Genes on distal chromosome 18 determine vulnerability to excitotoxic neurodegeneration following status epilepticus, but not striatal neurodegeneration induced by quinolinic acid

Previous studies have provided evidence that a quantitative trait locus (QTL) on the distal part of chromosome 18 (chr18) is a major determinant of vulnerability to hippocampal neurodegeneration following kainic acid (KA)-induced seizures in inbred mouse strains. We assessed excitotoxic vulnerability in two congenic, “genome tagged” mouse strains carrying segments of either distal or proximal/medial chr18 from vulnerable DBA/2J mice on a resistant C57BL/6 background. Systemic KA injections triggered brain-wide neurodegeneration in the distal chr18 congenic strain, and specifically in the hilus of the dentate gyrus, but not in CA3. In contrast, the proximal/medial chr18 congenic strain exhibited enhanced degeneration in CA1 and CA3, but little neurodegeneration elsewhere. Both strains exhibited low levels of QUIN-induced striatal neurodegeneration comparable to what is seen in C57BL/6 mice. These results suggest that gene(s) on distal chr18 are important determinants of vulnerability to KA-induced hippocampal neurodegeneration, but not QUIN-induced striatal neurodegeneration.     

Dissociation of seizure traits in inbred strains of mice using the flurothyl kindling model of epileptogenesis

Previous seizure models have demonstrated genetic differences in generalized seizure threshold (GST) in inbred mice, but the genetic control of epileptogenesis is relatively unexplored. The present study examined, through analysis of inbred strains of mice, whether the seizure characteristics observed in the flurothyl kindling model are under genetic control. Eight consecutive, daily generalized seizures were induced by flurothyl in mice from five inbred strains. Following a 28-day rest period, mice were retested with flurothyl. The five strains of mice demonstrated inter-strain differences in GST, decreases in GST across seizure trials, and differences in the behavioral seizure phenotypes expressed. Since many of the seizure characteristics that we examined in the flurothyl kindling model were dissociable between C57BL/6J and DBA/2J mice, we analyzed these strains in detail. Unlike C57BL/6J mice, DBA/2J mice had a lower GST on trial 1, did not demonstrate a decrease in GST across trials, nor did they show an alteration in seizure phenotype upon flurothyl retest. Surprisingly, [C57BL/6J × DBA/2J] F1-hybrids had initial GST on trial 1 and GST decreases across trials similar to what was found for C57BL/6J, but they did not undergo the alteration in behavioral seizure phenotype that had been observed for C57BL/6J mice. Our data establish the significance of the genetic background in flurothyl-induced epileptogenesis. The [C57BL/6J × DBA/2J] F1-hybrid data demonstrate that initial GST, the decrease in GST across trials, and the change in seizure phenotype differ from the characteristics of the parental strains, suggesting that these phenotypes are controlled by independent genetic loci.     

Multifaceted strain-specific effects in a mouse model of depression and of antidepressant reversal

Etiopathogenesis of depression and the cause of insensitivity to treatment remain poorly understood, although genetic makeup has been established as a contributing factor. The isogenicity of inbred mouse strains provides a useful tool for investigating the link between genes and behavior or drug response. Hence, our aim was to identify inbred mouse strains (among A/J, BALB/c, C3H, C57BL/6, CBA, DBA and FVB) sensitive to a 9-week period of unpredictable chronic mild stress (UCMS) and, from the fifth week onward, to the reversal effect of an antidepressant (AD) (imipramine, 20 mg/kg/day i.p.) on various depression-related changes: physical, behavioral and neuroendocrine states. UCMS induced a significant deterioration of the coat state (in all the strains), blunted emotional reactivity in the novelty-suppressed feeding (NSF) test (A/J, BALB/c, C57BL/6), and changes in the level of fecal corticosterone metabolites (BALB/c, C57BL/6, DBA, FVB). Imipramine treatment reversed the UCMS-induced alterations of the coat state (BALB/c, DBA), in the NSF test (A/J, BALB/c, C57BL/6) and in fecal corticosterone metabolites (BALB/c, C57BL/6). C3H, CBA and FVB mice were irresponsive to imipramine treatment. It is noteworthy that UCMS-induced physical or behavioral changes occurred without hypothalamo–pituitary–adrenal (HPA) axis alterations in some strains (A/J, C3H, CBA), although the AD-induced reversal of these changes in BALB/c and C57BL/6 was associated with HPA axis normalization. Finally, UCMS is shown to discriminate various alterations and to replicate in a strain-dependent manner diverse profiles reminiscent of human disease subtypes. UCMS may thus enable the selection of strains suitable for investigating specific depression-related features and could be an appropriate model for identifying genetic factors associated with increased vulnerability, specific symptoms of affective disorders, and AD resistance.     

