivation that is less complete than for Nav1.6 channels in STN neurons or for Nav1.1 or Nav1.2 channels. Differences between Nav1.6 channels in different types of neurons could reflect expression of different accessory beta subunits, which affect channel gating (Qu et al. 2001). In addition, regulation of native resurgent current by phosphorylation (Grieco et al. 2002) and of inactivation of heterologously expressed Nav1.6 channels by calcium/calmodulin (Herzog et al. 2003) have both been reported, and either of these mechanisms could underlie functional differences in currents formed by Nav1.6 channels in different cells. In principle, the presence of the blocking particle hypothesized to underlie resurgent current might result in less steady-state persistent current than would otherwise be the case; however, detailed modeling of resurgent current suggests that block by this particle is steeply voltage dependent (Raman and Bean 2001) and is weak over the voltage range where persistent current is prominent (–60 to –30 mV). Thus there is no fundamental inconsistency between the presence of a large resurgent current and a large persistent sodium current.
If the conventional inactivation of Nav1.6 channels in Purkinje neurons is less complete than that of Nav1.6 channels in STN neurons, the channels in Purkinje neurons also would be predicted to produce a larger resurgent current relative to transient current (because a greater fraction of channels can be blocked by the "resurgent particle" in competition with conventional inactivation). This idea is consistent with transient current being reduced 40% in both Purkinje and STN neurons that lack Nav1.6 but resurgent current being reduced much more in Purkinje neurons than in STN neurons.
Resurgent current in STN neurons is faster to rise and faster to decay with Nav1.6 present. Such a difference would be expected if the hypothetical blocking particle binds more weakly to Nav1.6 channels than non-Nav1.6 channels. More rapid unbinding of the particle from channels during repolarization would result in a faster rise of resurgent current, and biasing the equilibrium between open and blocked states in favor of open states would result in a faster decay of resurgent current because channels are delivered more quickly to conventional inactivated states. Such a kinetic difference also would tend to produce a larger peak resurgent current and may account in part for the greater effectiveness of Nav1.6 channels in producing such current. However, the most important factor in this regard is likely to be a slower rate of conventional inactivation in Nav1.6 channels than non-Nav1.6 channels (at least during strong depolarizations), allowing a larger fraction of channels to bind the "resurgent" blocking particle before undergoing conventional inactivation (Grieco and Raman 2004).
In principle, a reduction in the magnitude of resurgent current relative to transient current (as seen in both STN and Purkinje neurons) might be expected to be accompanied by a reduction in the magnitude of the slow phase of decay of transient current. In fact, however, the relative contribution of the slow time constant of inactivation showed little change in med compared with wild-type mice in either STN or Purkinje neurons (while the speed of decay of this component changed in med Purkinje cells but not STN neurons). Interpretation of the kinetics of overall transient current is difficult because it reflects the combined current of all the sodium channels present, including any that do not form resurgent current. Even a single-channel type with only conventional activation can have inactivation kinetics with two time constants (Spampanato et al. 2001). Thus although the decay kinetics of overall transient current could be fit reasonably well by two time constants, the slow time constant probably reflects not just the unbinding of the "resurgent" blocking particle but also conventional inactivation of a complex mixture of multiple channel types, some of which may be upregulated in response to loss of Nav1.6.
It was surprising that the firing properties of STN neurons studied in brain slice were so little different in homozygous med mutants compared with wild-type littermates. Neither the frequency of spontaneous firing nor the frequency of firing in response to current injection were different. The only clear difference was a reduction of burst firing after switching off of large current injections that induced depolarization block; this reduction of burst firing in STN neurons is reminiscent of a reduction in all-or-none burst firing (fewer spikes per burst stimulated with brief current injections) seen in Purkinje neurons from Nav1.6-null mice (Raman et al. 1997). Resurgent current immediately after a spike is expected to promote firing of a subsequent spike, both as a result of the inward resurgent current itself and because the flow of this current appears to be associated with rapid recovery from inactivation (Raman and Bean 2001; Raman et al. 1997). The number of spikes in a burst may be very sensitive to the level of resurgent current since spike success or failure depends on whether the sum of a number of large currents following a spike is barely net inward or barely net outward (Swensen and Bean 2003).
