Leslie K. Sprunger

Altered Subthreshold Sodium Currents and Disrupted Firing Patterns in Purkinje Neurons of Scn8a Mutant Mice

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.

Marshall Syndrome Associated with a Splicing Defect at the COL11A1 Locus

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.

The Sodium Channel Scn8a Is the Major Contributor to the Postnatal Developmental Increase of Sodium Current Density in Spinal Motoneurons

Sodium currents were recorded from motoneurons that were isolated from mice at postnatal days 0–8 (P0–P8) and maintained in culture for 12–24 hr. Motoneurons from normal mice exhibited a more than threefold increase in peak sodium current density from P0 to P8. For mice lacking a functional Scn8a sodium channel gene, motoneuronal sodium current density was comparable at P0 to that of normal mice but failed to increase from P0 to P8. The absence of Scn8asodium channels is associated with the phenotype “motor end plate disease,” which is characterized by a progressive neuromuscular failure and is fatal by 3–4 postnatal weeks. Thus, it appears that the development and function of mature motoneurons depends on the postnatal induction of Scn8a expression.

Allelic mutations of the sodium channel SCN8A reveal multiple cellular and physiological functions

Allelic mutations of Scn8a in the mouse have revealed the range of neurological disorders that can result from alternations of one neuronal sodium channel. Null mutations produce the most severe phenotype, with motor neuron failure leading to paralysis and juvenile lethality. Two less severe mutations cause ataxia, tremor, muscle weakness, and dystonia. The electrophysiological effects have been studied at the cellular level by recording from neurons from the mutant mice. The data demonstrate that Scn8a is required for the complex spiking of cerebellar Purkinje cells and for persistent sodium current in several classes of neurons, including some with pacemaker roles. The mouse mutations of Scn8a have also provided insight into the mode of inheritance of channelopathies, and led to the identification of a modifier gene that affects transcript splicing. These mutations demonstrate the value of mouse models to elucidate the pathophysiology of human disease.

Sodium Channels and Neurological Disease: Insights from Scn8a Mutations in the Mouse

The human genome contains 10 voltage-gated sodium channel genes, 7 of which are expressed in neurons of the CNS and PNS. The availability of human genome sequences and high-throughput mutation screening methods make it likely that many human disease mutations will be identified in these genes in the near future. Mutations of Scn8a in the mouse demonstrate the broad spectrum of neurological disease that can result from different alleles of the same sodium channel gene. Null mutations of Scn8a produce motor neuron failure, loss of neuromuscular transmission, and lethal paralysis. Less severe mutations result in ataxia, tremor, muscle weakness, and dystonia. The effects of Scn8a mutations on channel properties have been studied in the Xenopusoocyte expression system and in neurons isolated from the mutant mice. The Scn8a mutations provide insight into the mode of inheritance, effect on neuronal sodium currents, and role of modifier genes in sodium channel disease, highlighting the ways in which mouse models of human mutations can be used in the future to understand the pathophysiology of human disease.

Ion Channel Mutations in Mouse Models of Inherited Neurological Disease

Analysis of the molecular defects in mouse mutants can identify candidate genes for human neurological disorders. During the past 2 years, mutations in sodium channels, calcium channels and potassium channels have been identified by positional cloning of the spontaneous mouse mutants motor endplate disease, tottering, lethargic and weaver. The phenotypes of four allelic mutations identified in the sodium channel gene Scn8a range from ataxia and muscle weakness through severe dystonia and progressive paralysis, indicating that human mutations in this gene could be associated with a variety of clinical syndromes. Mutations of the calcium channel subunits beta 4 in the lethargic mouse and alpha 1A in the tottering mouse have specific effects on cerebellar function. Targeted mutation of ligand-gated ion channels has also been used to generate new models of neurological disease. We will review these recent achievements and their implications for human neurological disease. The mouse studies indicate that mutations in ion channel genes are likely to be responsible for a broad spectrum of clinical phenotypes in human neurological disorders.

