Congenic strains provide evidence that a mapped locus on chromosome 15 influences excitotoxic cell death

Abstract

Inbred strains of mice differ in their susceptibility to excitotoxin-induced cell death, but the genetic basis of individual variation is unknown. Prior studies with crosses of the FVB/NJ (seizure-induced cell death susceptible) mouse and the seizure-induced cell death resistant mouse, C57BL/6J, showed the presence of three quantitative trait loci (QTLs), named seizure-induced cell death 1 (Sicd1) to Sicd3. To better localize and characterize the Sicd2 locus, two reciprocal congenic mouse strains were created. While the B6.FVB-Sicd2 congenic mouse was without effect on modifying susceptibility to seizure-induced excitotoxic cell death, the FVB.B6-Sicd2 congenic mouse, in which the chromosome (Chr) 15 region of C57BL/6J was introgressed into FVB/NJ, showed reduced seizure-induced excitotoxic cell death following kainate administration. Phenotypic comparison between FVB and the congenic FVB.B6-Sicd2 strain confirmed that the Sicd2 interval harbors gene(s) conferring strong protection against seizure-induced excitotoxic cell death. Interval-specific congenic lines (ISCLs) that encompass Sicd2 on Chr 15 were generated and were used to fine-map this QTL. Resultant progeny were treated with kainate and examined for the extent of seizure-induced cell death in order to deduce the Sicd2 genotypes of the recombinants through linkage analysis. All of the ISCLs exhibited reduced cell death associated with the C57BL/6J phenotype; however, ISCL-2 showed the most dramatic reduction in seizure-induced cell death in both area CA3 and in the dentate hilus. These findings confirm the existence of polymorphic loci within the reduced critical region of Sicd2 that regulate the severity of seizure-induced cell death.

Fig. 1. Schematic diagram of chromosome 15 in FVB.B6-Sicd2 congenic mice

Genetic map illustrating the introgressed chromosomal segment in the congenic strain FVB.B6-Sicd2. The first abbreviation (FVB) refers to recipient and the second abbreviation (B6) refers to donor. Darkly shaded regions reflect genomic contribution from the donor strain, C57BL/6J (B6) and lightly shaded regions reflect genomic contribution from the background, FVB/NJ (FVB) strain. DNA microsatellite markers used for genomic introgression are shown to the left of the chromosome. Numbers to the far left represent the genetic map position (http://www.informatics.jax.org/) and are given in centimorgans (cM). Sicd, seizure-induced cell death.

Fig. 2. Confirmation that the FVB.B6-Sicd2 congenic strain captures a gene(s) that influences susceptibility to seizure-induced cell death

(a) Data represent neuronal damage scores for the entire right and left hippocampus (in arbitrary units, mean ± SEM) for FVB.B6-Sicd2 congenic (n=46), B6.FVB-Sicd2 congenic (n=39), FVB/NJ (n=14) and C57BL/6J strains (n=14). Note that modulation of susceptibility to seizure-induced cell death was only statistically significant in FVB.B6-Sicd2 mice. (b) Quantitative analysis of neuronal density in hippocampal subfields following kainic acid (KA) administration to FVB.B6-Sicd2 congenic (n=41) and FVB mice (n=5). A strain-dependent difference in cell loss in area CA3 was observed at 7 days following KA administration. Data represent the mean ± SEM. B6, C57BL/6J; DG, dentate gyrus; Sicd, seizure-induced cell death.

Fig. 3. Kainate-induced cell death susceptibility is reduced in the FVB.B6-Sicd2 congenic strain. Susceptibility to kainate-induced cell death in the FVB/NJ (FVB) background strain and FVB.B6-Sicd2 congenic strain at 7 days following systemic administration of kainic acid

Low-magnification Fluoro-Jade B staining (a and c) and high magnification Fluoro-Jade B staining (b and d) of horizontal sections through the hippocampus (box denotes area of high magnification). Note the significant reduction of cell loss (decreased Fluoro-Jade fluorescence) in representative sections from the FVB.B6-Sicd2 congenic mice (c,d). CA1 and CA3 hippocampal subfields; H, dentate hilus. Scale bars: a and c, 750 μm; b and d, 100 μm. B6, C57BL/6J; Sicd, seizure-induced cell death.

