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1Department of Human Genetics and 2Unit for Laboratory Animal Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan, and 3Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois
Submitted 9 November 2005; accepted in final form 5 May 2006
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ABSTRACT |
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subunit NaV1.6, which is widely expressed throughout the nervous system. Global null mutations that eliminate Scn8a in all cells result in severe motor dysfunction and premature death, precluding analysis of the physiological role of NaV1.6 in different neuronal types. To test the effect of cerebellar NaV1.6 on motor coordination in mice, we used the Cre-lox system to eliminate Scn8a expression exclusively in Purkinje neurons (Purkinje KO) and/or granule neurons (granule KO). Whereas granule KO mice had only minor behavioral defects, adult Purkinje KO mice exhibited ataxia, tremor, and impaired coordination. These disorders were exacerbated in double mutants lacking Scn8a in both Purkinje and granule cells (double KO). In Purkinje cells isolated from adult Purkinje KO and double KO but not granule KO mice, the ratio of resurgent-to-transient tetrodotoxin- (TTX)-sensitive Na current amplitudes decreased from 
15 to 5%. In cerebellar slices, Purkinje cell spontaneous and maximal firing rates were reduced 10-fold and twofold relative to control in Purkinje KO and double KO but not granule KO mice. Additionally, short-term plasticity of high-frequency parallel fiber EPSCs was altered relative to control in Purkinje KO and double KO but not granule KO mice. These data suggest that the specialized kinetics of Purkinje Na channels depend directly on Scn8a expression. The loss of these channels leads to a decrease in Purkinje cell firing rates as well as a modification of the synaptic properties of afferent parallel fibers, with the ultimate consequence of disrupting motor behavior. |
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INTRODUCTION |
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subunits NaV1.1, NaV1.2, and NaV1.6, encoded by the genes Scn1a, Scn2a, and Scn8a, are the major channels carrying Na currents underlying action potentials in mature mammalian central neurons. Most neurons express more than one of these subunits, raising the question of whether these channels make distinct contributions to signaling. Mice with global null mutations of Scn1a, Scn2a, or Scn8a, in which the expression of any one type of Na channel is eliminated throughout the nervous system, each exhibit a different disorder, demonstrating that the channels are not functionally redundant in vivo (Burgess et al. 1995
; Planells-Cases et al. 2000; Yu et al. 2004). Because all these global null mutations are lethal, however, it has not been possible to dissect the different roles of these Na channels in specific adult behaviors.
A salient characteristic of spontaneous mutations of Scn8a is that various motor disorders develop during the second postnatal week, most of which progressively worsen until the animals die at around 3 wk of age (Hamann et al. 2003; Kearney et al. 2002; Meisler and Kearney 2005; Meisler et al. 2004). Because NaV1.6 replaces NaV1.2 in myelinated axons during this period of postnatal development (Boiko et al. 2001, 2003; Caldwell et al. 2000), changes in action potential conduction in peripheral motoneurons probably contribute to the pathology of global Scn8a null mice. Other aspects of the motor disorders are likely to arise from inappropriate signaling through brain regions that regulate coordination, such as the cerebellum and basal ganglia. In cerebellar Purkinje cells of Scn8a null mice, for example, TTX-sensitive resurgent and steady-state Na currents decrease disproportionately relative to transient current, changing the kinetics of total Na currents in Purkinje cells (Raman and Bean 1997). These altered kinetics decrease the rate of action potential firing measured in isolated Purkinje cells at room temperature (Khaliq et al. 2003), which may contribute in part to the observed phenotype. In global Scn8a null mice, however, the Scn8a gene is absent in all cells, throughout development, making it difficult to infer how the presence or absence of NaV1.6 from Purkinje cells directly affects either cellular properties or motor output.
To evaluate the relationship between motor control and the effects of Scn8a-dependent Na currents on Purkinje cell firing, we used a "floxed" allele of Scn8a (Levin and Meisler 2004) to generate mice lacking NaV1.6 exclusively in either Purkinje or cerebellar granule cells. We also generated double mutants lacking NaV1.6 in both cell types. In addition to assessing motor behavior in normal and mutant adult mice, we recorded Na currents in isolated Purkinje cells, action potential firing in cerebellar slices and trains of EPSCs at parallel fiber to Purkinje synapses. The results support the idea that the NaV1.6 channel carries resurgent Na currents and permits high-frequency firing by Purkinje neurons. Additionally, expression of this channel in Purkinje cells, but not granule cells, can regulate the pattern of parallel fiber short-term plasticity. These changes in intrinsic and synaptic activity, which can be induced by loss of Scn8a from Purkinje cells exclusively, are sufficient to produce ataxia, tremor, and poor motor coordination.
METHODS
Mice
All procedures were carried out in accordance with institutional guidelines. Mice with a conditional "floxed" allele of Scn8a (B6;129-Scn8atm1Mm) contain loxP sites flanking the first coding exon (exon 1), which is deleted by exposure to Cre recombinase (Cre; Fig. 1A). Deletion of exon 1 in Purkinje cells utilized Pcp2-Cre transgenic mice (Jackson Laboratory No. 004146) with the Cre cDNA inserted into exon 4 of a 3-kb Pcp2 (L7) minigene (Barski et al. 2000). Deletion in granule cells utilized BAC6-Cre transgenic mice with Cre regulated by the GABAA receptor 6 subunit promoter (Aller et al. 2003). In crosses with the ROSA26 Cre reporter line (Soriano 1999), we confirmed expression of Pcp2-Cre in Purkinje cells only and expression of 6-Cre in granule cells with occasional positive cells in the cerebellar nuclei, pontine nuclei, and cortex (not shown).
