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.     

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.     

The α<Subscript>1</Subscript>,α<Subscript>2</Subscript>, and α<Subscript>3</Subscript> Subunits of GABA<Subscript>A</Subscript> Receptors: Comparison in Seizure-Prone and -Resistant Mice and during Development

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in brain, opens chloride channels through actions on GABAA receptors. We now report base and amino acid sequences of the alpha 1, alpha 2, and alpha 3 subunits from GABAA receptors of audiogenic seizure-prone (DBA/2J) and -resistant (C57BL/6J) inbred strains of mice. Inbreeding had fixed different alleles of the alpha 1 subunit in the two strains, giving five base differences in the cDNAs. None of these affected amino acid sequence, but one did create a NsiI restriction site potentially useful in mapping genomic DNA. No base or amino acid sequence differences between the strains were detected for the other two subunits. Northern blots revealed no apparent strain differences in message levels for these three subunits in whole brains of the mice at 3 weeks of age, the peak of seizure susceptibility in DBA/2J, but did reveal distinct regional and developmental patterns of expression among the subunits in mouse brain.     

Audiogenic seizure proneness requires the contribution of two susceptibility loci in mice

Juvenile mice of the DBA/2J strain undergo generalised seizures when exposed to a high-intensity auditory stimulus. Genetic analysis identified three different loci underlying this audiogenic seizure proneness (ASP)—Asp1, Asp2 and Asp3 on chromosomes 12, 4 and 7, respectively. Asp1 is thought to have the strongest influence, and mice with only Asp1 derived from the DBA/2J strain are reported to exhibit ASP. The aim of this study was to characterise more accurately the contributions of the Asp1 and Asp3 loci in ASP using congenic strains. Each congenic strain contains a DBA/2J-derived interval encompassing either Asp1 or Asp3 on a C57BL/6J genetic background. A double congenic C57BL/6J strain containing both Asp loci derived from DBA/2J was also generated. Here, we report that DBA/2J alleles at both of these Asp loci are required to confer ASP because congenic C57BL/6 mice harbouring DBA/2J alleles at only Asp1 or Asp3 do not exhibit ASP, whereas DBA/2J alleles at both loci resulted in increased susceptibility for audiogenic seizure in double congenic C57BL/6 mice.     

FOS and FOSB expression in the medial preoptic nucleus pars compacta of maternally active C57BL/6J and DBA/2J mice

C57BL/6J and DBA/2J inbred mice differ in aspects of maternal behavior and in the morphology of the medial preoptic nucleus (MPO), suggesting a possible association. DBA/2J mice have a compact subnucleus in the MPO, the MPOpc, that is sexually dimorphic and absent in C57BL/6J mice. To determine whether MPOpc cells are activated by maternal behavior, FOS and FOSB immunohistochemistry was performed on brain sections of C57BL/6J and DBA/2J mothers following the return of their pups after a separation of 2 days. In both light and dark phases of the daily cycle, stimulation of DBA/2J mothers evoked an increase in FOS- and FOSB-immunoreactivity in the MPOpc. Stimulated C57BL/6J mice, which lack the MPOpc, did not show an increase in cellular activity in the corresponding MPO region. Cells immediately lateral to the MPOpc were activated by pup stimulation, in both strains. These results suggest that MPOpc cells are active during maternal behavior, and that strain differences in maternal behavior are related to anatomical differences in the MPO.     

