Expression of sodium channel Nav1.6 in cholinergic myenteric neurons of guinea pig proximal colon.

We wished to establish the functional identity of Na(v)1.6-expressing myenteric neurons of the guinea pig proximal colon by determining the extent of colocalization of Na(v)1.6 and selected neurochemical markers. Na(v)1.6-like immunoreactivity (-li) was primarily localized to the hillock and initial segments of myenteric neurons located near junctions with internodal fiber tracts. Immunoreactivity for Na(v)1.6 was co-localized with choline-acetyltransferase-li, representing 96% of Na(v)1.6-immunoreactive neurons; about 5% of these neurons showed co-localization with calretinin-li, but none with substance-P-li. Cholinergic neurons expressing Na(v)1.6 were amongst the smallest (somal area <300 mum(2)) of all cholinergic myenteric neurons observed. Only three of 234 Na(v)1.6-immunoreactive neurons exhibited nNOS-li, and none co-localized with calbindin-li. These data suggest that Na(v)1.6 is expressed in a small uniform population of cholinergic myenteric neurons that lie within the guinea pig proximal colon and that are likely to function as excitatory motor neurons.

Expression of the sodium channel Na(v)1.2 in chemically identified myenteric neurons in the guinea pig.

Our purpose was to identify Na(v)1.2-expressing myenteric neurons of the small and large intestine of the guinea pig by using antibodies directed against Na(v)1.2 and selected neurochemical markers. Na(v)1.2-like immunoreactivity (-li) co-localized with immunoreactivity for choline acetyltransferase in all regions, representing 45%-67% of Na(v)1.2-positive neurons. Na(v)1.2-li co-localized with immunoreactivity for the neural form of nitric oxide synthase more frequently in the colon (20% of neurons exhibiting Na(v)1.2-li) than in the ileum (8%). Co-localization of Na(v)1.2-li with immunoreactivity for a form of neurofilament (NF145) was infrequently observed in the ileum and colon. Enkephalin-immunoreactive cell bodies co-localized with Na(v)1.2-li in all regions. Few myenteric cell bodies immunoreactive for neuropeptide Y were observed in the ileum, but all co-localized with Na(v)1.2-li. This and our previous data suggest that Na(v)1.2 is widely expressed within the guinea pig enteric nervous system, including the three main classes of myenteric neurons (sensory, motor, and interneurons), and is involved in both excitatory and inhibitory pathways. Notable exceptions include the excitatory motor neurons to the longitudinal smooth muscle, the ascending interneurons of the ileum, and the myenteric neurons immunoreactive for NF145, few of which are immunoreactive for Na(v)1.2.

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 Xenopus oocyte 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.

D1/D5 dopamine receptor activation differentially modulates rapidly inactivating and persistent sodium currents in prefrontal cortex pyramidal neurons.

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 voltage-dependent Na(+) currents, particularly persistent Na(+) currents. However, this conjecture is based on indirect evidence. To provide a direct test of this hypothesis, we combined voltage-clamp 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(+) 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(+) current was unaffected by D(1)/D(5) receptor activation-suggesting that rapidly inactivating and persistent Na(+) currents arise in part from different channels. Single-cell RT-PCR profiling showed that pyramidal neurons coexpressed three alpha-subunit mRNAs (Nav1.1, 1.2, and 1.6) that code for the Na(+) channel pore. In neurons from Nav1.6 null mice the persistent Na(+) currents were significantly smaller than in wild-type neurons. Moreover, the residual persistent currents in these mutant neurons-which 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(+) current in PFC pyramidal neurons arising from Nav1.1/1.2 Na(+) channels but does not modulate effectively the persistent component of the Na(+) current that is attributable to Nav1.6 Na(+) channels.

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.

Dystonia associated with mutation of the neuronal sodium channel Scn8a and identification of the modifier locus Scnm1 on mouse chromosome 3.

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.

Exon organization, coding sequence, physical mapping, and polymorphic intragenic markers for the human neuronal sodium channel gene SCN8A.

The voltage-gated sodium channel SCN8A is associated with inherited neurological disorders in the mouse that include ataxia, dystonia, severe muscle weakness, and paralysis. We report the complete coding sequence and exon organization of the human SCN8A gene. The predicted 1980 amino acid residues are distributed among 28 exons, including two pairs of alternatively spliced exons. The SCN8A protein is evolutionarily conserved, with 98.5% amino acid sequence identity between human and mouse. Consensus sites for phosphorylation of serine/threonine and tyrosine residues are present in cyoplasmic loop domains. The polymorphic (CA)n microsatellite marker D12S2211, with PIC = 0.68, was isolated from intron 10C of SCN8A. Single nucleotide polymorphisms in intron 19 and exon 22 were also identified. We localized SCN8A to chromosome band 12q13.1 by physical mapping on a YAC contig. The cDNA clone CSC-1 was reported by others to be a cardiac-specific sodium channel, but sequence comparison demonstrates that it is derived from exon 24 of human SCN8A. The genetic information described here will be useful in evaluating SCN8A as a candidate gene for human neurological disease.

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 Scn8a sodium 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.

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.

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.

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.

Effects of charybdotoxin on K+ channel (KV1.2) deactivation and inactivation kinetics.