Strain background influences neurotoxicity and behavioral abnormalities in mice expressing the tetracycline transactivator

The tet-off system has been widely used to create transgenic models of neurological disorders including Alzheimer's, Parkinson's, Huntington's, and prion disease. The utility of this system lies in the assumption that the tetracycline transactivator (TTA) acts as an inert control element and does not contribute to phenotypes under study. Here we report that neuronal expression of TTA can affect hippocampal cytoarchitecture and behavior in a strain-dependent manner. While studying neurodegeneration in two tet-off Alzheimer's disease models, we unexpectedly discovered neuronal loss within the dentate gyrus of single transgenic TTA controls. Granule neurons appeared most sensitive to TTA exposure during postnatal development, and doxycycline treatment during this period was neuroprotective. TTA-induced degeneration could be rescued by moving the transgene onto a congenic C57BL/6J background and recurred on reintroduction of either CBA or C3H/He backgrounds. Quantitative trait analysis of B6C3 F2 TTA mice identified a region on Chromosome 14 that contains a major modifier of the neurodegenerative phenotype. Although B6 mice were resistant to degeneration, they were not ideal for cognitive testing. F1 offspring of TTA C57BL/6J and 129X1/SvJ, FVB/NJ, or DBA/1J showed improved spatial learning, but TTA expression caused subtle differences in contextual fear conditioning on two of these backgrounds, indicating that strain and genotype can interact independently under different behavioral settings. All model systems have limitations that should be recognized and mitigated where possible; our findings stress the importance of mapping the effects caused by TTA alone when working with tet-off models.     

Hippocampal injury,atrophy, synaptic reorganization,and epileptogenesis after perforant pathway stimulation‐induced status epilepticus in the mouse

Prolonged dentate granule cell discharges produce hippocampal injury and chronic epilepsy in rats. In preparing to study this epileptogenic process in genetically altered mice, we determined whether the background strain used to generate most genetically altered mice, the C57BL/6 mouse, is vulnerable to stimulation‐induced seizure‐induced injury. This was necessary because C57BL/6 mice are reportedly resistant to the neurotoxic effects of kainate‐induced seizures, which we hypothesized to be related to strain differences in kainate's effects, rather than genetic differences in intrinsic neuronal vulnerability. Bilateral perforant pathway stimulation‐induced granule cell discharge for 4 hours under urethane anesthesia produced degeneration of glutamate receptor subunit 2 (GluR2)‐positive hilar mossy cells and peptide‐containing interneurons in both FVB/N (kainate‐vulnerable) and C57BL/6 (kainate‐resistant) mice, indicating no strain differences in neuronal vulnerability to seizure activity. Granule cell discharge for 2 hours in C57BL/6 mice destroyed most GluR2‐positive dentate hilar mossy cells, but not peptide‐containing hilar interneurons, indicating that mossy cells are the neurons most vulnerable to this insult. Stimulation for 24 hours caused extensive hippocampal neuron loss and injury to the septum and entorhinal cortex, but no other detectable damage. Mice stimulated for 24 hours developed hippocampal sclerosis, granule cell mossy fiber sprouting, and chronic epilepsy, but not the granule cell layer hypertrophy (granule cell dispersion) produced by intrahippocampal kainate. These results demonstrate that perforant pathway stimulation in mice reliably reproduces the defining features of human mesial temporal lobe epilepsy with hippocampal sclerosis. Experimental studies in transgenic or knockout mice are feasible if electrical stimulation is used to produce controlled epileptogenic insults. J. Comp. Neurol. 515:181–196, 2009. © 2009 Wiley‐Liss, Inc.     

In vivo and in vitro glycogenic effects of methionine sulfoximine are different in two inbred strains of mice