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). Crossing Scn8aflox/+ mice with Pcp2-Cre mice or 6-Cre mice generated Scn8aflox/+,Cre+ mice, which were then crossed with Scn8aflox/flox mice to produce Purkinje Scn8a knockout mice ("Purkinje KO"; Scn8aflox/flox,Pcp2-Cre+), granule Scn8a KO mice ("granule KO"; Scn8aflox/flox,6-Cre+), and Cre-negative control mice (Scn8aflox/flox). Double Scn8a KO mice ("double KO"; Scn8aflox/flox,Pcp2-Cre+,6-Cre+) were generated from the cross (Scn8aflox/flox,Pcp2-Cre+ X Scn8aflox/+,6-Cre+). Offspring were recovered in predicted Mendelian ratios. As described for other constructs (Zhao et al. 2001), expression of Cre transgenes in the male germline resulted in occasional transmission of the deleted allele Scn8adel by Scn8aflox,Cre+ males. We observed no phenotypic differences between Scn8aflox/del,Cre+ and Scn8aflox/flox,Cre+ mice, which were combined for analysis.
Wild-type, floxed, and deleted alleles of Scn8a were genotyped by PCR of genomic DNA with primer pair F2/R2 (Levin and Meisler 2004). Cre transgenes were genotyped with the Cre-specific forward primer 5'-ACT TAG CCT GGG GGT AAC TAA ACT-3' and reverse primer 5'GGT ATC TCT GAC CAG AGT CAT CCT 3'. Mice carrying both Cre transgenes were genotyped with two primer pairs: a forward primer from the Pcp2-Cre promoter region, 5'CAA TGT CTG ACC AAA TAC CAC CAC 3', with the Cre reverse primer 5'GCT GGA TAG TTT TTA CTG CCA GAC 3', and a forward primer upstream of the first coding exon of the 6 GABAA receptor gene, 5'-AGA TAC CAC TGC TTT CCA GAT TTC 3', with the Cre reverse primer 5'ATT TCC GGT TAT TCA ACT TGC ACC 3'.
Southern blot and RT-PCR
Southern blotting was performed on genomic DNA prepared from dissected cerebellum from 6- to 10-mo-old mice after digestion with BstXI (New England Biolabs, Beverly, MA). The 0.3-kb hybridization probe (Fig. 1A) with 150 bp of flanking sequence upstream of exon 1 and 150 bp downstream of exon 1 was generated by PCR of genomic DNA from Scn8adel/del mice using primer pair F2/R2 (Levin and Meisler 2004). RNA was isolated from dissected cerebellum. RT-PCR was carried out with forward primer in the 5'UTR (5'TCA TGC CAA CTT CAC AGA TCT ACC 3') (Drews et al. 2005) and reverse primer in exon 4 (5'CAG AAA CCT CTG GCG ATG ATT TTC 3'). Specific amplification of transcripts lacking exon 1 was obtained with the reverse primer from exon 4 and an allele-specific forward primer spanning the junction between the 5'UTR and exon 2 (5' TAC CCA CTG AGT TTG GAG AAG ACT 3').
Histology
Mice (8–16 wk old) were perfused with phosphate buffered saline followed by 10% neutral buffered formalin or 4% paraformaldehyde. Brains were embedded in paraffin. Hematoxylin and eosin staining was performed on 3-µm parasagittal sections. For immunohistochemistry, 40-µm sections were permeabilized for 2 h with 0.1M Tris buffer (pH 7.6) containing 0.1% Triton X-100, 10% normal goat serum, and 0.05% BSA and incubated with monoclonal anti-calbindin D28K (1:2000; clone CB-955: Sigma) for 48 h at 4°C. Immunoreactivity was visualized with an anti-mouse-Alexa 488 secondary antibody (1:300; Molecular Probes). Images were captured with an Olympus FV-500 confocal microscope in The University of Michigan Microscopy and Image Analysis Laboratory.
Motor function
Rotarod performance was evaluated as described (Clark et al. 1997) with three trials per day on four consecutive days and was carried out blind to genotype. The animal was placed on the rotarod (Ugo Basile, Varese, Italy), which accelerated linearly from 4 to 40 rpm during the first 5 min of each trial, followed by 40 rpm for 5 min. The latency to fall was recorded for each animal. P values were calculated with paired two-tailed t-test with unequal variance. Motor strength was evaluated with a hanging wire apparatus (Crawley 2000). For the vertical hanging wire test, mice were placed on the parallel bars of a standard mouse box lid. For the horizontal test, mice were placed on a 12 x 20-cm wire grid with 0.7 cm spacing. The apparatus was gently shaken three times and held vertically or inverted horizontally; time to fall was recorded.
Recordings from isolated cells.
