Microarray-assisted fine mapping of quantitative trait loci on chromosome 15 for susceptibility to seizure-induced cell death in mice.

Prior studies with crosses of the FVB/NJ (FVB; seizure-induced cell death-susceptible) mouse and the C57BL/6J (B6; seizure-induced cell death-resistant) mouse revealed the presence of a quantitative trait locus (QTL) on chromosome 15 that influenced susceptibility to kainic acid-induced cell death (Sicd2). In an earlier study, we confirmed that the Sicd2 interval harbors gene(s) conferring strong protection against seizure-induced cell death through the creation of the FVB.B6-Sicd2 congenic strain, and created three interval-specific congenic lines (ISCLs) that encompass Sicd2 on chromosome 15 to fine-map this locus. To further localise this Sicd2 QTL, an additional congenic line carrying overlapping intervals of the B6 segment was created (ISCL-4), and compared with the previously created ISCL-1-ISCL-3 and assessed for seizure-induced cell death phenotype. Whereas all of the ISCLs showed reduced cell death associated with the B6 phenotype, ISCL-4, showed the most extensive reduction in seizure-induced cell death throughout all hippocampal subfields. In order to characterise the susceptibility loci on Sicd2 by use of this ISCL and identify compelling candidate genes, we undertook an integrative genomic strategy of comparing exon transcript abundance in the hippocampus of this newly developed chromosome 15 subcongenic line (ISCL-4) and FVB-like littermates. We identified 10 putative candidate genes that are alternatively spliced between the strains and may govern strain-dependent differences in susceptibility to seizure-induced excitotoxic cell death. These results illustrate the importance of identifying transcriptomics variants in expression studies, and implicate novel candidate genes conferring susceptibility to seizure-induced cell death.

Strain differences in seizure-induced cell death following pilocarpine-induced status epilepticus.

Mouse strains differ from one another in their susceptibility to seizure-induced excitotoxic cell death. Previously, we have demonstrated that mature inbred strains of mice show remarkable genetic differences in susceptibility to the neuropathological consequences of seizures in the kainate model of status epilepticus. At present, while the cellular mechanisms underlying strain-dependent differences in susceptibility remain unclear, some of this variation is assumed to have a genetic basis. However, it remains unclear whether strain differences in susceptibility to seizure-induced cell death observed following kainate administration are observed following systemic administration of other chemoconvulsants. In rodents, the cholinomimetic convulsant pilocarpine is widely used to induce status epilepticus (SE), followed by hippocampal damage and spontaneous recurrent seizures, resembling temporal lobe epilepsy. This model has initially been described in rats, but is increasingly used in mice. We characterized neuronal pathologies after pilocarpine-induced status epilepticus (SE) in eight inbred strains of mice focusing on the hippocampus. A ramping-up dose protocol for pilocarpine was used and behavior was monitored for 4-5 h. While we did not observe any significant differences in seizure latency or duration to pilocarpine among the inbred strains, we did observe a significant difference in susceptibility to the neuropathological consequences of pilocarpine-induced SE. Of the eight genetically diverse mouse strains screened for pilocarpine-induced status, BALB/cJ and BALB/cByJ were the only two strains that were resistant to the neuropathological consequences of seizure-induced cell death. Additional studies of these murine strains may be useful for investigating genetic influences on pilocarpine-induced status epilepticus.

Effect of age on kainate-induced seizure severity and cell death.