We investigated the relationship between brain glycogen anabolism and methionine sulfoximine (MSO)-induced seizures in two inbred mouse strains that presented differential susceptibility to the convulsant. CBA/J was considered a MSO-high-reactive strain and C57BL/6J a MSO-low-reactive strain. Accordingly, the dose of MSO needed to induce seizures in CBA/J mice is lower than that in C57BL/6J mice, and CBA/J mice which had seizures, died during the first convulsion. In addition, the time--course of the MSO effect is faster in CBA/J mice than that in C57BL/6J mice. Analyses were performed in C57BL/6J and CBA/J mice after administration of 75 (subconvulsive dose) and 40 mg/kg of MSO (subconvulsive dose, not lethal dose), respectively. In the preconvulsive period, MSO induced an increase in the brain glycogen content of C57BL/6J mice only. Twenty-four hours after MSO administration, the brain glycogen content increased in both strains. The activity and expression of fructose-1,6-bisphosphatase, the last key enzyme of the gluconeogenic pathway, were increased in MSO-treated C57BL/6J mice as compared to control mice, at all experimental time points, whereas they were increased in CBA/J mice only 24 h after MSO administration. These latter results correspond to CBA/J mice that did not have seizures. Interestingly, the differences observed in vivo were consistent with results in primary cultured astrocytes from the two strains. This data suggests that the metabolism impairment, which was not a consequence of seizures, could be related to the difference in seizure susceptibility between the two strains, depending on their genetic background.     

Genetic strain differences in platelet aggregation of laboratory mice

To investigate the physiological role of novel genes and proteins in platelet activation, various knockout mice have been produced. A number of standard inbred mouse strains each possessing genetically unique characters such as high tumor generation, hyperglycemia or hyperlipidemia, have been bred. In breeding knockout mice for investigation of specific physiological functions, appropriate selection of parental or backcross strains is necessary. Thus, examination of strain-specific platelet characteristics is important. In the present study, platelet aggregation responses of 13 laboratory mouse strains, 129/Sv, A, AKR, BALB/c, C3H/He, C57BL/6J, CBA, DBA/1, DBA/2, ddY, FVB, ICR, and NZW, and the diabetic strain C57BL/KsJ db/db, were compared. Marked strain differences were observed in ADP- and collagen-induced platelet aggregation. The highest responses with both were seen in AKR/J and NZW/N, whereas the lowest were seen in DBA/2 and DBA/1. There was a 5-fold difference in the platelet aggregation threshold index (PATI) for ADP-induced PRP aggregation between AKR/J (0.6 microM) and DBA/2 (3.0 microM). With whole blood aggregation, the highest response was seen in AKR, whereas the lowest was seen in DBA/2 and DBA/1. The present study demonstrated that there is considerable strain difference in platelet aggregation among laboratory mice, which should be taken into account in backcrossing knockout strains.     

Strain comparisons and developmental profile of the delta subunit of the murine GABAA receptor.

GABAA receptors are multisubunit inhibitory chloride channels in the brain which open in response to binding of gamma-aminobutyric acid (GABA) and are thought to be involved in some forms of seizures. We compare the sequence and expression of the GABAA receptor delta subunit in audiogenic seizure prone (DBA/2J) and seizure resistant (C57BL/6J) inbred strains of mice and also report this subunit's postnatal developmental profile. We did not detect any unique features in the delta subunits of DBA/2J mice which might explain their seizure susceptibility, but did detect in some clones from both DBA/2J mice and C57BL/6J mice an unusual substitution of His for a conserved Tyr in the delta subunit's first putative transmembrane region.     

Development of a model of seizure-induced hippocampal injury with features of programmed cell death in the BALB/c mouse

Although mice are amenable to gene knockout, they have not been exploited in the setting of seizure-induced neurodegeneration due to the resistance to injury of key mouse strains. We refined and developed models of seizure-induced neuronal death in the C57BL/6 and BALB/c strains by focally evoking seizures using intra-amygdala kainic acid. Seizures in adult male BALB/c mice, or C57BL/6 mice as reference, caused ipsilateral death of CA1 and CA3 neurons within the hippocampus. Termination of seizures by lorazepam was more effective than diazepam in both strains, largely restricting neuronal loss to the CA3 sector. Electroencephalography (EEG) recordings defined injurious and non-injurious seizure patterns, which could not be separated adequately by behavioral observation alone. Degenerating neurons in the hippocampus were positive for DNA fragmentation and approximately a third of these exhibited morphologic features of programmed cell death. Western blot analysis revealed the cleavage of caspase-8 after seizures in both strains. These data refine our C57BL/6 model and establish a companion model of focally evoked limbic seizures in the BALB/c mouse that provides further evidence for activation of programmed cell death after seizures.     

Strain comparisons and developmental profile of the delta subunit of the murine GABAA Receptor

GABA_A receptors are multisubunit inhibitory chloride channels in the brain which open in response to binding of -γ-aminobutyric acid (GABA) and are thought to be involved in some forms of seizures. We compare the sequence and expression of the GABA_A receptor δ subunit in audiogenic seizure prone (DBA/2J) and seizure resistant (C57BL/6J) inbred strains of mice and also report this subunit's postnatal developmental profile. We did not detect any unique features in the 6 subunits of DBA/2J mice which might explain their seizure susceptibility, but did detect in some clones from both DBA/2J mice and C57BL/6J mice an unusual substitution of His for a conserved Tyr in the δ subunit's first putative transmembrane region.     