Purkinje cells were acutely isolated from 7- to 8-wk-old control and littermate Purkinje, granule, or double KO mice (Grieco et al. 2002; Raman et al. 1997; Regan 1991), and neurons recovered in the recording chamber in Tyrode's solution (which contained, in mM, 150 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, and 10 glucose; pH 7.4 with NaOH). Whole cell voltage-clamp recordings were made from Purkinje cell bodies, 1–6 h after trituration. Cells were maintained at 22–24°C rather than at elevated temperatures to improve the quality of voltage clamp of voltage-gated Na currents. Patch pipettes (1.8–3.5 M
) were wrapped with Parafilm to reduce capacitance and were filled with (in mM) 108 CsCH3SO3, 9 NaCl, 1.8 MgCl2, 9 HEPES, 1.8 EGTA, 63 sucrose, 4 MgATP, and 0.3 Tris-GTP; pH 7.3 with CsOH. Data were recorded with an Axopatch 200B amplifier and pClamp 9.0 software (Axon Instruments). Series resistance was compensated >80%. Recordings were made in extracellular solutions controlled with gravity-driven flow pipes containing Tyrode's solution, 10 mM TEACl, and 300 µM CdCl2 ±300 nM tetrodotoxin (TTX). TTX-sensitive Na currents were obtained by subtraction. Cells were held at –90 mV. Transient current was evoked by step depolarizations and resurgent current was evoked by step repolarizations following 5- or 10-ms steps to +30 mV (Raman and Bean 1997) as resurgent current amplitudes vary by <2% across this range of conditioning step durations (Raman and Bean 2001). Both durations were used in all genotypes.
Slice recordings
Mice (7–8 wk old) were anesthetized with halothane and perfused with artificial cerebrospinal fluid (ACSF; which contained in mM: 123.75 NaCl, 3.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, and 10 glucose, 4°C) and then decapitated (Khaliq and Raman 2005). Parasagittal cerebellar slices (200 µm) were incubated in ACSF (32°C) bubbled with 95% O2-5% CO2 for >1 h. Patch pipettes (3–5 M) were filled with (in mM) 108 KCH3SO3, 9 NaCl, 1.8 MgCl2, 0.9 EGTA, 9 HEPES, 14 Tris-creatine phosphate, 4 MgATP, and 0.3 Tris GTP; pH 7.3 with KOH. Whole cell recordings from Purkinje cells were made near physiological temperature (33–35°C) with an Axoclamp 2B amplifier (for action potentials) or an Axopatch 200B amplifier (for EPSCs) and pClamp 8.0 software (Axon Instruments) within 6 h of incubation. GABAA receptors were blocked with 10 µM SR95531. For current-clamp recordings, after spontaneous firing was recorded, cells were silenced by holding near –65 mV, and firing was evoked by 400-ms depolarizing current steps. Current amplitudes were increased until the cell failed to fire throughout the step and instead entered depolarization block. The maximal sustained firing rate was recorded as the rate achieved on the step preceding entry into depolarization block. For voltage-clamp recordings, cells were filled with the Cs-based solution used in recordings of isolated cells, to which 600 µM QX-314 was added to block Na channels. Cells were held at –70 mV, and parallel fiber EPSCs were evoked with a theta glass stimulating electrode placed 50–100 µm from the cell body in the molecular layer. The theta glass pipette was filled with 145 mM NaCl and 10 mM HEPES, (pH 7.4 with NaOH). The stimulation and ground electrodes were controlled by an IsoFlex stimulus isolation unit (A.M.P.I., Jerusalem, Israel). Stimuli were delivered for 100 µs and the intensity was adjusted from 1 to 8 V to evoke EPSPs of an initial amplitude between 150 and 350 pA. Trains were evoked with a Master 8 controller (A.M.P.I.). Drugs were obtained from Sigma-Aldrich except TTX (Alomone Labs, Jerusalem). Data were analyzed with IgorPro 4.06 (WaveMetrics, Lake Oswego, OR). For analysis of EPSC trains, EPSC amplitude was measured as the peak EPSC relative to the decaying baseline of the preceding EPSC. Statistical significance was assessed with Student's unpaired two-tailed t-test, except for the EPSC data, which were analyzed with a repeated-measures univariate ANOVA (SPSS).
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RESULTS |
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Mice homozygous for the floxed allele of Scn8a produce normal levels of NaV1.6 protein in the absence of Cre recombinase (Cre), but in the presence of Cre recombinase, Scn8a mRNA and protein are not detectable (Levin and Meisler 2004). We crossed Scn8aflox/flox mice with mice expressing Cre under the control of a Purkinje-specific promoter (Pcp2-Cre) (Barski et al. 2000) and a granule-specific promoter (6-Cre) (Aller et al. 2003) to generate Purkinje KO mice, granule KO mice, and double KO mice. Based on the onset of Cre expression in these lines (Aller et al. 2003; Barski et al. 2000), NaV1.6 protein levels are expected to be normal during prenatal development and to decrease gradually after P7.
To verify deletion of Scn8a exon 1 in adult cerebellum, genomic DNA was isolated from mouse cerebella and examined by Southern blotting with a probe to sequences flanking exon 1. In control DNA a 1.7-kb hybridizing fragment is predicted, and deletion of exon 1 reduces the size of the hybridizing fragment to 1.2 kb (Fig. 1A). Cerebellar DNA from Scn8aflox/flox controls contained only the 1.7-kb fragment (Fig. 1B, lane 1), while DNA from Scn8aflox/del heterozygous controls contained the 1.7- and 1.2-kb fragments in equal abundance (Fig. 1B, lane 5). In granule KO and double KO mice, the 1.2-kb fragment was more abundant (Fig. 1B, lanes 3 and 4), consistent with deletion of exon 1 in a majority of granule cells, which constitute 80% of cells in the cerebellum (Altman and Bayer 1997; Herculano-Houzel and Lent 2005). Given that fewer than 0.15% of cerebellar neurons are Purkinje cells (Altman and Bayer 1997), it was not surprising that the 1.2-kb fragment could not be detected in Purkinje KO mice (Fig. 1B, lane 2).