Previous work in Purkinje and STN neurons has suggested that resurgent current also helps generate the rapid spontaneous firing that is typical of these cell types (Do and Bean 2003; Khaliq et al. 2003), so a reduction in resurgent current by an average of 63% would be expected to result in a significantly lower frequency of firing if there were no compensatory changes in other currents. However, it is very plausible that other currents do change in the absence of Nav1.6 in a manner to favor faster pacemaking. In Purkinje neurons from med mice, Khaliq and colleagues (2003) showed that the voltage dependence of a component of potassium current is altered so as to be less readily activated (midpoint shifted in the depolarizing direction) and that input resistance of the cells is increased; their modeling showed that both changes tend to promote faster firing. There is also an upregulation in the magnitude of both T- and P-type calcium currents in Purkinje neurons of med mice (A. Swensen and B. P. Bean, unpublished results). If similar changes in input resistance, voltage-activated potassium currents, or calcium currents occur in STN neurons, they could produce faster pacemaking than would otherwise be the case.
Another possibility relates to the fact that the current-clamp experiments were done with STN neurons in brain slice, while changes in sodium current under voltage clamp were studied in acutely dissociated cell bodies (necessary to ensure adequate voltage control). In both Purkinje neurons and retinal ganglion neurons, Nav1.6 channels are clustered at high density at the initial segment of the axon by a mechanism involving ankyrin-G (Boiko et al. 2003; Jenkins and Bennett 2001), and in layer 5 pyramidal neurons and subicular neurons, spike formation occurs first in the axon, 
30 µm away from the soma (Colbert and Johnson 1996; Stuart et al. 1997). There is also evidence that axonal channels have different voltage dependence, requiring smaller depolarizations for activation (Colbert and Pan 2002). Thus the current-clamp behavior of cells in brain slice may depend most strongly on the properties and density of sodium channels in the axon. Previous experiments relating changes in sodium current to changes in firing characteristics of Purkinje neurons used dissociated neurons for current-clamp as well as voltage-clamp experiments, allowing direct comparison between the two sets of measurements. Unfortunately, we were unable to obtain good current-clamp recordings from STN neurons dissociated from med mice; the cells generally were not robust enough to sustain spontaneous activity (unlike their behavior when studied in brain slice), so it was not feasible to examine the current-clamp properties in dissociated neurons.
Whatever the explanation for the relatively minor changes in current-clamp behavior of STN neurons from med mice, the experiments suggest that the functional role of STN neurons may be considerably less affected by loss of Nav1.6 channels than that of Purkinje neurons and that changes in STN neuron function probably play little role in the motor deficits of Nav1.6-null mice. In this regard, an important limitation of our study is that expression of Na channels changes during development, and our analysis was of necessity confined to relatively young animals. Nav1.6-null animals die at 3 wk, probably in connection to the replacement of Nav1.2 by Nav1.6 that normally occurs at this time in nodes of Ranvier (Boiko et al. 2001). Thus it is possible that in fully mature mice, elimination of Nav1.6 channels would have more dramatic consequences for the function of STN neurons, especially if this could be studied in the absence of compensatory changes in expression of other channels.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. P. Bean, Dept. of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115 (E-mail: bruce_bean{at}hms.harvard.edu).
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REFERENCES |
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Beurrier C, Congar P, Bioulac B, and Hammond C. Subthalamic nucleus neurons switch from single-spike activity to burst-firing mode. J Neurosci 19: 599–609, 1999.
Bevan MD and Wilson CJ. Mechanisms underlying spontaneous oscillation and rhythmic firing in rat subthalamic neurons. J Neurosci 19: 7617–7628, 1999.
Boiko T, Rasband MN, Levinson SR, Caldwell JH, Mandel G, Trimmer JS, and Matthews G. Compact myelin dictates the differential targeting of two sodium channel isoforms in the same axon. Neuron 30: 91–104, 2001.[CrossRef][ISI][Medline]
Boiko T, Van Wart A, Caldwell JH, Levinson SR, Trimmer JS, and Matthews G. Functional specialization of the axon initial segment by isoform-specific sodium channel targeting. J Neurosci 23: 2306–2313, 2003.