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.     

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.     

Synaptosomal high-affinity noradrenaline uptake does not differ between mice susceptible (DBA/2J) and resistant (C57 BL/6) to audiogenic seizures

Abnormalities in noradrenaline-mediated neurotransmission have been advocated as a basis of the age-related susceptibility of DBA/2J mice to generalised convulsions induced by auditory stimulation. We have measured the kinetics of synaptosomal high-affinity noradrenaline uptake in 5 brain regions of DBA/2J mice at ages before, during and after their maximal susceptibility to audiogenic seizures, and age-matched C57 BL/6 mice, a strain resistant to audiogenic seizures at all ages. No differences were found between the two strains of mice in any of the brain regions studied. Abnormalities of high-affinity noradrenaline uptake do not contribute to audiogenic seizure susceptibility of DBA/2J mice.     

Neurospecific S-100 protein content in brains of different mouse strains

Total whole brain concentrations of S-100 protein and of its water-soluble fraction were determined in 11 inbred mouse straine: DBA/2J, AKR/J, CBA/Lac, C57BL/6J, C57BL/6J-Ay, C3H/He, C3H/f, DD, A/He, BALB/cLac, CC57BR/Mv, and in cerebral cortex, cerebellum and hippocampus in DBA/2J, AKR/J and CBA/Lac strains. Highly significant differences in the concentrations of the water-soluble S-100 protein were found between some strains. Slight differences were found in total S-100 protein content in whole brains between the strains (0.01 less that P less than 0.05). The DBA/2J mice had the highest brain S-100 protein content, and were characterized by a higher learning rate in shuttle-box as compared to CBA/Lac and AKR/J mice, who had a low content of this neurospecific protein.     

Genetic variance contributes to naltrexone-induced inhibition of sucrose intake in inbred and outbred mouse strains

The study of genetic variance in opioid receptor antagonism of sucrose and other forms of sweet intake has been limited to reductions in sweet intake in mice that are opioid receptor-deficient or lacking either pre-pro-enkephalin or beta-endorphin. Marked genetic variance in inbred mouse strains has been observed for sucrose intake across a wide array of concentrations in terms of sensitivity, magnitude, percentages of kilocalories consumed as sucrose and compensatory chow intake. The present study examined potential genetic variance in systemic naltrexone's dose-dependent (0.01-5 mg/kg) and time-dependent (5-120 min) ability to decrease sucrose (10%) intake in eleven inbred (A/J, AKR/J, BALB/cJ, CBA/J, C3H/HeJ, C57BL/6J, C57BL/10J, DBA/2J, SJL/J, SWR/J, 129P3/J) and one outbred (CD-1) mouse strains. A minimum criterion sucrose intake (1 ml) under vehicle treatment, designed to avoid "floor effects" of antagonist treatment was not achieved in three (A/J, AKR/J, CBA/J) inbred mouse strains. Marked genetic variance in naltrexone's ability to inhibit sucrose intake was observed in the remaining strains with the greatest sensitivity observed in the C57BL/10J and C57BL/6J strains, intermediate sensitivity in BALB/cJ, C3H/HeJ, CD-1 and DBA/2J mice, and the least sensitivity in 129P3/J, SWR/J and SJL/J strains with a 7.5-36.5 fold range of greater effects in the ID(50) of naltrexone-induced inhibition in C57BL/10J relative to the three less-sensitive strains across the time course. Naltrexone primarily affected the maintenance, rather than the initiation of intake in BALB/cJ, CD-1, C3H/HeJ, DBA/2J and SJL/J mice, but significantly reduced sucrose intake at higher doses across the time course in C57BL/6J, C57BL/10J and 129P3/J mice. Whereas SWR/J mice failed to display any significant reduction in sucrose intake at any time point following any of the naltrexone doses, naltrexone's maximal magnitude of inhibitory effects was small (35-40%) in 129P3/J and SJL/J mice, moderate ( approximately 50%) in BALB/cJ, C3H/HeJ, CD-1 and DBA2/J mice, and profound (70-80%) in C57BL/6J and C57BL/10J mice. Indeed, the latter two strains displayed significantly greater percentages of naltrexone-induced inhibition of sucrose intake than virtually all other strains. These data indicate the importance of genetic variability in opioid modulation of sucrose intake.     

