George G. Smith

Fine mapping of a seizure susceptibility locus on mouse Chromosome 1: nomination of Kcnj10 as a causative gene.

Previous quantitative trait loci (QTL) mapping studies document that the distal region of mouse Chromosome (Chr) 1 contains a gene(s) that is in large part responsible for the difference in seizure susceptibility between C57BL/6 (B6) (relatively seizure-resistant) and DBA/2 (D2) (relatively seizure-sensitive) mice. We now confirm this seizure-related QTL (Szs1) using reciprocal, interval-specific congenic strains and map it to a 6.6-Mb segment between Pbx1 and D1Mit150. Haplotype conservation between strains within this segment suggests that Szs1 may be localized more precisely to a 4.1-Mb critical interval between Fcgr3 and D1Mit150. We compared the coding region sequences of candidate genes between B6 and D2 mice using RT-PCR, amplification from genomic DNA, and database searching and discovered 12 brain-expressed genes with SNPs that predict a protein amino acid variation. Of these, the most compelling seizure susceptibility candidate is Kcnj10. A survey of the Kcnj10 SNP among other inbred mouse strains revealed a significant effect on seizure sensitivity such that most strains possessing a haplotype containing the B6 variant of Kcnj10 have higher seizure thresholds than those strains possessing the D2 variant. The unique role of inward-rectifying potassium ion channels in membrane physiology coupled with previous strong association between ion channel gene mutations and seizure phenotypes puts even greater focus on Kcnj10 in the present model. In summary, we confirmed a seizure-related QTL of large effect on mouse Chr 1 and mapped it to a finely delimited region. The critical interval contains several candidate genes, one of which, Kcnj10, exhibits a potentially important polymorphism with regard to fundamental aspects of seizure susceptibility.

Differential susceptibility to seizures induced by systemic kainic acid treatment in mature DBA/2J and C57BL/6J mice

Summary: Mature DBA/2J (D2) and C57BL/6J (B6) mice aged P.10 weeks were studied to determine susceptibility to behavioral seizures induced by kainic acid (KA) and the possible influence exerted by differences in metabolism and blood‐brain barrier (BBB) transport. Mice were observed for 4 h after subcutaneous (s.c.) KA injection. Behavioral seizure parameters included latency to first seizure (clonus), latency to tonic/clonic seizure, and latency to status epilepticus (SE). At a KA dose of 25 mg/kg, 80% of D2 mice exhibited tonicklonic seizures, whereas all B6 mice remained seizure‐free. At 30 mg/kg, tonic/ clonic seizures were observed in 100% of D2 mice and 25% of B6 mice. Of D2 mice exhibiting at least one clonic seizure in response to KA at a dose of 25 mg/kg, 50% entered SE and eventually died. Administration of [3H]KA (6.6 × 106dpm) at doses of 25 mg/kg (convulsive) or 11.1 μ/g (nonconvulsive) to mice of both strains resulted in similar levels of radioactivity in cortex, hippocampus, and cerebellum 30 and 60 min after injection. Bioconversion of [3H]KA to a radiolabeled brain metabolite in vivo could not be documented in mice from either strain. Results confirm previously reported differences between D2 and B6 mice in their relative susceptibility to seizures induced by systemic KA administration and suggest that these differences are not related to strain‐specific variation in metabolism or BBB transport of KA. Further studies of these two strains of mice may be useful for investigating genetic influences upon seizure susceptibility.

Mapping murine loci for seizure response to kainic acid

Mature DBA/2J (D2) mice are very sensitive to seizures induced by various chemical and physical stimuli, whereas C57BL/6J (B6) mice are relatively seizure resistant. We have conducted a genome-wide search for quantitative trait loci (QTLs) influencing the differential sensitivity of these strains to kainic acid (KA)-induced seizures by studying an F2 intercross population. Parental, F1, and F2 mice (8–10 weeks of age) were injected subcutaneously with 25 mg/kg of KA and observed for 3 h. Latencies to focal and generalized seizures and status epilepticus were recorded and used to calculate an overall seizure score. Results of seizure testing indicated that the difference in susceptibility to KA-induced seizures between D2 and B6 mice is a polygenic phenomenon with at least 65% of the variance due to genetic factors. First-pass genome screening (10-cM marker intervals) in F2 progeny (n = 257) documented a QTL of moderate effect on Chromosome (Chr) 1 with a peak LOD score of 5.5 (17% of genetic variance explained) localized between D1Mit30 and D1Mit16. Provisional QTLs of small effect were detected on Chr 11 (D11Mit224–D11Mitl4), 15 (D15Mit6–D15Mit46) and 18 (D18Mit9–D18Mitl44). Multiple locus models generally confirmed the Mapmaker/QTL results and also provided evidence for another QTL on Chr 4 (D4Mit9). Multilocus analysis of seizure severity suggested that additional loci on Chrs 5 (D5Mit11), 7 (D7Mit66), and 15 (D15Nds2) might also contribute to KA-induced seizure response. Overall, our results document a complex genetic determinism for KA-induced seizures in these mouse strains with contributions from as many as eight QTLs.