Burgess DL, Kohrman DC, Galt J, Plummer NW, Jones JM, Spear B, and Meisler MH. Mutation of a new sodium channel gene, Scn8a, in the mouse mutant motor endplate disease. Nat Genet 10: 461–465, 1995.[CrossRef][ISI][Medline]
Caldwell JH, Schaller KL, Lasher RS, Peles E, and Levinson SR. Sodium channel Nav1.6 is localized at nodes of ranvier, dendrites, and synapses. Proc Natl Acad Sci USA 97: 5616–5620, 2000.
Catterall WA. From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26: 13–25, 2000.[CrossRef][ISI][Medline]
Colbert CM and Johnston D. Axonal action-potential initiation and Na+ channel densities in the soma and axon initial segment of subicular pyramidal neurons. J Neurosci 16: 6676–6686, 1996.
Colbert CM and Pan E. Ion channel properties underlying axonal action potential initiation in pyramidal neurons. Nat Neurosci 5: 533–538, 2002.[CrossRef][ISI][Medline]
Do MTH and Bean BP. Subthreshold sodium current and pacemaking of subthalamic neurons: modulation by slow inactivation. Neuron 39: 109–120, 2003.[CrossRef][ISI][Medline]
Felts PA, Yokoyama S, Dib-Hajj S, Black JA, and Waxman SG. Sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1): different expression patterns in developing rat nervous system. Brain Res Mol Brain Res 45: 71–82, 1997.[Medline]
Garcia KD, Sprunger LK, Meisler MH, and Beam KG. The sodium channel Scn8a is the major contributor to the postnatal developmental increase of sodium current density in spinal motoneurons. J Neurosci 18: 5234–5239, 1998.
Goldin AL. Resurgence of sodium channel research. Annu Rev Physiol 63: 871–894, 2001.[CrossRef][ISI][Medline]
Grieco TM, Afshari FS, and Raman IM. A role for phosphorylation in the maintenance of resurgent sodium current in cerebellar purkinje neurons. J Neurosci 22: 3100–3107, 2002.
Grieco TM and Raman IM. Production of resurgent current in Nav1.6-null Purkinje neurons by slowing sodium channel inactivation with 
-pompilidotoxin. J Neurosci 24: 35–42, 2004.
Hamann M, Meisler MH, and Richter A. Motor disturbances in mice with deficiency of the sodium channel gene Scn8a show features of human dystonia. Exp Neurol 184: 830–838, 2003.[CrossRef][ISI][Medline]
Herzog RI, Liu C, Waxman SG, and Cummins TR. Calmodulin binds to the C terminus of sodium channels Nav1.4 and Nav1.6 and differentially modulates their functional properties. J Neurosci 23: 8261–8270, 2003.
Jenkins SM and Bennett V. Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments. J Cell Biol 155: 739–746, 2001.
Kearney JA, Buchner DA, De Haan G, Adamska M, Levin SI, Furay AR, Albin RL, Jones JM, Montal M, Stevens MJ, Sprunger LK, and Meisler MH. Molecular and pathological effects of a modifier gene on deficiency of the sodium channel Scn8a (Na(v)1.6). Hum Mol Genet 11: 2765–2775, 2002.
Khaliq ZM, Gouwens NW, and Raman IM. The contribution of resurgent sodium current to high-frequency firing in Purkinje neurons: an experimental and modeling study. J Neurosci 23: 4899–4912, 2003.
Kohrman DC, Harris JB, and Meisler MH. Mutation detection in the med and medJ alleles of the sodium channel Scn8a: unusual splicing due to a minor class AT-AC intron. J Biol Chem 271: 17576–17581, 1996.
Krzemien DM, Schaller KL, Levinson SR, and Caldwell JH. Immunolocalization of sodium channel isoform NaCh6 in the nervous system. J Comp Neurol 420: 70–83, 2000.[CrossRef][ISI][Medline]
Maurice N, Tkatch T, Meisler M, Sprunger LK, and Surmeier DJ. D1/D5 dopamine receptor activation differentially modulates rapidly inactivating and persistent sodium currents in prefrontal cortex pyramidal neurons. J Neurosci 21: 2268–2277, 2001.