Therefore to confirm deletion of exon 1 in the Purkinje KO mice, we examined Scn8a transcripts by RT-PCR of cerebellar RNA with a forward primer in the 5' UTR and a reverse primer in exon 4. As expected, RNA from Scn8aflox/flox control generated a 650-bp RT-PCR product containing exon 1, whereas RNA from Scn8aflox/del heterozygous controls produced a 325 bp product lacking exon 1 in addition to the 650 bp product (Fig. 1C). Both fragments were amplified from cerebellar RNA from Purkinje, granule, and double KO mice, indicating that the deleted transcript was present in mice of all three genotypes (Fig. 1C). The transcript lacking exon 1 was also detected with an allele-specific forward primer that spanned the junction between the 5'UTR and exon 2 that is present only in the deleted transcript. The allele-specific RT-PCR product was obtained from all knockout mice (Fig. 1D), verifying deletion of exon 1 in Purkinje and granule cells.
Cerebellar morphology has been reported to be normal in mice with global loss of Scn8a expression (Dick et al. 1986; Sprunger et al. 1999). Consistent with these reports, no severe cerebellar malformations were detected in brains from 8-wk-old Purkinje, granule, and double KO mice. Foliation was normal, and layering and cellular organization showed no obvious defects (Fig. 2A), suggesting that the cerebellum developed normally and that postnatal knockout of Scn8a did disrupt gross cerebellar structure. Calbindin staining indicated that the Purkinje cells in the mutant mice retained the regular orientation of dendrites present in control animals (Fig. 2B). The only abnormality noted was scattered calbindin staining in the granule cell layer of Purkinje KO and double KO but not control or granule KO mice (Fig. 2B, arrows); such labeling has previously been described in the Scn8amed-jo (A1071T) mice and may reflect axonal swelling (Dick et al. 1985).
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The KO mice were generally vigorous and exhibited no signs of the dystonia, paralysis, or juvenile lethality characteristic of global Scn8a mutants (Meisler et al. 2004). By 6–8 wk of age, however, Purkinje KO mice developed a mildly ataxic, waddling gait that appeared to result from poor hindlimb coordination. The ataxia resembled that of pcd (Purkinje cell degeneration) mutant mice, which lose most Purkinje cells by P28 (Grüsser-Cornehls and Bäurle 2001). Granule KO mice, in contrast, had no visible motor impairment. Severe ataxia was evident in double KO mice; both forelimbs and hindlimbs were affected, resulting in a slow, jerky, wobbling gait, with tails held erect. At 5–6 wk of age, double KO mice also developed a tremor that was most pronounced during movement. The gait of mice with each genotype is shown in the Supplemental Video.
Motor coordination was assessed quantitatively with the rotarod test. Control mice improved markedly (P < 0.001) during four daily sessions of training (Fig. 3). Although granule KO mice did not differ from controls on day 1 (P > 0.5), their rate of improvement was slower (Fig. 3). The initial performance of Purkinje and double KO mice was impaired, and they demonstrated little improvement during the training period (Fig. 3).
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; Sprunger et al. 1999). Na currents in Purkinje cells of cell-specific Scn8a KO mice
Na channels of normal Purkinje neurons produce transient currents on depolarization and resurgent currents on repolarization. To test how these currents were affected by loss of Scn8a expression in either Purkinje cells only, granule cells only, or both, we recorded voltage-clamped transient and resurgent Na currents from Purkinje neurons acutely isolated from adult mice. Recordings were made in physiological (155 mM) Na to increase resurgent current amplitudes (Afshari et al. 2004) and to facilitate detection of the small, NaV1.6-independent resurgent currents predicted to be present in Scn8a-lacking Purkinje cells (Grieco and Raman 2004). In Purkinje cells from control mice, voltage steps from –90 to 0 mV evoked peak transient currents of –6.2 ± 0.9 nA (n = 12; Fig. 4, A and B). Purkinje cells from all three KO mice had robust transient currents, with amplitudes that were not significantly different from control (Purkinje KO, –4.3 ± 0.7 nA, n = 13, P = 0.14; granule KO, –3.9 ± 0.7 nA, n = 6, P = 0.08; double KO, –4.6 ± 0.8 nA, n = 5, P = 0.24; Fig. 4, A and B). As in 2- to 3-wk-old global Scn8a null mice (Raman et al. 1997), however, Scn8a loss had a greater effect on resurgent than on transient current as made evident by measuring the ratio of resurgent current at –30 mV to transient current at 0 mV (Fig. 4, A and C). Specifically, in Purkinje cells with unaltered Scn8a expression, steps from +30 to –30 mV evoked a resurgent current of –868 ± 123 pA (control) and –561 ± 105 pA (granule KO), yielding resurgent-to-transient current ratios of 15 ± 2 and 17 ± 4% (P = 0.7). In contrast, in Scn8a-KO Purkinje cells, resurgent current was only –181 ± 29 pA (Purkinje KO) and –261 ± 59 pA (double KO), giving current ratios of 5 ± 1 and 6 ± 1% (P < 0.001, P = 0.002). These results not only provide evidence that resurgent current remains a property of normal Purkinje neurons into adulthood but also suggest that loss of this component of current in mutant Purkinje neurons is a direct consequence of loss of Scn8a expression.