Rat strain and age differences in kainic acid induced seizures

This study reports comparative dose-response data for kainic acid (KA) induced seizures in juvenile (35-40 days old) and adult (70-90 days old) Wistar-Furth (WF), Fisher 344 (F344), Sprague-Dawley (SD) and Long-Evans Hooded (LEH) rats. Juvenile male WF (n = 51), F344 (n = 55), SD (n = 60), LEH (n = 50) and adult male WF (n = 48), F344 (n = 52), SD (n = 52), LEH (n = 53) rats were given KA 6, 8, 10, 12 or 14 mg/kg, sc. As previously demonstrated adult WF and F344 rats showed the greatest sensitivity and most reliable convulsant responses to kainic acid; SD and LEH rats were less sensitive and showed more variable convulsant responses. Regardless of strain, all juvenile rats exhibited greater sensitivity and less variable convulsant response to KA compared to adults. This was most evident in juvenile SD and LEH rats. Results suggest that while seizure sensitivity to KA decreases with age, genetic factors may regulate the expression of this resistance.

Mouse strain variation in maximal electroshock seizure threshold.

Maximal electroshock seizure threshold (MEST) is a classical measure of seizure sensitivity with a wide range of experimental applications. We determined MEST in nine inbred mouse strains and one congenic strain using a procedure in which mice are given one shock per day with an incremental (1 mA) current increase in each successive trial until a maximal seizure (tonic hindlimb extension) is elicited. C57BL/6J and DBA/2J mice exhibited the highest and lowest MEST, respectively, with the values of other strains falling between these two extremes. The relative rank order of MEST values by inbred strain (highest to lowest) is as follows: C57BL/6J > CBA/J = C3H/HeJ > A/J > Balb/cJ = 129/SvIMJ = 129/SvJ > AKR/J > DBA/2J. Results of experiments involving a single electroconvulsive shock given to separate groups of mice at different current intensities suggest that determination of MEST by the method used is not affected by repeated sub-maximal seizures. Overall, results document a distinctive mouse strain distribution pattern for MEST. Additionally, low within strain variability suggests that environmental factors which affect quantification of MEST are readily controlled under the conditions of this study. We conclude that MEST represents a useful tool for dissecting the multifactorial nature of seizure sensitivity in mice.

Genetic influences on electrical seizure threshold.

C57BL/6J (B6) and DBA/2J (D2) mice have been characterized previously as seizure-resistant and seizure-sensitive, respectively, a distinction based primarily upon a differential response to the convulsant effects of various drugs. In the present study, electroconvulsive shock (ECS) was used to assess maximal electroshock threshold (MET) in B6, D2 and hybrid mice. Results revealed that D2 mice have a significantly lower MET compared to B6 mice. There was also a significant gender effect for B6 and F2 mice with females exhibiting a lower MET compared to males. METs for F1 and F2 intercross mice were intermediate between the two parental strains. The difference in variance between F2 and F1 generation mice indicated that about three-quarters of the total variance is due to genetic influence. Taken together, results of this study suggest that the large difference in MET between B6 and D2 mice is a highly heritable trait which may yield to genetic dissection through use of quantitative trait locus mapping.

Acute Cocaine-Induced Seizures: Differential Sensitivity of Six Inbred Mouse Strains

Mature male and female mice from six inbred stains were tested for susceptibility to behavioral seizures induced by a single injection of cocaine. Cocaine was injected ip over a range of doses (50–100 mg/kg) and behavior was monitored for 20 minutes. Seizure end points included latency to forelimb or hindlimb clonus, latency to clonic running seizure and latency to jumping bouncing seizure. A range of strain specific sensitivities was documented with A/J and SJL mice being most sensitive and C57BL/6J most resistant. DBA/2J, BALB/cByJ and NZW/LacJ strains exhibited intermediate sensitivity. EEG recordings were made in SJL, A/J and C57BL/6J mice revealing a close correspondence between electrical activity and behavior. Additionally, levels of cocaine determined in hippocampus and cortex were not different between sensitive and resistant strains. Additional studies of these murine strains may be useful for investigating genetic influences on cocaine-induced seizures.