Immunochemical studies of brain glutamate decarboxylase and GABA-transaminase of six inbred strains of mice

Six different inbred strains of mice (C57BL/6J, CBA/CaJ, CE/J, DBA/2J, LP/J and RF/J) were compared in terms of specific activities and immunochemical properties of brain L-glutamate decarboxylase (GAD) and gamma-aminobutyrate transaminase (GABA-T), the enzymes responsible for the synthesis and degradation of GABA, respectively. GAD from the brains of the different strains was indistinguishable on the basis of specific activities, double diffusion tests, immunoelectrophoresis and inhibition by antibody. However, microcomplement fixation tests showed GAD from DBA and C57BL mice to be most distinctly different from GAD extracted from the Swiss mouse, from which the original antigen was prepared and that the enzyme from the CE, LP and RF also differed. Similar fixation curves were obtained for the GAD from CBA and Swiss mice. GABA-T from the different strains was indistinguishable on the basis of all the tests employed.     

Differential sensitivity to bicuculline in three inbred mouse strains

We examined the inbred mouse strains DBA/2Ibg, C57BL/6Ibg, and C3H/2Ibg for differences in susceptibility to bicuculline-induced seizures, as well as to bicuculline-induced epileptiform activity recorded in the CA1 pyramidal cell layer of hippocampal slices. For susceptibility to seizure onset the strain rank order was (most to least susceptible): C3H = DBA greater than C57. The rank order for sensitivity to effects of bicuculline on tonic seizure latency and on hippocampal epileptiform activity were identical: C3H greater than DBA = C57. It is suggested that mechanisms underlying the development of bicuculline-induced epileptiform events in the hippocampal slice may be similar to those involved in the development of tonic seizures measured in vivo.     

Genetic basis of kainate-induced excitotoxicity in mice: phenotypic modulation of seizure-induced cell death

Excitotoxicity, a process in which excessive excitation of glutamate receptors results in cell death, has been implicated in a number of neurological disorders. However, the genetic characteristics and molecular mechanisms that can modulate the extent of cell death are unclear. Previously, we had reported that the extent of excitotoxic cell death is conferred by differences in the genetic background of several mouse strains. As a first step in the identification of loci that can modulate the extent of excitotoxin-induced cell death, we tested C57BL/6 and FVB/N mice, their F1 hybrids and backcross progeny for differences in apparent excitotoxic cell death induced by kainic acid (KA). While no strain dependent differences in seizure duration were observed, phenotypic analysis of cell death indicated that C57BL/6 mice showed no seizure-induced cell death, while FVB/N mice exhibited extensive cell death. Studies of seizure-induced cell death in hybrid and backcross progeny revealed an association between seizure-induced cell death and genotype. Mice from the F1 cross exhibited little to no seizure-induced cell death, indicative that the extent of cell death is conferred as a dominant genetic trait. Phenotypic assessment of cell death in backcross progeny suggests that differences in apparent cell death are conferred by a single gene locus. These findings implicate genetic factors in individual differences in excitotoxin-induced cell death.     

C57BL/6 strain is most susceptible to cerebral ischemia following bilateral common carotid occlusion among seven mouse strains: selective neuronal death in the murine transient forebrain ischemia

Rats and gerbils have been used widely to investigate the molecular mechanism of selective neuronal death following transient global ischemia. Recently, the availability of transgenic mice has enabled us to examine the involvement of specific gene products in various pathophysiological conditions. However, there has been only limited information about the experimental model of cerebral ischemia in mice, particularly in regard of selective neuronal death. We examined whether bilateral carotid occlusion produced global forebrain ischemia in seven common mouse strains including C57BL/6, ICR, BALB/c, C3H, CBA, ddY and DBA/2, based on neurological signs, histological findings and cortical microcirculatory as well as India ink perfusion patterns. The C57BL/6 strain was found to be the most susceptible among seven strains. All C57BL/6 mice died within 6 h after permanent bilateral carotid occlusion. After transient bilateral carotid occlusion for 20 min, more than 90% of C57BL/6 mice showed typical neurological signs such as torsion of the neck and rolling fits, and developed selective neuronal death in the hippocampus and caudoputamen. Hypothermia prevented the neuronal death. Visualization of brain vasculature by India ink perfusion indicated that the susceptibility of the mice after bilateral carotid occlusion depended mainly on the degree of anastomosis between carotid and basilar arteries. Our results showed the feasibility of investigating selective neuronal death in transgenic mice with simple temporary occlusion of both common carotid arteries, when those from the C57BL/6 strain or inbred transgenic mice from other strains with the C57BL/6 strain in a back-cross manner are used.