While the onset and extent of epilepsy increases in the aged population, the reasons for this increased incidence remain unexplored. The present study used two inbred strains of mice (C57BL/6J and FVB/NJ) to address the genetic control of age-dependent neurodegeneration by building upon previous experiments that have identified phenotypic differences in susceptibility to hippocampal seizure-induced cell death. We determined if seizure induction and seizure-induced cell death are affected differentially in young adult, mature, and aged male C57BL/6J and FVB/NJ mice administered the excitotoxin, kainic acid. Dose response testing was performed in three to four groups of male mice from each strain. Following kainate injections, mice were scored for seizure activity and brains from mice in each age group were processed for light microscopic histopathologic evaluation 7 days following kainate administration to evaluate the severity of seizure-induced brain damage. Irrespective of the dose of kainate administered or the age group examined, resistant strains of mice (C57BL/6J) continued to be resistant to seizure-induced cell death. In contrast, aged animals of the FVB/NJ strain were more vulnerable to the induction of behavioral seizures and associated neuropathology after systemic injection of kainic acid than young or middle-aged mice. Results from these studies suggest that the age-related increased susceptibility to the neurotoxic effects of seizure induction and seizure-induced injury is regulated in a strain-dependent manner, similar to previous observations in young adult mice.

Diurnal variation in the elimination rate of human growth hormone (GH): the half-life of serum GH is prolonged in the evening, and affected by the source of the hormone, as well as by body size and serum estradiol.

The half-lives of endogenous and exogenous (biosynthetic monomeric) GH were compared in the morning and evening in healthy young men (n = 10). In group A, a bolus of GHRH was injected either at 0800 or at 2000 h, whereas in group B hGH was injected iv after suppression of endogenous GH by somatostatin. GH was sampled every 10 min and the t1/2 for GH was determined by deconvolution analysis (two compartments). The GH elimination half-life was shorter in the morning: for endogenous GH, t1/2 was 23 +/- 1.1 min (mean +/- SE) in the morning compared to 26 +/- 1.7 min in the evening (P < 0.02). T1/2 correlated negatively with estradiol (r = -0.78; P < 0.01) and positively with sex hormone-binding globulin (r = 0.71; P < 0.03). The half-life of exogenous 22-kilodalton GH was shorter compared to endogenous GH (P < 0.002), and diurnal variation was even more pronounced: t1/2 was 14 +/- 1.0 min in the morning and 19 +/- 1.0 min in the evening (P < 0.01). These effects were not due to differences in GH distribution volumes. The half-life of exogenous GH was significantly affected by weight (r = -0.8; P < 0.01) and height (r = 0.67; P < 0.05). We conclude that in young males, the rate of GH disappearance from the circulation depends on both diurnal mechanisms as well as the source or structural composition of the hormone. Body size and sex steroids contribute to the variability of GH clearance in healthy man.

Genetic influences on cellular reactions to brain injury: activation of microglia in denervated neuropil in mice carrying a mutation (Wld(S)) that causes delayed Wallerian degeneration.

This study examines the relationship between the appearance of degenerative changes in synaptic terminals and axons and the activation of microglia in denervated neuropil regions of normal mice of the C57BL/6 strain and mutant mice (Wld(S)), in which Wallerian degeneration is substantially delayed. The time course of degenerative changes in synaptic terminals and axons was assessed using selective silver staining. Microglial cells were identified by immunostaining for Mac-1, a monoclonal antibody to the CR3 complement receptor, and by histochemical staining for nucleoside diphosphatase (NDPase). Increased argyrophilia, indicative of degenerative changes, was evident as early as 1 day postlesion in normal mice, but was not seen until 6-8 days in mice with the Wld(S) mutation. Microglial activation in normal C57BL/6 mice was evident by 24 hours postlesion, as evidenced by increased immunostaining for Mac-1, increased histochemical staining for NDPase, and morphological changes indicative of an activated phenotype (short, thick processes). Quantitative evaluation of immunostaining for Mac-1 revealed that peak activation occurred between 2 and 6 days postlesion with a return to a quiescent phenotype by 12 days. In contrast, the microglial response was significantly delayed and prolonged in mice bearing the Wld(S) mutation. Activated microglia were not seen within the deafferented area until 6 to 8 days postlesion and peak activation occurred between 12 and 20 days postlesion. These data suggest that the response of microglia in denervated neuropil zones is triggered by the same types of degenerative changes that cause increased argyrophilia as detected by selective silver staining methods.