Mouse strain differences in locomotor,sensitisation and rewarding effect of heroin; association with alterations in MOP‐r activation and dopamine transporter binding

There is growing agreement that genetic factors play an important role in the risk to develop heroin addiction, and comparisons of heroin addiction vulnerability in inbred strains of mice could provide useful information on the question of individual vulnerability to heroin addiction. This study examined the rewarding and locomotor‐stimulating effects of heroin in male C57BL/6J and DBA/2J mice. Heroin induced locomotion and sensitisation in C57BL/6J but not in DBA/2J mice. C57BL/6J mice developed conditioned place preference (CPP) to the highest doses of heroin, while DBA/2J showed CPP to only the lowest heroin doses, indicating a higher sensitivity of DBA/2J mice to the rewarding properties of heroin vs C57BL/6J mice. In order to investigate the neurobiological substrate underlying some of these differences, the effect of chronic ‘intermittent’ escalating dose heroin administration on the opioid, dopaminergic and stress systems was explored. Twofold higher μ‐opioid receptor (MOP‐r)‐stimulated [³⁵S]GTPγS binding was observed in the nucleus accumbens and caudate of saline‐treated C57BL/6J mice compared with DBA/2J. Heroin decreased MOP‐r density in brain regions of C57BL/6J mice, but not in DBA/2J. A higher density of dopamine transporters (DAT) was observed in nucleus accumbens shell and caudate of heroin‐treated DBA/2J mice compared with heroin‐treated C57BL/6J. There were no effects on D₁ and D₂ binding. Chronic heroin administration decreased corticosterone levels in both strains with no effect of strain. These results suggest that genetic differences in MOP‐r activation and DAT expression may be responsible for individual differences in vulnerability to heroin addiction.     

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.     

Differential involvement of the dorsal hippocampus in passive avoidance in C57bl/6J and DBA/2J mice

The inferior performance of DBA/2 mice when compared to C57BL/6 mice in hippocampus-dependent behavioral tasks including contextual fear conditioning has been attributed to impaired hippocampal function. However, DBA/2J mice have been reported to perform similarly or even better than C57BL/6J mice in the passive avoidance (PA) task that most likely also depends on hippocampal function. The apparent discrepancy in PA versus fear conditioning performance in these two strains of mice was investigated using an automated PA system. The aim was to determine whether these two mouse strains utilize different strategies involving a different contribution of hippocampal mechanisms to encode PA. C57BL/6J mice exhibited significantly longer retention latencies than DBA/2J mice when tested 24 h after training irrespective of the circadian cycle. Dorsohippocampal NMDA receptor inhibition by local injection of the selective antagonist DL-2-amino-5-phosphonovaleric acid (AP5, 3.2 microg/mouse) before training resulted in impaired PA retention in C57BL/6J but not in DBA/2J mice. Furthermore, nonreinforced pre-exposure to the PA system before training caused a latent inhibition-like reduction of retention latencies in C57BL/6J, whereas it improved PA retention in DBA/2J mice. These pre-exposure experiments facilitated the discrimination of hippocampal involvement without local pharmacological intervention. The results indicate differences in PA learning between these two strains based on a different NMDA receptor involvement in the dorsal hippocampus in this emotional learning task. We hypothesize that mouse strains can differ in their PA learning performance based on their relative ability to form associations on the basis of unisensory versus multisensory contextual/spatial cues that involve hippocampal processing.     

Finding the right motivation: genotype-dependent differences in effective reinforcements for spatial learning

Memory impairments of DBA/2J mice have been frequently reported in spatial and emotional behavior tests. However, in some memory tests involving food reward, DBA/2J mice perform equally well to C57BL/6J mice or even outperform them. Thus, it is conceivable that motivational factors differentially affect cognitive performance of different mouse strains. Therefore, spatial memory of DBA/2J and C57BL/6J mice was investigated in a modified version of the Barnes maze (mBM) test with increased complexity. The modified Barnes maze test allowed using either aversive or appetitive reinforcement, but with identical spatial cues and motor requirements. Both mouse strains acquired spatial learning in mBM tests with either reinforcement. However, DBA/2J mice learned slower than C57BL/6J mice when aversive reinforcement was used. In contrast, the two strains performed equally well when appetitive reinforcement was used. The superior performance in C57BL/6J mice in the aversive version of the mBM test was accompanied by a more frequent use of the spatial strategy. In the appetitive version of the mBM test, both strains used the spatial strategy to a similar extent. The present results demonstrate that the cognitive performance of mice depends heavily on motivational factors. Our findings underscore the importance of an effective experimental design when assessing spatial memory and challenges interpretations of impaired hippocampal function in DBA/2J mice drawn on the basis of behavior tests depending on aversive reinforcement.     