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), we recorded action potentials of Purkinje cells in cerebellar slices at 33–35°C. Like the amplitude of resurgent current, firing rates depended strongly on whether Scn8a was present in Purkinje cells. Cells were hyperpolarized to –65 mV to silence spontaneous firing and action potentials were evoked with depolarizing current steps. Cells from control mice responded to 1.3-nA steps by firing at rates >150 spikes/s (Fig. 5, A and B), with maximum firing rates of 227 ± 31 spikes/s (Khaliq and Raman 2005). Most control Purkinje cells (9/10) were also spontaneously active, firing at a mean rate of 71 ± 15 spikes/s (Fig. 5C). In granule KO mice, the firing of Purkinje cells was nearly identical to control, with spontaneous and maximal rates of 86 ± 14 and 230 ± 10 spikes/s (n = 5, P = 0.5, P = 0.9; Fig. 5C). In both Purkinje and double KO mice, however, both spontaneous and maximal firing rates were decreased relative to control (Fig. 5C; Purkinje KO, 7 ± 5 and 93 ± 19 spikes/s, n = 12, P = 0.002, 0.002; double KO, 5 ± 5 and 148 ± 6 spikes/s, n = 7, P = 0.002, 0.03). The approximately twofold decrease in the slope of the input/output curve in Purkinje and double KO mice (Fig. 5B) suggests that the number of Purkinje cell spikes evoked by any given amount of synaptic excitation will be decreased relative to control and that the inhibitory drive onto target cells in the cerebellar and vestibular nuclei is likely to be reduced.
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). Interestingly, although these changes do not restore normal excitability, they tend to promote firing and therefore appear to compensate partially for the disrupted Na currents. To test whether the loss of Scn8a expression might induce changes at the circuit as well as at the cellular level, we recorded parallel fiber excitatory postsynaptic currents (EPSCs) in Purkinje cells from control and cell-specific Scn8a KO mice. In control animals, single EPSCs decayed with a single exponential time constant of 8.5 ± 0.1 ms (n = 7). The decay time was unchanged in granule and double KO mice (8.1 ± 0.09, n = 7, P = 0.77; 8.5 ± 0.01, n = 5, P = 0.98) but tended to be briefer in Purkinje KOs 5.8 ± 0.04; n = 5, P = 0.06). Trains of EPSCs were evoked at 10, 50, 100, and 200 Hz, and currents were normalized to the peak of the first EPSC in the train (Fig. 6A). To detect differences in EPSCs that were more likely to depend on release properties than postsynaptic kinetics, we analyzed the amplitude of each EPSC relative to the current just before the preceding stimulus artifact (Fig. 6B). At all frequencies, control and granule KO EPSC amplitudes tended to overlap, whereas EPSCs from Purkinje and double KO mice had larger relative EPSC amplitudes, particularly at higher stimulus frequencies, as a result of more facilitation and/or less depression. Analysis of EPSC amplitudes indicated a significant interaction of genotype and stimulus number at all four stimulus rates [10, 50, 100, 200 Hz: F(57,380) = 2.18, 2.32, 1.74, 2.98; P < 0.001, <0.001, <0.01, <0.001]. Relative to control, at 10 Hz, facilitation was significantly greater in double KO mice [F(19,190) = 3.06, P < 0.001], and at 50 Hz, facilitation was greater and steady-state depression was reduced in Purkinje KO mice [F(19,190) = 5.50, P < 0.001]. At 100 and 200 Hz, Purkinje and double KO EPSCs facilitated more than controls [100 Hz, 200 Hz: Purkinje versus control, F(19,190) = 2.30, 4.85; P < 0.01, < 0.001; double vs. control, F(19,190) = 2.92, 2.46, P < 0.001, <0.01]. Short-term plasticity in the granule KO mice, however, was statistically indistinguishable from controls [10, 50, 100, and 200 Hz: F(19,228) = 0.98, 1.61, 0.27, 1.45; P = 0.49, 0.06, 0.99, 0.11]. These results indicate that the most significant changes in granule-to-Purkinje synaptic transmission occurred when Purkinje rather than granule cells lacked Scn8a, suggesting that the activity of Purkinje cells regulates the strength of their afferents.
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DISCUSSION |
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). Two observations suggest that NaV1.6 protein persists for several weeks after Cre activation: at 3 wk of age, the ratio of resurgent-to-transient current is normal in Purkinje cells from Purkinje KO mice (n = 6, data not shown), and the onset of ataxia is not seen before 6–8 wk of age. Direct effects of Scn8a KO
Scn8a loss in Purkinje cells led to changes in voltage-gated Na currents that were nearly identical in Purkinje and double KO mice, namely, a significant decrease in the amplitude of resurgent relative to transient currents. Although the recordings only represented Na current from the soma and possibly part of the axon initial segment, action potential firing in Purkinje neurons is driven largely by Na channels in this region of the cell (Khaliq and Raman 2006). Notably, the disruptions in the cell-specific mutant mice are indistinguishable from the changes previously described in global Scn8a null mice (Raman et al. 1997), despite several differences in the animals: the measurements reported here were made in adult mice, these mice had developed normally until about P7, and the knockout of Scn8a was restricted to one or two cell types. Previous reports from Scn8amedJ mice indicate that transcript levels of Scn1a and Scn2a remain constant in the absence of Scn8a (Kearney et al. 2002); however, the retention of substantial transient current in Purkinje cells lacking Scn8a suggests that these neurons may increase their surface expression of other Na channel subunits after loss of NaV1.6. Such an increase would suggest that Purkinje cells rely primarily on intrinsic mechanisms, possibly through feedback from some aspect of their own firing rates, to regulate levels of Na channel expression (e.g., Golowasch et al. 1999). Alternatively, NaV1.6 channels might normally contribute only modestly to total transient currents in Purkinje cell somata, although this possibility seems unlikely given the robust transient currents seen in expression studies (Smith et al. 1998).