Expression and distribution of TTX-sensitive sodium channel alpha subunits in the enteric nervous system

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 Nav1.2, Nav1.3, Nav1.6, and Nav1.7 mRNA was detected in small and large intestinal preparations from guinea pigs. Expression of Nav1.1 mRNA as well as Nav1.1‐like immunoreactivity (‐li) were not observed in any intestinal region investigated. Nav1.2‐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. Nav1.3‐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. Nav1.6‐li was primarily observed in the initial axonal segment of colonic myenteric neurons. Nav1.7‐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 Nav1.2‐li cell bodies colocalized with calbindin‐li while colocalization with calretinin‐li was rare. In contrast, 22% of Nav1.3‐li cell bodies colocalized with calretinin‐li but colocalization with calbindin‐li was not observed. In the colon, both Nav1.2‐li and Nav1.3‐li cell bodies frequently colocalized with either calretinin‐li or calbindin‐li. Nav1.2‐li cell bodies also colocalized with the majority of NeuN‐li cells in the small and large intestine. These data suggest that Nav1.1 may not be highly expressed in the ENS, but that Nav1.2, Nav1.3, and Nav1.6, and possibly Nav1.7, have broadly important and distinct functions in the ENS. J. Comp. Neurol. 486:117–131, 2005.

Reduced spontaneous activity in the dorsal cochlear nucleus of Scn8a mutant mice.

Spontaneous activity was recorded in the dorsal cochlear nucleus of brain slices from mice homozygous for the med-J and jolting mutations in the neuronal sodium channel alpha-subunit Scn8a. Densities of spontaneously active neurons in slices from both mutants were significantly lower than in control slices. Spontaneous firing patterns with bursts of action potentials were recorded from approximately 50% of the neurons in control slices, but the typical bursting patterns were not observed in neurons of med-J and jolting mouse slices. The results suggest that this voltage-gated sodium channel is essential for the spontaneous bursting firing of cochlear nucleus cartwheel neurons. This mutant animal model may be useful for the study of the functional roles of cochlear nucleus neurons.

High-resolution mapping of the sodium channel modifier Scnm1 on mouse chromosome 3 and identification of a 1.3-kb recombination hot spot

Variation between inbred strains of mice can be used to identify modifier genes affecting the susceptibility to inherited disease. The medJ allele of the sodium channel Scn8a contains a splice site mutation that results in sodium channel deficiency. The severity of the neurological disorder is determined by the modifier locus Scnm1. The wild-type allele of the modifier results in correct splicing of 10% of Scn8amedJ pre-mRNA and a dystonic phenotype. The susceptible allele of the modifier in strain C57BL/6J results in 5% correctly spliced transcripts and a lethal phenotype. A mapping cross with C3H using 26 new markers and 2304 affected F2 animals localized the modifier gene to a 950-kb interval on mouse chromosome 3. Fine mapping of recombination breakpoints revealed a recombination hot spot of 1.3 kb. The ratio of genetic to physical distance in the hot spot is 85 cM/Mb, two orders of magnitude higher than the mouse genome average of 0.5 cM/Mb. The role of the modifier in other disorders in human and mouse can be tested with linked markers described here.

Neural Proliferation and Restoration of Neurochemical Phenotypes and Compromised Functions Following Capsaicin-Induced Neuronal Damage in the Nodose Ganglion of the Adult Rat

We previously reported that neuronal numbers within adult nodose ganglia (NG) were restored to normal levels 60 days following the capsaicin-induced destruction of nearly half of the neuronal population. However, the nature of this neuronal replacement is not known. Therefore, we aimed to characterize neural proliferation, neurochemical phenotypes, and functional recovery within adult rat NG neurons following capsaicin-induced damage. Sprague-Dawley rats received intraperitoneal injections of capsaicin or vehicle solution, followed by 5-bromo-2-deoxyuridine (BrdU) injections to reveal cellular proliferation. NG were collected at multiple times post-treatment (up to 300 days) and processed for immunofluorescence, RT-PCR, and dispersed cell cultures. Capsaicin-induced cellular proliferation, indicated by BrdU/Ki-67-labeled cells, suggests that lost neurons were replaced through cell division. NG cells expressed the stem cell marker, nestin, indicating that these ganglia have the capacity to generate new neurons. BrdU-incorporation within β-III tubulin-positive neuronal profiles following capsaicin suggests that proliferating cells matured to become neurons. NG neurons displayed decreased NMDAR expression up to 180-days post-capsaicin. However, both NMDAR expression within the NG and synaptophysin expression within the central target of NG neurons, the NTS, were restored to pre-injury levels by 300 days. NG cultures from capsaicin-treated rats contained bipolar neurons, normally found only during development. To test the functional recovery of NG neurons, we injected the satiety molecule, CCK. The effect of CCK on food intake was restored by 300-days post-capsaicin. This restoration may be due to the regeneration of damaged NG neurons or generation of functional neurons that replaced lost connections.