Sodium currents and action potentials were characterized in Purkinje neurons from ataxic mice lacking expression of the sodium channel Scn8a. Peak transient sodium current was approximately 60% of that in normal mice, but subthreshold sodium current was affected much more. Steady-state current elicited by voltage ramps was reduced to approximately 30%, and resurgent sodium current, an unusual transient current elicited on repolarization following strong depolarizations, was reduced to 8%-18%. In jolting mice, with a missense mutation in Scn8a, steady-state and resurgent current were also reduced, with altered voltage dependence and kinetics. Both spontaneous firing and evoked bursts of spikes were diminished in cells from null and jolting mice. Evidently Scn8a channels carry most subthreshold sodium current and are crucial for repetitive firing.

Dopamine (DA) is a well established modulator of prefrontal cortex (PFC) function, yet the cellular mechanisms by which DA exerts its effects in this region are controversial. A major point of contention is the consequence of D 1 DA receptor activation. Several studies have argued that D 1 receptors enhance the excitability of PFC pyramidal neurons by augmenting voltagedependent Na ϩ currents, particularly persistent Na ϩ currents. However, this conjecture is based on indirect evidence. To provide a direct test of this hypothesis, we combined voltageclamp studies of acutely isolated layer V-VI prefrontal pyramidal neurons with single-cell RT-PCR profiling. Contrary to prediction, the activation of D 1 or D 5 DA receptors consistently suppressed rapidly inactivating Na ϩ currents in identified corticostriatal pyramidal neurons. This modulation was attenuated by a D 1 /D 5 receptor antagonist, mimicked by a cAMP analog, and blocked by a protein kinase A (PKA) inhibitor. In the same cells the persistent component of the Na ϩ current was unaffected by D 1 /D 5 receptor activation-suggesting that rapidly inactivating and persistent Na ϩ currents arise in part from different channels. Single-cell RT-PCR profiling showed that pyramidal neurons coexpressed three ␣-subunit mRNAs (Nav1.1, 1.2, and 1.6) that code for the Na ϩ channel pore. In neurons from Nav1.6 null mice the persistent Na ϩ currents were significantly smaller than in wild-type neurons. Moreover, the residual persistent currents in these mutant neuronswhich are attributable to Nav1.1/1.2 channels-were reduced significantly by PKA activation. These results argue that D 1 /D 5 DA receptor activation reduces the rapidly inactivating component of Na ϩ current in PFC pyramidal neurons arising from Nav1.1/1.2 Na ϩ channels but does not modulate effectively the persistent component of the Na ϩ current that is attributable to Nav1.6 Na ϩ channels.

Marshall syndrome is a rare, autosomal dominant skeletal dysplasia that is phenotypically similar to the more common disorder Stickler syndrome. For a large kindred with Marshall syndrome, we demonstrate a splice-donor-site mutation in the COL11A1 gene that cosegregates with the phenotype. The G+1-->A transition causes in-frame skipping of a 54-bp exon and deletes amino acids 726-743 from the major triple-helical domain of the alpha1(XI) collagen polypeptide. The data support the hypothesis that the alpha1(XI) collagen polypeptide has an important role in skeletal morphogenesis that extends beyond its contribution to structural integrity of the cartilage extracellular matrix. Our results also demonstrate allelism of Marshall syndrome with the subset of Stickler syndrome families associated with COL11A1 mutations.

Scn8a encodes an abundant, widely distributed voltage-gated sodium channel found throughout the central and peripheral nervous systems. Mice with different mutant alleles of Scn8a provide models of the movement disorders ataxia, dystonia, tremor and progressive paralysis. We previously reported that the phenotype of the hypomorphic allele of Scn8a, medJ, is dependent upon an unlinked modifier locus, Scnm1. Strain C57BL/6J carries a sensitive allele of the modifier locus that results in juvenile lethality. We now provide evidence that the modifier acts on the splicing efficiency of the mutant splice donor site. Mutant mice display either 90% or 95% reduction in the proportion of correctly spliced mRNA, depending on modifier genotype. The abundance of the channel protein, Na(v)1.6, is also reduced by an order of magnitude in medJ mice, resulting in delayed maturation of nodes of Ranvier, slowed nerve conduction velocity, reduced muscle mass and reduction of brain metabolic activity. medJ mice provide a model for the physiological effects of sodium channel deficiency and the molecular mechanism of bigenic disease.