The alteration of Na current kinetics in Purkinje neurons significantly reduced action potential firing rates in both Purkinje and double KO mice. These results are qualitatively consistent with reports of disrupted firing by Purkinje cells in global Scn8a null mice (Harris et al. 1992; Khaliq et al. 2003; Raman et al. 1997) but provide more quantitative and physiologically relevant information about spiking, as they come from intracellular recordings from intact Purkinje cells at near physiological temperature. Not only was spontaneous firing virtually abolished in the absence of NaV1.6 in Purkinje cells, but current injections did not restore firing to normal rates. These data lend further support to the conclusions from experimental and modeling studies, which indicate that, in the absence of resurgent kinetics, increases in transient or persistent Na current are unable to restore normal high-frequency firing patterns (Khaliq et al. 2003).
The reduction in firing rate is likely to have significant downstream consequences, particularly producing a decrease in the IPSC frequency in target neurons in the cerebellar and vestibular nuclei. In Lurcher mutant mice in which Purkinje cells degenerate, apparent compensation for the reduced inhibitory drive occurs at synapses onto target cells, via upregulation of the GABA receptor expression or sensitivity (Garin et al. 2002; Linnemann et al. 2004). Whether or not similar changes occur in the Purkinje and double KO animals, the motor defects suggest that any downstream strengthening of inhibition was insufficient to compensate fully for the approximately twofold decrease in firing rates. Moreover, despite the high convergence of Purkinje cells onto target neurons, the circuit apparently did not, or could not, compensate for the reduced activity in individual cells by recruitment of a larger number of synchronously active Purkinje neurons.
Related results come from ataxic mutant mice in which firing rates of cerebellar nuclear cells were increased by SK channel knockout as a model for decreased synaptic inhibition (Shakkottai et al. 2004). Likewise, in mice with global mutations in P/Q-type Ca channels, modest changes in Purkinje cell firing patterns are sufficient to impair motor coordination (Walter et al. 2006).
Indirect effects of the loss of Scn8a
Despite evidence that resurgent kinetics directly influence firing rate in Purkinje cells, previous studies of global Scn8a null mice also indicate that changes in Na channel expression can induce changes in other intrinsic currents, which appear homeostatic, though incompletely so (Khaliq et al. 2003). The increase in short-term facilitation of parallel fiber EPSCs in Purkinje and double KO mice, which is also suggestive of a compensatory response, suggests that such secondary effects are not restricted to Purkinje cells themselves. In fact, the observation that changes in synaptic dynamics follow from a loss of Na current that regulates firing rates is consistent with the highly plastic nature of the cerebellar circuit (Ito 2000; McCormick et al. 1985; Medina and Mauk 1999; Medina et al. 2002; Thompson 1986). A variety of mechanisms have been described in which increases in (postsynaptic) Purkinje firing decreases the strength of (presynaptic) parallel fiber excitation, both in the short term, via endocannabinoid signaling (Kreitzer and Regehr 2001), as well as in the long term, via nitric oxide (Lev-Ram et al. 1995). Consistent with the ability of firing to feed back negatively on synaptic excitation, the decrease in firing by Scn8a KO Purkinje cells led to an increased facilitation, occasionally accompanied by less steady-state depression, of parallel fiber EPSCs. Given the tendency of granule cells to fire in high-frequency bursts (Chadderton et al. 2004; Hartmann and Bower 2001), an increased facilitation is expected to increase the excitatory drive per afferent.
The recordings of parallel fiber EPSCs also allow inferences to be made about the contribution of Scn8a to firing in granule cells. Interestingly, despite evidence that granule cells express NaV1.6 in axons (Schaller and Caldwell 2000, 2003), the loss of Scn8a in granule neurons did not significantly affect action-potential-evoked neurotransmitter release. In fact, the similarity of the EPSCs trains suggests that conduction failures did not occur more often than in control, except possibly at 200 Hz, for which a mild increase in depression evident. This observation suggests that, at least in this range of stimulation frequencies, the NaV1.6 protein does not play a special role in permitting rapid firing in granule cells, similar to subthalamic nuclear neurons and cerebellar nuclear cells (Aman and Raman 2005; Do and Bean 2004). While the normal EPSCs are consistent with the normal gait of the granule KO mice, the animals' degraded performance on the rotarod task, along with the exacerbated motor defects in double KO relative to Purkinje KO mice, nevertheless suggests that more subtle but significant changes in signaling indeed occurred as a consequence of the loss of Scn8a from granule cells.