Enhanced but delayed axonal sprouting of the commissural/associational pathway following a combined entorhinal cortex/fimbria fornix lesion.

From previous lesion studies of the hippocampus it has been reported that axons of the commissural/associational pathway expand their termination zone in the molecular layer of the dentate gyrus by 20-25% in response to loss of input from the entorhinal cortex. However, although much is known about the response of the commissural/associational pathway with regard to extent, latency, and speed of the reinnervation response following an entorhinal cortex lesion, little is known about how the loss of additional afferent systems might modulate this response. To address this issue, we examined at 14, 30, and 45 days postlesion, the sprouting of commissural/associational afferents following either a unilateral fimbria fornix transection, a unilateral entorhinal cortex lesion, or combined lesions of both the entorhinal cortex and the fimbria fornix. Loss of septal innervation to the hippocampus was assessed using the cholinesterase stain, whereas sprouting from the commissural/associational pathway was determined from Holmes fiber-stained sections. In addition, the Timms stain was used to examine the time course of the loss of terminal fields of the various zinc-containing afferent systems within the hippocampus. Following the removal of input to the hippocampus via the fimbria fornix transection, there was no evidence of sprouting of the commissural/associational fibers into the deafferented portion of the dentate gyrus. In contrast, rats receiving an entorhinal cortex lesion showed a significant increase (28%) in the width of the commissural/associational fiber plexus that was present by 14 days postlesion. By comparison, the magnitude of the expansion of the commissural/associational fiber plexus was significantly larger after lesioning both the entorhinal cortex and the fimbria than after the entorhinal cortex lesion alone (45% vs. 28%). In addition, the expansion of the commissural/associational fiber plexus was not increased at 14 days postlesion but was significantly increased at 30 days postlesion. The delay in the sprouting of the commissural/associational pathway coincided with the time course of loss of zinc-containing fibers in the outer molecular layer of the dentate gyrus as assessed with the Timms stain. These results suggest that the magnitude and time course for the sprouting of axons from the commissural/associational pathway into the partially deafferented hippocampus of the adult rat is lesion dependent and that the effect of the loss of input from the entorhinal cortex can be modulated and enhanced by the concomitant depletion of input from the fimbria fornix.

Complications associated with genetic background effects in models of experimental epilepsy.

To elucidate the genetic influences contributing to susceptibility to seizure disorders, researchers have long used selected lines and inbred strains of rodents. In recent years, the use of genetically altered mice as models of complex human disease has revolutionized biomedical research into the genetics of disease pathogenesis and potential therapeutic interventions. In particular, the study of transgenic and gene-deleted (knockout) mice can provide important insights into the in vivo function and interaction of specific gene products. While a variety of inbred mouse mutations have been used to directly evaluate the genetic basis of seizure disorders, data obtained from such genetically altered mice must be interpreted carefully. An increasing number of scientific articles have reported that the phenotype of a given single gene mutation in mice can be modulated by the genetic background of the inbred strain in which the mutation is maintained. This effect is attributable to so-called modifier genes, which act in combination with the causative gene. In this review, the author points out the importance of considering the genetic background of the strain used to create these animal models, the potential problems with interpretation of phenotype, and solutions to selecting an appropriate mouse model of experimental epilepsy. Despite these potential limitations, knockout mice provide a powerful tool for understanding the genetic and neurobiological mechanisms contributing to experimental epilepsy.

Comparison of RPTP zeta/beta, phosphacan, and trkB mRNA expression in the developing and adult rat nervous system and induction of RPTP zeta/beta and phosphacan mRNA following brain injury.