Of particular interest for voltage-gated K+ channels are the effects of membrane voltage and pharmacologic agents on channel kinetics. We have characterized in detail properties of Kv1.2 channel expressed in oocytes as the basis for investigation of its structure-function relationships. This channel exhibited a voltage-dependent rate of activation with a V1/2 of -21 mV. Voltage-dependent steady-state inactivation overlapped the activation curve with half-maximal inactivation occurring at -22 mV. Dendrotoxin inhibited channel activation with an IC50 of 8.6 nM at + 35 mV. Charybdotoxin also blocked this K+ channel (IC50 = 5.6 nM). While dendrotoxin block was not affected by channel activation, charybdotoxin exhibited additional accumulation of block following activation, which was relieved with a time constant of 0.5 s upon repolarization of the membrane. The deactivation of this channel was accelerated in the presence of charybdotoxin while not significantly affected by dendrotoxin.

Regulation of interleukin-6 expression in porcine immune cells.

Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates the immune response, acute-phase reaction, and hematopoiesis. As a first step in studying the actions of IL-6 in pigs, the regulation of IL-6 expression was examined in various swine cells, including a fibroblast cell line, peripheral blood mononuclear cells (PBMC), and alveolar macrophages. IL-6 expression in transformed swine testicular (TST) fibroblasts was enhanced by TNF and IL-1 beta and to a lesser extent by poly(I).(C) and LPS. IL-6 was induced in porcine PBMC by either LPS or PHA; however, the combination of LPS plus PHA resulted in maximal IL-6 expression. Furthermore, in PBMC cells separated by adherence, LPS was a more potent inducer than PHA in adherent cells, whereas PHA was more potent in nonadherent cells. Alveolar macrophages collected from different pigs could be divided into low and high responders with respect to IL-6 induction by LPS. IL-6 mRNA induction by LPS could be detected in only 6 of 20 donor animals. Other inflammatory cytokines (IL-8, IL-beta, and TNF) were readily induced by LPS in alveolar macrophages from both low and high responders. Treatment of low-responder alveolar macrophages with conditioned medium containing IFN-gamma did not significantly alter the capacity of these macrophages to synthesize IL-6 mRNA in response to LPS. Comparison of IL-6 production capacity by the cell types in this study revealed the following order: PBMC = high-responder alveolar macrophages >> TST.cells > low-responder alveolar macrophages. Thus, PBMC appear to be quantitatively the most significant source of IL-6 in swine on a per cell basis.

Playground equipment injuries in a large, urban school district.

We studied the epidemiologic features of playground equipment-related injuries occurring in a large, urban school district over a two-year period. Nurses in each of the district's 68 elementary schools completed self-coded reporting forms on all injuries meeting standardized criteria. A total of 511 equipment-related injuries were reported, an incidence of 8.9 injuries per 1,000 student-years. One-fourth of the injuries were severe, and climbing equipment was disproportionately represented among playground equipment associated with injuries. Extreme variability was found among school-specific rates of equipment injury, with schools at the two extremes separated by as much as a 40-fold difference in incidence. Two school characteristics--smaller student enrollments and the presence of alternative educational programs--were significantly associated with higher equipment-related injury rates.

Epidemiology of injuries in a large, urban school district.

Injuries represent the single greatest threat to the health and well-being of US children. A large number of childhood injuries are sustained in schools, yet little is currently known of the epidemiologic features of school-related injuries. A surveillance of injuries occurring in a large, urban school district during a 2-year period was conducted. Nurses in each of the district's 96 schools completed reporting forms on all injuries meeting standardized criteria, and both principals and nurses completed questionnaires on school characteristics that were judged potentially important to the injury rate in individual schools. A total of 5,379 injuries were reported, among the district's 55,000 students, for an overall injury rate of 49 injuries/1,000 student-years. Injury rates were higher for boys than girls at all age levels. Self-caused and sports-related injuries comprised nearly half of all those reported, and 14% were related to use of playground or sports equipment. Eighteen percent of injuries were severe, and playground- and equipment-related injuries were significantly more likely to be severe (P less than .001). Rates of injury among individual schools varied markedly, with schools at the two extremes separated by a 25-fold difference in rates. Higher overall injury rates were found in schools with longer hours, alternative educational programs, less experienced school nurses, and lower student-to-staff ratios (P less than .0001).

Death and the pediatric house officer revisited.

Thirty-six third-year pediatric residents at four Western university training programs were interviewed individually and retrospectively about the magnitude of their clinical experience in managing the treatment of chronically ill and dying children, as well as the psychosocial educational curriculum of their training program as it pertained to these experiences. The residents managed an average of 35 dying children during their first 2 1/2 years of pediatric residency. They imparted the news of a potentially fatal disease to an average of 33 families during this same time span. There was a disparity between the magnitude of the clinical experience and the time and emphasis on these issues in the residency curriculum. The implications of these findings for an improved educational curriculum in the psychosocial care of dying children are discussed.

An analysis of physician-parent communication in pediatric prenatal interviews.

Thirty-three prenatal interviews done in the offices of four practicing pediatricians were audio-recorded, transcribed, and analyzed for content by two separate methods. Interviews with the individual pediatricians varied markedly in length and were significantly longer if the father was present. Pediatricians took charge of the interviews by asking more questions than parents and by talking twice as much as parents. A relatively small number of topics (medical care of the infant, infant feeding, business aspects of the pediatrician's practice, and history of the current pregnancy) occupied a majority of the interview time. Although interviews with individual pediatricians tended to include a standard set of topics, any given visit was individualized to address parents' issues. Follow-up interviews with mothers showed that they were highly satisfied with the visits and perceived the doctors as having been warm and friendly. Mothers' highly favorable reactions were achieved in spite of the fact that interview style was generally doctor-active/parent-passive, indicating that, in some medical settings, this approach may be quite appropriate.