Earlier mouse models of cerebellar defects are characterized by significant changes in cellular organization and/or neuronal number, e.g., loss of Purkinje cells in the Lurcher (Lc), nervous (nv), and pcd mutants, and loss of granule cells in weaver (wv) and staggerer (sg) mice (Grüsser-Cornehls and Bäurle 2001). Selective mutation of Scn8a in cerebellar neurons replicates some of the defects of global Scn8a mutants while minimally perturbing nervous system development and cellular organization. These mice promise to be useful for further analysis of normal and pathological signaling through cerebellar circuits.
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ACKNOWLEDGMENTS |
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6-Cre mice. |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. H. Meisler, Dept. of Human Genetics, 4909 Buhl Box 0618, University of Michigan, Ann Arbor, MI 48109-0618 (E-mail: meislerm{at}umich.edu)
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REFERENCES |
|---|
|
Aller MI, Jones A, Merlo D, Paterlini M, Meyer AH, Amtmann U, Brickley S, Jolin HE, McKenzie AN, Monyer H, Farrant M, and Wisden W. Cerebellar granule cell Cre recombinase expression. Genesis 36: 97–103, 2003.[CrossRef][Web of Science][Medline]
Altman J and Bayer SA. Development of the Cerebellar System: In Relation to its Evolution, structure, and Functions. Boca Raton, FL: CRC, 1997.
Aman TK and Raman IM. Differential regulation of Na channel availability by long-lived inactivated states in Purkinje and cerebellar nuclear neurons. Soc Neurosci Abstr 151.6, 2005.
Barski JJ, Dethleffsen K, and Meyer M. Cre recombinase expression in cerebellar Purkinje cells. Genesis 28: 93–98, 2000.[CrossRef][Web of Science][Medline]
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][Web of Science][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][Web of Science][Medline]
Caldwell JH, Schaller KL, Lasher RS, Peles E, and Levinson SR. Sodium channel Na(v)1.6 is localized at nodes of ranvier, dendrites, and synapses. Proc Natl Acad Sci USA 97: 5616–5620, 2000.
Chadderton P, Margrie TW, and Hausser M. Integration of quanta in cerebellar granule cells during sensory processing. Nature 428: 856–860, 2004.[CrossRef][Medline]
Clark HB, Burright EN, Yunis WS, Larson S, Wilcox C, Hartman B, Matilla A, Zoghbi HY, and Orr HT. Purkinje cell expression of a mutant allele of SCA1 in transgenic mice leads to disparate effects on motor behaviors, followed by a progressive cerebellar dysfunction and histological alterations. J Neurosci 17: 7385–7395, 1997.
Crawley JN. What's Wrong With My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice. New York: Wiley-Liss, 2000.
Dick DJ, Boakes RJ, and Harris JB. A cerebellar abnormality in the mouse with motor end-plate disease. Neuropathol Appl Neurobiol 11: 141–147, 1985.[Web of Science][Medline]
Dick DJ, Boakes RJ, Candy JM, Harris JB, and Cullen MJ. Cerebellar structure and function in the murine mutant "jolting. " J Neurol Sci 76: 255–267, 1986.[CrossRef][Web of Science][Medline]
Do MTH and Bean BP. Sodium currents in subthalamic nucleus neurons from Nav1.6-null mice. J Neurophysiol 92: 726–733, 2004.
Drews VL, Lieberman AP, and Meisler MH. Multiple transcripts of sodium channel SCN8A [Na(V)1.6] with alternative 5'- and 3'-untranslated regions and initial characterization of the SCN8A promoter. Genomics 85: 245–257, 2005.[CrossRef][Web of Science][Medline]
Garin N, Hornung JP, and Escher G. Distribution of postsynaptic GABA(A) receptor aggregates in the deep cerebellar nuclei of normal and mutant mice. J Comp Neurol 447: 210–217, 2002.[CrossRef][Web of Science][Medline]
Golowasch J, Abbott LF, and Marder E. Activity-dependent regulation of potassium currents in an identified neuron of the stomatogastric ganglion of the crab Cancer borealis. J Neurosci 19: RC33, 1999.
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 beta-pompilidotoxin. J Neurosci 24: 35–42, 2004.
Grusser-Cornehls U and Baurle J. Mutant mice as a model for cerebellar ataxia. Prog Neurobiol 63: 489–540, 2001.[CrossRef][Web of Science][Medline]
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 2003.
Harris JB, Boakes RJ, and Court JA. Physiological and biochemical studies on the cerebellar cortex of the murine mutants "jolting" and "motor end-plate disease. " J Neurol Sci 110: 186–194, 1992.[CrossRef][Web of Science][Medline]
Hartmann MJ and Bower JM. Tactile responses in the granule cell layer of cerebellar folium crus IIa of freely behaving rats. J Neurosci 21: 3549–3563, 2001.
Herculano-Houzel S and Lent R. Isotropic fractionator: a simple, rapid method for the quantification of total cell and neuron numbers in the brain. J Neurosci 25: 2518–2521, 2005.
Ito M. Mechanisms of motor learning in the cerebellum. Brain Research 886: 237–245, 2000.[CrossRef][Web of Science][Medline]
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.
Khaliq ZM and Raman IM. Axonal propagation of simple and complex spikes in cerebellar Purkinje neurons. J Neurosci 25: 454–463, 2005.
Khaliq ZM and Raman IM. Relative contributions of axonal and somatic Na channels to action potential initiation in cerebellar Purkinje neurons. J Neurosci 26: 1935–1944, 2006.