The receptor protein tyrosine phosphatase (RPTP) zeta/beta and a major isoform, phosphacan, a chondroitin sulfate proteoglycan that contains the RPTP zeta/beta extracellular domain but not the transmembrane and intracellular phosphatase domains, are expressed abundantly in the nervous system, primarily by astroglia. Because of similarities in the expression patterns of RPTP zeta/beta and the receptor tyrosine kinase TrkB, we investigated whether RNAs encoding these proteins were co-localized during development, which would suggest that these molecules might functionally interact in vivo. By in-situ hybridization, we noted extensive areas of overlap in the expression of trkB and RPTP zeta/beta mRNAs in the developing peripheral and central nervous systems. Analysis with a probe specific for the catalytic TrkB isoform suggested that RPTP zeta/beta and non-catalytic trkB mRNAs were co-expressed in particular regions of the nervous system while the catalytic trkB and RPTP zeta/beta transcripts were also, but to a lesser extent. RPTP zeta/beta and phosphacan expression were extremely similar, differing particularly in the level of expression in the ventricular and subventricular zones, hippocampus, and ependyma. Furthermore, both RPTP zeta/beta and phosphacan mRNAs were found in several subsets of neurons as well as astrocytes. Following CNS injury, we observed robust induction of RPTP zeta/beta mRNA in areas of axonal sprouting, and of both RPTP zeta/beta and phosphacan mRNAs in areas of glial scarring, implying that the encoded proteins and the cell adhesion molecules and extracellular matrix proteins to which they bind may contribute to recovery from injury and perhaps regulation of axonal regrowth in the nervous system.

Seizure-induced neuronal death is associated with induction of c-Jun N-terminal kinase and is dependent on genetic background.

Previous studies have shown that expression of c-Jun protein, as well as the c-Jun amino-terminal kinase (JNK) group of mitogen-activated protein kinases, may play a critical role in the pathogenesis of glutamate neurotoxicity. In order to define the molecular cascade that leads to c-Jun activation following excitotoxic injury and delineate whether induction of protein synthesis is related to cell death signaling cascades or those changes associated with increased seizure activity, we examined the expression of JNK-1, as well as its substrate, c-Jun and N-terminal phosphorylated c-Jun following kainic acid (KA) administration in two strains of mice. In the present study, we assessed the immunohistochemical expression of these proteins at time points between 2 h and 7 days, in excitotoxic cell death-resistant (C57BL/6) and -susceptible (FVB/N) mouse strains that were systemically injected with saline or kainic acid. No strain-related differences in the immunohistochemical expression of any of the proteins were observed in intact control mice. However, following KA administration, the magnitude and period of induction of JNK-1 protein was associated with impending cell death, while increased phosphorylation of c-Jun protein was associated with resistance to cell death. In contrast, expression of c-Jun protein does not appear to be a reliable indicator of impending cell death, as it was expressed in resistant and vulnerable subfields in mice susceptible to kainate injury. These results provide the first evidence that JNK-1 expression may be involved in producing the neuronal cell death response following excitotoxin-induced injury.

Helicobacter pylori infection: prevalence and clinical relevance in a large company.

Although gastrointestinal (GI) illnesses account for considerable sick absenteeism, there have been few workplace studies of GI disorders. We determined the prevalence of Helicobacter pylori infection by serology and assessed its relation to upper GI tract complaints, personal ulcer history, and family history of stomach cancer in 6,143 employees (mean age, 40.4 years) at BASF's main chemical production facilities in Ludwigshafen, Germany. Employees were recruited during occupational health clinic visits (n = 4,488) and through broad communications efforts (n = 1,655). Participation among clinic attendees was 66%, and this recruitment method was particularly effective in reaching shift employees. Positive immunoglobulin G (IgG) serology (38.2%), ulcers (4.9%), nonulcer dyspepsia (20.4%), and a family history of stomach cancer (6.1%) were common occurrences in this work setting. Further diagnostic evaluation and eradication therapy was recommended for 795 employees (12.9%), based on a combination of positive serology and either upper GI tract complaints or family stomach cancer history, and has been completed for 541 employees. A weak but consistent association was seen between positive serology and cigarette smoking, and shift work was found to be associated with positive serology, but not with ulcer or nonulcer dyspepsia occurrence.

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

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.

The relevance of individual genetic background and its role in animal models of epilepsy.