Meisler MH, Kearney J, Escayg A, MacDonald BT, and Sprunger LK. Sodium channels and neurological disease: insights from Scn8a mutations in the mouse. Neuroscientist 7: 136–145, 2001.[Abstract]
Mercer JN, Chan CS, and Surmeier DJ. Sodium channels in neurons of the globus pallidus. Soc Neurosci Abstr 706.12, 2003.
Neher E. Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol 207: 123–131, 1992.[ISI][Medline]
Pan F and Beam KG. The absence of resurgent sodium current in mouse spinal neurons. Brain Res 849: 162–168, 1999.[CrossRef][ISI][Medline]
Qu Y, Curtis R, Lawson D, Gilbride K, Ge P, DiStefano PS, Silos-Santiago I, Catterall WA, and Scheuer T. Differential modulation of sodium channel gating and persistent sodium currents by the beta1, beta2, and beta3 subunits. Mol Cell Neurosci 18: 570–580, 2001.[CrossRef][ISI][Medline]
Raman IM and Bean BP. Resurgent sodium current and action potential formation in dissociated cerebellar Purkinje neurons. J Neurosci 17: 4517–26, 1997.
Raman IM and Bean BP. Inactivation and recovery of sodium currents in cerebellar Purkinje neurons: evidence for two mechanisms. Biophys J 80: 729–37, 2001.
Raman IM, Gustafson AE, and Padgett D. Ionic currents and spontaneous firing in neurons isolated from the cerebellar nuclei. J Neurosci 20: 9004–9016, 2000.
Raman IM, Sprunger LK, Meisler MH, and Bean BP. Altered subthreshold sodium currents and disrupted firing patterns in Purkinje neurons of Scn8a mutant mice. Neuron 19: 881–891, 1997.[CrossRef][ISI][Medline]
Schaller KL and Caldwell JH. Developmental and regional expression of sodium channel isoform NaCh6 in the rat central nervous system. J Comp Neurol 420: 84–97, 2000.[CrossRef][ISI][Medline]
Schaller KL and Caldwell JH. Expression and distribution of voltage-gated sodium channels in the cerebellum. Cerebellum 2: 2–9, 2003.[CrossRef][ISI][Medline]
Smith MR, Smith RD, Plummer NW, Meisler MH, and Goldin AL. Functional analysis of the mouse Scn8a sodium channel. J Neurosci 18: 93–102, 1998.
Song WJ, Baba Y, Otsuka T, and Murakami F. Characterization of Ca2+ channels in rat subthalamic nucleus neurons. J Neurophysiol 84: 2630–2637, 2000.
Spampanato J, Escayg A, Meisler MH, and Goldin AL. Functional effects of two voltage-gated sodium channel mutations that cause generalized epilepsy with febrile seizures plus type 2. J Neurosci. 21: 7481–7490, 2001.
Sprunger LK, Escayg A, Tallaksen-Greene S, Albin RL, and Meisler MH. Dystonia associated with mutation of the neuronal sodium channel Scn8a and identification of the modifier locus Scnm1 on mouse chromosome 3. Hum Mol Genet 8: 471–479, 1999.
Stuart G, Schiller J, and Sakmann B. Action potential initiation and propagation in rat neocortical pyramidal neurons. J Physiol 505: 617–632, 1997.[CrossRef][ISI][Medline]
Swensen AM and Bean BP. Ionic mechanisms of burst firing in dissociated Purkinje neurons. J Neurosci 23: 9650–9663, 2003.
Vega-Saenz de Miera EC, Rudy B, Sugimori M, and Llinas R. Molecular characterization of the sodium channel subunits expressed in mammalian cerebellar Purkinje cells. Proc Natl Acad Sci USA 94: 7059–7064, 1997.
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0.05; **, P 
. Plotted is mean – SD for wild-type (
) and mean + SD (
) for med neurons. n = 17 (STN wild-type), 8 (STN med), 6 (Purkinje wild-type), 10 (Purkinje med).