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.     

GABA-gated chloride ion influx and receptor binding studies in C57BL6J and DBA2J mice

GABA-gated chloride ion influx was measured in brain 'microsac' preparations of young (20-22-day-old) and older (40-42-day-old) C57BL6J and DBA2J mice. The young DBA2J mice are susceptible to audiogenic seizures. GABA sensitivity was reduced in young DBA2J mice as compared to age-matched C57BL6J mice or older mice of either strain. Age and strain differences in ligand binding to GABA/benzodiazepine receptor complex and glutamate receptor could not account for this finding. These results provide evidence for a defect in GABA-gated chloride ion influx in audiogenic seizure-susceptible DBA2J mice.     

Comparison of seizure phenotype and neurodegeneration induced by systemic kainic acid in inbred, outbred, and hybrid mouse strains

We assessed inbred, outbred and hybrid mouse strains for susceptibility to seizures and neurodegeneration induced by systemic administration of kainic acid (KA). Each strain showed a unique pattern of susceptibility to seizures as assessed by the dose necessary to induce continuous tonic clonic seizures, progression through six seizure levels, the number of mice that failed to satisfy seizure criteria, and seizure-induced mortality. In general, the C57BL/6, ICR, FVB/N, and BALB/c strains were resistant to seizures while the C57BL/10, DBA/2 J, and F1 C57BL/6*CBA/J strains were vulnerable. Neuronal cell death was quantified in four subfields of the hippocampus: CA3, the hilus of the dentate gyrus, CA1, and the dentate granule cell layer. Neurodegeneration was also semiquantitatively assessed in other brain regions including the neocortex, striatum, thalamus, hypothalamus and amygdala. Although there was variability in the extent of cell death within strains, there were significant differences in the amount of hippocampal cell death between strains and also different patterns of neurodegeneration in affected brain areas. In general, the C57BL/6, C57BL/10, and F1 C57BL/6*CBA/J strains were resistant to neurodegeneration while the FVB/N, ICR and DBA/2 J strains were vulnerable. The BALB/c strain was unique in that neurodegeneration was confined to the hippocampus. Consistent with previous findings, the resistant neurodegeneration phenotype was dominant in an F1 cross of resistant and vulnerable inbred strains. Our results, using a large number of mouse strains, definitively demonstrate that a mouse strain's seizure phenotype is not related to its neurodegeneration phenotype.     

Basal levels and alcohol-induced changes in nociceptin/orphanin FQ, dynorphin, and enkephalin levels in C57BL/6J mice

In order to investigate the involvement of the opioid and nociceptin/orphanin FQ (N/OFQ) system in alcohol drinking behaviour, N/OFQ and the opioid peptides dynorphin B (DYNB) and Met-enkephalin-Arg(6) Phe(7) (MEAP) were examined in the alcohol-preferring C57BL/6J mice. Basal peptide levels were compared in the brain and the pituitary gland with basal levels in the alcohol-avoiding DBA/2J mice. Furthermore, the effects of chronic alcohol self-administration on peptides were studied in the C57BL/6J mice. Compared to the DBA/2J mice, C57BL/6J mice had low immunoreactive (ir) levels of DYNB and MEAP in the nucleus accumbens, the hippocampus, and the substantia nigra, low ir-DYNB levels in the striatum and low ir-MEAP levels in the frontal cortex. Higher ir-DYNB levels in the pituitary gland and in the periaqueductal gray (PAG) and higher ir-N/OFQ levels in the frontal cortex and in the hippocampus were detected in C57BL/6J mice compared to the DBA/2J mice. After 4 weeks of voluntary alcohol consumption, only minor changes in steady-state peptide levels were identified. However, 5 days after the alcohol-drinking period, lower levels of all peptides were detected in the ventral tegmental area and ir-DYNB levels were also lower in the amygdala and in the substantia nigra. Twenty-one days after cessation of alcohol self-administration, the opioid peptides in alcohol-consuming C57BL/6J mice were lower in the PAG, the N/OFQ was lower in the frontal cortex and DYNB was higher in the amygdala and substantia nigra as compared to control C57BL/6J mice. This study demonstrates strain differences between C57BL/6J mice and DBA/2J mice that could contribute to divergent drug-taking behaviour, and it also demonstrates time- and structure-specific changes in neuropeptide levels after alcohol self-administration in the C57BL/6J mice.     