Growing evidence has indicated that genetic factors contribute to the etiology of seizure disorders. Most epilepsies are multifactorial, involving a combination of additive and epistatic genetic variables. However, the genetic factors underlying epilepsy have remained unclear, partially due to epilepsy being a clinically and genetically heterogeneous syndrome. Similar to the human situation, genetic background also plays an important role in modulating both seizure susceptibility and its neuropathological consequences in animal models of epilepsy, which has too often been ignored or not been paid enough attention to in published studies. Genetic homogeneity within inbred strains and their general amenability to genetic manipulation have made them an ideal resource for dissecting the physiological function(s) of individual genes. However, the inbreeding that makes inbred mice so useful also results in genetic divergence between them. This genetic divergence is often unaccounted for but may be a confounding factor when comparing studies that have utilized distinct inbred strains. The purpose of this review is to discuss the effects of genetic background strain on epilepsy phenotypes of mice, to remind researchers that the background genetics of a knockout strain can have a profound influence on any observed phenotype, and outline the means by which to overcome potential genetic background effects in experimental models of epilepsy.

Neuroprotection by glutamate receptor antagonists against seizure-induced excitotoxic cell death in the aging brain.

We previously have identified phenotypic differences in susceptibility to hippocampal seizure-induced cell death among two inbred strains of mice. We have also reported that the age-related increased susceptibility to the neurotoxic effects of seizure-induced injury is regulated in a strain-dependent manner. In the present study, we wanted to begin to determine the pharmacological mechanism that contributes to variability in the response to the neurotoxic effects of kainate. Thus, we compared the effects of the NMDA receptor antagonist, MK-801 and of the AMPA receptor antagonist NBQX on hippocampal damage in the kainate model of seizure-induced excitotoxic cell death in young, middle-aged, and aged C57BL/6 and FVB/N mice, when given 90 min following kainate-induced status epilepticus. Following kainate injections, mice were scored for seizure activity and brains from mice in each age and antagonist group were processed for light microscopic histopathologic evaluation 7 days following kainate administration to evaluate the severity of seizure-induced injury. Administration of MK-801 significantly reduced the extent of hippocampal damage in young, mature and aged FVB/N mice, while application of NBQX was only effective at attenuating cell death in young and aged mice throughout all hippocampal subfields. Our results suggest that both NMDA and non-NMDA receptors are involved in kainate-induced cell death in the mouse and suggest that aging may differentially affect the ability of neuroprotectants to protect against hippocampal damage. Differences in the effectiveness of these two antagonists could result from differential regulation of glutamatergic neurotransmitter systems or ion channel specificity.

Galanin receptor 1 deletion exacerbates hippocampal neuronal loss after systemic kainate administration in mice.

BACKGROUND: Galanin is a neuropeptide with a wide distribution in the central and peripheral nervous systems and whose physiological effects are mediated through three G protein-coupled receptor subtypes, GalR1, GalR2, and GalR3. Several lines of evidence indicate that galanin, as well as activation of the GalR1 receptor, is a potent and effective modulator of neuronal excitability in the hippocampus. METHODOLOGY/PRINCIPAL FINDINGS: In order to test more formally the potential influence of GalR1 on seizure-induced excitotoxic cell death, we conducted functional complementation tests in which transgenic mice that exhibit decreased expression of the GalR1 candidate mRNA underwent kainate-induced status epilepticus to determine if the quantitative trait of susceptibility to seizure-induced cell death is determined by the activity of GalR1. In the present study, we report that reduction of GalR1 mRNA via null mutation or injection of the GalR1 antagonist, galantide, prior to kainate-induced status epilepticus induces hippocampal damage in a mouse strain known to be highly resistant to kainate-induced neuronal injury. Wild-type and GalR1 knockout mice were subjected to systemic kainate administration. Seven days later, Nissl and NeuN immune- staining demonstrated that hippocampal cell death was significantly increased in GalR1 knockout strains and in animals injected with the GalR1 antagonist. Compared to GalR1-expressing mice, GalR1-deficient mice had significantly larger hippocampal lesions after status epilepticus. CONCLUSIONS/SIGNIFICANCE: Our results suggest that a reduction of GalR1 expression in the C57BL/6J mouse strain renders them susceptible to excitotoxic injury following systemic kainate administration. From these results, GalR1 protein emerges as a new molecular target that may have a potential therapeutic value in modulating seizure-induced cell death.