Kreitzer AC and Regehr WG. Cerebellar depolarization-induced suppression of inhibition is mediated by endogenous cannabinoids. J Neurosci 21: RC174, 2001.
Levin SI and Meisler MH. Floxed allele for conditional inactivation of the voltage-gated sodium channel Scn8a [Na(v)1.6]. Genesis 39: 234–239, 2004.[CrossRef][Web of Science][Medline]
Lev-Ram V, Makings LR, Keitz PF, Kao JP, and Tsien RY. Long-term depression in cerebellar Purkinje neurons results from coincidence of nitric oxide and depolarization-induced Ca2+ transients. Neuron 15: 407–415, 1995.[CrossRef][Web of Science][Medline]
Linnemann C, Sultan F, Pedroarena CM, Schwarz C, and Thier P. Lurcher mice exhibit potentiation of GABA(A)-receptor-mediated conductance in cerebellar nuclei neurons in close temporal relationship to Purkinje cell death. J Neurophysiol 91: 1102–1107, 2004.
McCormick DA, Steinmetz JE, and Thompson RF. Lesions of the inferior olivary complex cause extinction of the classically conditioned eyeblink response. Brain Res 359: 120–130, 1985.[CrossRef][Web of Science][Medline]
Medina JF and Mauk MD. Simulations of cerebellar motor learning: computational analysis of plasticity at the mossy fiber to deep nucleus synapse. J Neurosci 19: 7140–7151, 1999.
Medina JF, Nores WL, and Mauk MD. Inhibition of climbing fibres is a signal for the extinction of conditioned eyelid responses. Nature 416: 330–333, 2002.[CrossRef][Medline]
Meisler MH and Kearney JA. Sodium channel mutations in epilepsy and other neurological disorders. J Clin Invest 115: 2010–2017, 2005.[CrossRef][Web of Science][Medline]
Meisler MH, Plummer NW, Burgess DL, Buchner DA, and Sprunger LK. Allelic mutations of the sodium channel SCN8A reveal multiple cellular and physiological functions. Genetica 122: 37–45, 2004.[CrossRef][Web of Science][Medline]
Planells-Cases R, Caprini M, Zhang J, Rockenstein EM, Rivera RR, Murre C, Masliah E, and Montal M. Neuronal death and perinatal lethality in voltage-gated sodium channel alpha(II)-deficient mice. Biophys J 78: 2878–2891, 2000.[Web of Science][Medline]
Raman IM and Bean BP. Resurgent sodium current and action potential formation in dissociated cerebellar Purkinje neurons. J Neurosci 17: 4517–4526, 1997.
Raman IM and Bean BP. Inactivation and recovery of sodium currents in cerebellar Purkinje neurons: evidence for two mechanisms. Biophys J 80: 729–737, 2001.[Web of Science][Medline]
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][Web of Science][Medline]
Regan LJ. Voltage-dependent calcium currents in Purkinje cells from rat cerebellar vermis. J Neurosci 11: 2259–2269, 1991.[Abstract]
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][Web of Science][Medline]
Schaller KL and Caldwell JH. Expression and distribution of voltage-gated sodium channels in the cerebellum. Cerebellum 2: 2–9, 2003.[CrossRef][Web of Science][Medline]
Shakkottai VG, Chou CH, Oddo S, Sailer CA, Knaus HG, Gutman GA, Barish ME, LaFerla FM, and Chandy KG. Enhanced neuronal excitability in the absence of neurodegeneration induces cerebellar ataxia. J Clin Invest 113: 582–590, 2004.[CrossRef][Web of Science][Medline]
Smith MR, Smith RD, Plummer NW, Meisler MH, and Goldin AL. Functional analysis of the mouse Scn8a sodium channel. J Neurosci 18: 6093–6102, 1998.
Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21: 70–71, 1999.[CrossRef][Web of Science][Medline]
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.
Thompson RF. The neurobiology of learning and memory. Science 233: 941–947, 1986.
Walter JT, Alvina K, Womack MD, Chevez C, and Khodakhah K. Decreases in the precision of Purkinje cell pacemaking cause cerebellar dysfunction and ataxia. Nat Neurosci 9: 389–397, 2006.[CrossRef][Web of Science][Medline]
Yu FH, Mantegazza M, Westenbroek RE, Robbins CA, Spain WJ, Burton KA, McKnight GS, Scheuer T, and Catterall WA. Deletion of the NaV1.1 channel: a mouse model for severe myoclonic epilepsy of infancy. Soc Neurosci Abstr 479.5, 2004.
Zhao L, Bakke M, Krimkevich Y, Cushman LJ, Parlow AF, Camper SA, and Parker KL. Steroidogenic factor 1 (SF1) is essential for pituitary gonadotrope function. Development 128: 147–154, 2001.[Abstract]
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ABSTRACT
INTRODUCTION
), BstXI restriction sites (B), and genotyping primers F2 and R2. B: Southern blot of BstXI-digested DNA from cerebellum of control and KO mice detects a 1.7-kb hybridizing fragment from the floxed allele and a 1.2-kb fragment from the deleted allele. C: RT-PCR of cerebellar RNA amplifies a 0.6-kb product from the full length transcript and a 0.3-kb fragment from transcripts lacking exon 1. D: allele-specific RT-PCR of transcripts lacking exon 1.
, calbindin-positive sites in the granule layer.