Transcriptional profiling of C57 and DBA strains of mice in the absence and presence of morphine

BackgroundThe mouse C57BL/6 (C57) and DBA/2J (DBA) inbred strains differ substantially in many aspects of their response to drugs of abuse. The development of microarray analyses represents a genome-wide method for measuring differences across strains, focusing on expression differences. In the current study, we carried out microarray analysis in C57 and DBA mice in the nucleus accumbens of drug-naïve and morphine-treated animals.ResultsWe identified mRNAs with altered expression between the two strains. We validated the mRNA expression changes of several such mRNAs, including Gnb1, which has been observed to be regulated by several drugs of abuse. In addition, we validated alterations in the enzyme activity of one mRNA product, catechol-O-methyltransferase (Comt). Data mining of expression and behavioral data indicates that both Gnb1 and Comt expression correlate with aspects of drug response in C57/DBA recombinant inbred strains. Pathway analysis was carried out to identify pathways showing significant alterations as a result of treatment and/or due to strain differences. These analyses identified axon guidance genes, particularly the semaphorins, as showing altered expression in the presence of morphine, and plasticity genes as showing altered expression across strains. Pathway analysis of genes showing strain by treatment interaction suggest that the phosphatidylinositol signaling pathway may represent an important difference between the strains as related to morphine exposure.ConclusionmRNAs with differing expression between the two strains could potentially contribute to strain-specific responses to drugs of abuse. One such mRNA is Comt and we hypothesize that altered expression of Comt may represent a potential mechanism for regulating the effect of, and response to, multiple substances of abuse. Similarly, a role for Gnb1 in responses to multiple drugs of abuse is supported by expression data from our study and from other studies. Finally, the data support a role for semaphorin signaling in morphine effects, and indicate that altered expression of genes involved in phosphatidylinositol signaling and plasticity might also affect the altered drug responses in the two strains.

Confirmation of a major QTL influencing oral morphine intake in C57 and DBA mice using reciprocal congenic strains

C57BL/6 (B6) and DBA/2 (D2) mice exhibit disparate behavior when tested for voluntary morphine intake in a two-bottle choice drinking paradigm with B6 mice consuming 10 times more drug than D2 mice. Previous genetic mapping studies identified a locus, Mop2, on the proximal part of chromosome 10 that explained over half of the genetic variance in this mouse model of opioid self-administration. We constructed a set of reciprocal congenic strains between B6 and D2 mice in which the proximal portion of chromosome 10 has been introgressed from one strain onto the background of the other. We tested mice from this pair of reciprocal strains together with progenitor B6 and D2 mice in a two-bottle choice drinking paradigm with morphine and quinine. The results showed that introgression of chromosome 10 alleles from the B6 strain onto a D2 genetic background increased voluntary morphine intake four-fold compared to progenitor D2 mice. Preference for morphine was also increased significantly in D2.B6-Mop2 mice compared to progenitor D2 mice. Conversely, introgression of chromosome 10 alleles from the D2 strain onto a B6 genetic background decreased morphine intake by half compared to progenitor B6 mice in B6.D2 -Mop2 mice; however, high morphine preference was maintained in this congenic strain most likely due to strong quinine aversion. When quinine was eliminated from the control bottle, morphine preference in B6.D2-Mop2 mice was decreased significantly relative to B6 and D2.B6-Mop2 mice. Overall, these data confirm the existence of a gene(s) on chromosome 10 proximal to D10Mit124 that has a strong influence on the difference in morphine drinking behavior between B6 and D2 mice.

Strain differences in convulsive response to the excitotoxin kainic acid.

We describe a strain of rats (Wistar-Furth) that is highly susceptible to the neurotoxic effects of kainic acid (KA) and presents a reliable and quantifiable (with low within-group variability) animal model of status epilepticus. Wistar-Furth rats are more sensitive and demonstrate a less variable convulsant response than Sprague-Dawley and Long-Evans rats when tested for total time in seizure activity, latency to onset of first seizure, latency to status epilepticus, seizure severity scores, and percentage exhibiting behavioral seizures and status epilepticus. Results suggest that significant heterogeneity exists in the rodent population with regard to neuronal sensitivity to an excitotoxic amino acid and indicate that strain differences are an important consideration in studies using KA.