Neuroprotection against excitotoxic brain injury in mice after ovarian steroid depletion.

Ovarian steroid hormones influence not only seizure phenomena, but also the neuronal cell death that follows. In the present study, we applied two models of ovarian steroid loss, ovariectomy and chemically-induced ovarian failure, to evaluate kainate-induced seizure activity and the susceptibility of hippocampal neurons to seizure-induced neurodegeneration. Young adult female FVB/NJ mice were ovariectomized with (OVX+E, n=6) or without (OVX, n=8) estrogen replacement. A separate group of females received the ovotoxin, 4-vinylcyclohexene diepoxide (VCD, n=8) to deplete ovarian follicles. Mice underwent kainate-induced status epilepticus and were evaluated for seizure activity (3 h) and delayed hippocampal neuronal injury (7 days). While there were no differences in latency or duration of severe seizures among control, OVX and VCD-treated mice, OVX+E mice exhibited seizures of a significantly longer duration. However, both VCD-induced ovarian failure and OVX led to a dramatic reduction in the extent of excitotoxic cell death, with slightly greater effects observed in VCD-treated mice. Estradiol administration to OVX mice also exerted a significant neuroprotective effect against kainate-induced cell death. These results support and extend earlier findings suggesting that the hormonal milieu may have differential effects on seizure susceptibility that are separate and distinct from those influencing hippocampal neuronal vulnerability. Collectively, these findings highlight the complex interactions among the loss of ovarian steroid hormones, estrogen replacement, seizures, and seizure-induced cell death.

Variation in Galr1 expression determines susceptibility to exocitotoxin-induced cell death in mice.

Inbred strains of mice differ in their susceptibility to excitotoxin-induced cell death, but the genetic basis of individual variation in differential susceptibility is unknown. Previously, we identified a highly significant quantitative trait locus (QTL) on chromosome 18 that influenced susceptibility to kainic acid-induced cell death (Sicd1). Comparison of susceptibility to seizure-induced cell death between reciprocal congenic lines for Sicd1 and parental background mice indicates that genes influencing this trait were captured in both strains. Two positional gene candidates, Galr1 and Mbp, map to 55 cM, where the Sicd1 QTL had been previously mapped. Thus, this study was undertaken to determine if Galr1 and/or Mbp could be considered as candidate genes. Genomic sequence comparison of these two functional candidate genes from the C57BL/6J (resistant at Sicd1) and the FVB/NJ (susceptible at Sicd1) strains showed no single-nucleotide polymorphisms. However, expression studies confirmed that Galr1 shows significant differential expression in the congenic and parental inbred strains. Galr1 expression was downregulated in the hippocampus of C57BL/6J mice and FVB.B6-Sicd1 congenic mice when compared with FVB/NJ or B6.FVB-Sicd1 congenic mice. A survey of Galr1 expression among other inbred strains showed a significant effect such that 'susceptible' strains showed a reduction in Galr1 expression as compared with 'resistant' strains. In contrast, no differences in Mbp expression were observed. In summary, these results suggest that differential expression of Galr1 may contribute to the differences in susceptibility to seizure-induced cell death between cell death-resistant and cell death-susceptible strains.

A quantitative trait locus on chromosome 18 is a critical determinant of excitotoxic cell death susceptibility.