Fig. 4. Histograms of seizure parameters in FVB.B6-Sicd2 and B6.FVB-Sicd2 congenic mice following kainic acid-induced status epilepticus

(a) Data represent latency scores in minutes (mean ± SEM) for FVB.B6-Sicd2 (n=42) and B6.FVB-Sicd2 congenic (n=39), FVB/NJ (FVB; n=14) and C57BL/6J (B6; n=14) strains. ANOVA testing revealed a significant reduction in latency between FVB.B6-Sicd2 and FVB strains. (b) Data represent the seizure duration scores in minutes (mean ± SEM) for FVB.B6-Sicd2 and B6.FVB-Sicd2 congenic and FVB and B6 strains. ANOVA testing revealed a significant increase in duration of severe seizures between FVB.B6-Sicd2 and FVB strains.

Fig. 5. Fine mapping of Sicd2 on Chromosome 15 with interval-specific congenic strains

Interval-specific congenic lines (ISCL) with varying B6-derived Chr 15 segments. The B6-derived interval is shown in black, and white denotes the interval containing the recipient, FVB/NJ, strain. Strains were produced as described in Materials and methods. Microsatellite DNA markers used for selecting interval-specific recombinants are shown to the left of the chromosome. Numbers to the far left represent the genetic map position (http://www.informatics.jax.org/) and are given in centimorgans (cM). Sicd, seizure-induced cell death.

Fig. 6. Seizure parameters of FVB/NJ (FVB) and interval-specific recombinant strains of the FVB.B6-Sicd2 congenic mice

(a) Latency to onset of first seizure among FVB (n=14) and interval-specific congenic lines (ISCL) 1–3 of FVB.B6-Sicd2 congenic mice is significantly reduced in ISCL-1 (n=37), ISCL-2 (n=29) and ISCL-3 (n=45) strains. Bars represent mean ± SEM. (b) Duration of severe seizures among FVB and ISCL1-3 of FVB.B6-Sicd2 congenic mice. Seizure duration was significantly increased in ISCL-1 and ISCL-3 strains as compared to FVB/NJ mice. Bars represent mean ± SEM.

Fig. 7. Kainate-induced cell death susceptibility phenotype of FVB/NJ (FVB) and interval-specific recombinant strains of the FVB.B6-Sicd2 congenic mice

Three interval-specific congenic lines (ISCLs 1-3) were developed and tested to attain higher resolution mapping of seizure-induced cell death (Sicd2). Strains were produced as described in Materials and Methods. (a) Susceptibility scores (cell damage score for the whole hippocampus) are given for the ISCL-1-3 (n=37 for ISCL-1; n=18 for ISCL-2; n=42 for ISCL-3) progeny (FVB.B6) and their FVBFVB littermates (n=16). Susceptibility to seizure-induced cell death was significantly less severe in ISCL-2 (FVB.B6) mice than their FVBFVB littermates. Bars represent mean ± SEM. (b) Quantitative analysis of neuronal density in hippocampal subfields following kainic acid (KA) administration to ISCL1-3 congenic lines (n=33 for ISCL-1; n=22 for ISCL-2; n=38 for ISCL-3) and FVB mice (n=16). Strain-dependent differences in cell loss in hippocampal subfields were observed at 7 days following KA administration. Data represent the mean ± SEM. B6, C57BL/6J; CA1 and CA3, hippocampal subfields; DG, dentate gyrus.

Fig. 8. Comparison of susceptibility to kainate-induced cell death in three interval-specific congenic line (ISCL) strains of the FVB.B6-Sicd2 congenic mice

(a,d,g) Low-magnification Fluoro-Jade staining of horizontal sections through the hippocampus in ISCL-1 (a), ISCL-2 (d), and ISCL-3 (g) at 7 days following systemic administration of kainic acid. High-magnification Fluoro-Jade staining (B,E,H) and Cresyl Violet staining (c,f,i) of horizontal sections through the hippocampus in ISCL-1 (b and c), ISCL-2 (e and f), and ISCL-3 (h and i). Note that a dramatic reduction in the extent of cell death is observed in ISCL-2 throughout area CA3 (d,e,f). CA1 and CA3, hippocampal subfields; H, dentate hilus. Scale bars: a,d,g, 750 μm; b,c,e,f,h,i, 100 μm. Sicd, seizure-induced cell death. High-magnification photomicrographs represent details of the boxed area of CA3 shown in a.