Impact of anaesthetics and analgesics on fetal growth in the mouse.

Common anaesthetic and analgesic agents used during pregnancy in mice have been observed to cause fetal growth restriction. We investigated the impact of therapeutic doses of three anaesthetics (ketamine/xylazine, isoflurane, and tribromoethanol) and two analgesics (buprenorphine and meloxicam) on fetal and placental growth. Pregnant mice were treated with one of these agents at fertilization (E0), attachment (E4), beginning of organogenesis (E6), end of organogenesis (E12), or during the logarithmic growth phase (E15), or they were placed into an untreated control group. At term (E18), fetal and placental growth were evaluated, morphological analyses were performed, and skeletal measurements were conducted. Fetal growth was reduced significantly (P < 0.01) by ketamine/xylazine treatment at E0, E4, E12, or E15, by isoflurane administered at E0 or E6, and by tribromoethanol administered at E6 or E12. Two-day treatment with buprenorphine beginning at E4 or E6, or with meloxicam at E0 also significantly reduced fetal growth (P < 0.01). Neither placental growth nor litter size was significantly affected by any of these agents. The occurrence of microphthalmia was nearly eight-fold higher (P < 0.05) in response to buprenorphine administration at E6 compared with controls. The length of the humerus was reduced at most gestation times in response to each of these agents and was correlated (P < 0.01) with fetal weight for ketamine/xylazine, tribromoethanol, and meloxicam. These data reveal patterns of acceptable and detrimental anaesthetic and analgesic use during fetal development and have refined our capability to provide recommendations for the use of these agents during pregnancy in the mouse.

Gene-environment interactions in a mutant mouse kindred with native airway constrictor hyperresponsiveness.

We mutagenized male BTBR mice with N-ethyl-N-nitrosourea and screened 1315 of their G3 offspring for airway hyperresponsiveness. A phenovariant G3 mouse with exaggerated methacholine bronchoconstrictor response was identified and his progeny bred in a nonspecific-pathogen-free (SPF) facility where sentinels tested positive for minute virus of mice and mouse parvovirus and where softwood bedding was used. The mutant phenotype was inherited through G11 as a single autosomal semidominant mutation with marked gender restriction, with males exhibiting almost full penetrance and very few females phenotypically abnormal. Between G11 and G12, facility infection eradication was undertaken and bedding was changed to hardwood. We could no longer detect airway hyperresponsiveness in more than 37 G12 offspring of 26 hyperresponsive G11 males. Also, we could not identify the mutant phenotype among offspring of hyperresponsive G8-G10 sires rederived into an SPF facility despite 21 attempts. These two observations suggest that both genetic and environmental factors were needed for phenotype expression. We suspect that rederivation into an SPF facility or altered exposure to pathogens or other unidentified substances modified environmental interactions with the mutant allele, and so resulted in disappearance of the hyperresponsive phenotype. Our experience suggests that future searches for genes that confer susceptibility for airway hyperresponsiveness might not be able to identify some genes that confer susceptibility if the searches are performed in SPF facilities. Experimenters are advised to arrange for multigeneration constancy of mouse care in order to clone mutant genes. Indeed, we were not able to map the mutation before losing the phenotype.

Impaired motor function in mice with cell-specific knockout of sodium channel Scn8a (NaV1.6) in cerebellar purkinje neurons and granule cells.

The Scn8a gene encodes the voltage-gated Na channel alpha subunit Na(V)1.6, 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 Na(V)1.6 in different neuronal types. To test the effect of cerebellar Na(V)1.6 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 approximately 15 to approximately 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.

Inactivation of sodium channel Scn8A (Na-sub(v)1.6) in Purkinje neurons impairs learning in Morris water maze and delay but not trace eyeblink classical conditioning.

To examine the isolated effects of altered currents in cerebellar Purkinje neurons, the authors used Scn8a-super(flox/flox), Purkinje cell protein-CRE (Pcp-CRE) mice in which Exon 1 of Scn8a is deleted only in Purkinje neurons. Twenty male Purkinje Scn8a knockout (PKJ Scn8a KO) mice and 20 male littermates were tested on the Morris water maze (MWM). Subsequently, half were tested in 500-ms delay and half were tested in 500-ms trace eyeblink conditioning. PKJ Scn8a KO mice were impaired in delay conditioning and MWM but not in trace conditioning. These results provide additional support for the necessary participation of cerebellar cortex in normal acquisition of delay eyeblink conditioning and MWM and raise questions about the role, if any, of cerebellar cortex in trace eyeblink conditioning.

Floxed allele for conditional inactivation of the voltage-gated sodium channel Scn8a (NaV1.6).

The sodium channel gene Scn8a encodes the channel NaV1.6, which is widely distributed in the central and peripheral nervous system. NaV1.6 is the major channel at the nodes of Ranvier in myelinated axons. Mutant alleles of mouse Scn8a result in neurological disorders including ataxia, tremor, paralysis, and dystonia. We generated a floxed allele of Scn8a by inserting loxP sites around the first coding exon. The initial targeted allele containing the neo-cassette was a severe hypomorph. In vivo deletion of the neo-cassette by Flp recombinase produced a floxed allele that generates normal expression of NaV1.6 protein. Ubiquitous deletion of the floxed exon by Cre recombinase in ZP3-Cre transgenic mice produced the Scn8a(del) allele. The null phenotype of Scn8a(del) homozygotes confirms the in vivo inactivation of Scn8a. Conditional inactivation of the floxed allele will make it possible to circumvent the lethality that results from complete loss of Scn8a in order to investigate the physiologic role of NaV1.6 in subpopulations of neurons.

Molecular and pathological effects of a modifier gene on deficiency of the sodium channel Scn8a (Na(v)1.6).

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.