C57BL/6J (B6) and FVB/NJ (FVB) mice are phenotypically distinct in their susceptibility to seizure-induced cell death after kainate administration. Previous studies using quantitative trait loci (QTLs) mapping established that the distal region of mouse chromosome 18 contains a gene(s) that is probably responsible for the difference in seizure-induced cell death susceptibility between two inbred strains, B6 and FVB, that are relatively resistant and susceptible, respectively, to seizure-induced cell death. The genetic locus has been mapped to a approximately 12-centimorgan region of chromosome 18, designated as seizure-induced cell death 1 (Sicd1). In order to confirm the Sicd1 QTL, we have developed congenic mouse strains containing the relevant donor segment from the resistant B6 strain on the susceptible FVB background, also referred to as the FVB.B6-Sicd1 congenic strain. Congenic and FVB littermate controls were tested in a seizure-induced cell death paradigm. The presence of B6 chromosome 18 alleles on an FVB genetic background conferred protection against seizure-induced cell death, as compared with FVB littermate controls. To further localize the Sicd1 QTL, new congenic lines carrying overlapping intervals of the B6 segment were created [interval-specific congenic lines (ISCLs)-1-4] and assessed for seizure-induced cell death phenotype. All of the ISCLs exhibited reduced cell death associated with the B6 phenotype, as compared with the parental FVB strain. The most dramatic of these, ISCL-4, showed a nearly four-fold reduction in the extent of seizure-induced cell death. This suggests that ISCL-4 contains the putative gene(s) of the Sicd1 QTL.

Modulation of cell death by mouse genotype: differential vulnerability to excitatory amino acid-induced lesions.

Previously we have demonstrated that mature inbred strains of mice differ significantly in their response to kainate-induced cell death. While both C57BL/6 and FVB/N mice exhibit similar seizure activity in response to kainate, only C57BL/6 mice can be characterized as resistant to kainate-induced cell death. To examine further the molecular pharmacological basis for this strain difference in hippocampal sensitivity, we assessed the ability of the ionotropic glutamate receptor agonists, kainic acid (KA), N-methyl-D-aspartate (NMDA), ibotenic acid (IBO), and quinolinic acid (QUIN), to promote excitotoxic damage. We examined seizure-related behavior and subsequent neurotoxicity in C57BL/6 and FVB/N mice following intrahippocampal administration of the kainate receptor agonist, KA, the NMDA receptor agonists NMDA or QUIN, or the NMDA and metabotropic glutamate receptor agonist, IBO. The time course and extent of cell death in mice were evaluated using Nissl and selective silver stains, and Fluoro-Jade, a fluorescent marker for dying neurons. In the present study, FVB/N mice were exquisitely sensitive to injection of KA at all doses, while susceptibility in C57BL/6 mice was dose dependent. In contrast, while hippocampal damage was present in both strains at all doses of QUIN, the extent of cell damage was significantly less in C57BL/6 mice at low doses (30 and 60 mM). Similarly, IBO administration resulted in differences in the extent of cell death when administered at the highest dose (126 mM). No strain-dependent differences in cell loss were observed following NMDA lesions. These results provide further evidence that susceptibility to excitotoxin-induced cell death is highly strain dependent and is kainate and NMDA receptor dependent.

Genetic determinants of susceptibility to excitotoxic cell death: implications for gene targeting approaches.

Recent studies have sought to identify the genes involved in excitotoxic neurodegeneration. Here we report that certain strains of mice, including strains that are used for gene targeting studies, do not exhibit excitotoxic cell death after kainic acid seizures. Kainic acid produced excitotoxic cell death in the CA3 and CA1 subfields of the hippocampus in 129/SvEMS and FVB/N mice, in the same pattern as described in rats. C57BL/6 and BALB/c mice exhibited excitotoxic cell death only at very high doses of kainate, and then only in a very restricted area, although they exhibited comparable seizures. Hybrids of 129/SvEMS x C57BL/6 mice created using embryonic stem cells from 129/SvEMS mice also did not exhibit excitotoxic cell death. These results demonstrate that C57BL/6 and BALB/c strains carry gene(s) that convey protection from glutamate-induced excitotoxicity. This differential susceptibility to excitotoxicity represents a potential complication for gene targeting studies.