No abstract

Vagal afferent neurons relay important information regarding the control of the gastrointestinal system. However, the ionic mechanisms that underlie vagal activation induced by sensory inputs are not completely understood. We postulate that transient receptor potential (TRP) channels and/or two-pore potassium (K2p) channels are targets for activating vagal afferents. In this study we explored the distribution of these channels in vagal afferents by quantitative PCR after a capsaicin treatment to eliminate capsaicin-sensitive neurons, and by single-cell PCR measurements in vagal afferent neurons cultured after retrograde labeling from the stomach or duodenum. We found that TRPC1/3/5/6, TRPV1-4, TRPM8, TRPA1, TWIK2, TRAAK, TREK1, and TASK1/2 were all present in rat nodose ganglia. Both lesion results and single-cell PCR results suggested that TRPA1 and TRPC1 were preferentially expressed in neurons that were either capsaicin sensitive or TRPV1 positive. Expression of TRPM8 varied dynamically after various manipulations, which perhaps explains the disparate results obtained by different investigators. Last, we also examined ion channel distribution with the A-type CCK receptor (CCK-R(A)) and found there was a significant preference for neurons that express TRAAK to also express CCK-R(A), especially in gut-innervating neurons. These findings, combined with findings from prior studies, demonstrated that background conductances such as TRPC1, TRPA1, and TRAAK are indeed differentially distributed in the nodose ganglia, and not only do they segregate with specific markers, but the degree of overlap is also dependent on the innervation target.

The mouse mutant medJ contains a splice site mutation in the neuronal sodium channel Scn8a that results in a very low level of expression. On a C57BL/6J genetic background, medJ homozygotes exhibit progressive paralysis and juvenile lethality. The C3H genetic background has an ameliorating effect, producing viable adults with a novel dystonic phenotype. The dystonic mice exhibit movement-induced, sustained abnormal postures of the trunk and limbs. A dominant modifier locus responsible for the difference between strains was mapped to a 4.5 +/- 1.3 cM interval on mouse chromosome 3. Our findings establish a role for ion channels in dystonia and demonstrate the impact of genetic background on its severity and progression. This new model suggests that SCN8A on chromosome 12q13 and SCNM1 on chromosome 1p21-1q21 may contribute to human inherited dystonia.

The expression and distribution of TTX-sensitive voltage-gated sodium channel (VGSC) alpha subunits in the enteric nervous system (ENS) has not been described. Using RT-PCR, expression of Na(v)1.2, Na(v)1.3, Na(v)1.6, and Na(v)1.7 mRNA was detected in small and large intestinal preparations from guinea pigs. Expression of Na(v)1.1 mRNA as well as Na(v)1.1-like immunoreactivity (-li) were not observed in any intestinal region investigated. Na(v)1.2-li was primarily observed within the soma of the majority of myenteric and submucosal neurons, although faint immunoreactivity was occasionally observed in ganglionic and internodal fibers. Na(v)1.3-li was observed in dendrites, soma, and axons in a small group of myenteric neurons, as well as in numerous myenteric internodal fibers; immunoreactivity was rarely observed in the submucosal plexus. Na(v)1.6-li was primarily observed in the initial axonal segment of colonic myenteric neurons. Na(v)1.7-li was observed in dorsal root ganglia neurons but not in the myenteric plexus of the small and large intestine. In the ileum, 37% of Na(v)1.2-li cell bodies colocalized with calbindin-li while colocalization with calretinin-li was rare. In contrast, 22% of Na(v)1.3-li cell bodies colocalized with calretinin-li but colocalization with calbindin-li was not observed. In the colon, both Na(v)1.2-li and Na(v)1.3-li cell bodies frequently colocalized with either calretinin-li or calbindin-li. Na(v)1.2-li cell bodies also colocalized with the majority of NeuN-li cells in the small and large intestine. These data suggest that Na(v)1.1 may not be highly expressed in the ENS, but that Na(v)1.2, Na(v)1.3, and Na(v)1.6, and possibly Na(v)1.7, have broadly important and distinct functions in the ENS.