The GABAA receptor γ1-subunit in seizure prone (DBA/2) and resistant (C57BL/6) mice

γ-Aminobutyric acid (GABA)_A receptors are the sites of action for many antiepileptic drugs such as benzodiazepines and barbiturates. We report the results of molecular cloning of the γ₁-subunit from seizure prone DBA/2J and resistant C57BL/6J inbred mice, and analyses of nucleotide sequences and expression of the γ₁-subunit messenger RNA (mRNA) in DBA/2 and C57BL/6 inbred mice. The mouse γ₁-subunit complementary DNA (cDNA) shares 98% similarity with that of the rat at the level of amino acid sequence. Northern blot hybridization indicates that the γ₁-subunit mRNA is expressed predominantly in areas other than the cerebral cortex and cerebellum and shows little change with postnatal development. No differences have been found for the subunit between DBA/2 and C57BL/6 mice either for nucleotide sequence or for level of expression of the subunit’s mRNA in whole brain by Northern blots at 3 weeks of age.     

Social approach-avoidance behavior of inbred mouse strains towards DBA/2 mice

Little is known about the genetics of social approach-avoidance behaviors. We measured social approach-avoidance of prepubescent female C57BL/6J, DBA/2J, FVB/NJ, AKR/J, A/J, and BALB/cJ mice towards prepubescent DBA/2J female mice. C57BL/6J mice showed the greatest predominance of approach, while BALB/cJ mice showed the greatest predominance of avoidance. Thus, this phenotype is affected by spontaneous genetic variation in mice and can be measured in an assay useful for future neurogenetic studies.     

Strain differences in the development of susceptibility to audiogenic seizures after acoustic priming but not after hearing loss

Either exposure to an initial auditory stimulus (IAS) or external ear plugging (EEP) was used to produce susceptibility to audiogenic seizures in C57BL/6Bg and DBA/1Bg mice. After the IAS, increments in seizure susceptibility occurred by 5 h in C57BL/6Bg mice and by 24 h in DBA/1Bg-ras mice, whereas after EEP, increments in seizure susceptibility occurred by 48 h in C57BL/6Bg and by 24 h in DBA/1Bg-ras mice. Because both the IAS and EEP produce hearing loss, the strain differences in the effect of the IAS on the development of susceptibility and the strain similarities in the effect of the EEP on the development of susceptibility support the hypothesis that acoustic priming in the C57BL/6Bg at 19 days of age involves another mechanism in addition to that of hearing loss and disuse supersensitivity. It was suggested elsewhere that the other mechanism is mediated by a post-IAS decrease in the concentration of brain γ-aminobutyric acid and requires brain protein synthesis for a brief period post-IAS.     

Effect of Milacemide on Audiogenic Seizures and Cortical (Na+, K+)-ATPase of DBA/2J Mice

Milacemide (MLM, CP 1552 S, 2-N-pentylaminoacetamide), a glycinamide derivative, is currently being evaluated clinically for antiepileptic activity. Anticonvulsant properties have been shown in various animal models, but the mechanism of action of MLM is unclear. We studied its activity in audiogenic seizures of DBA/2J mice. MLM was effective in inhibiting the convulsions induced by sound with a biphasic dose-effect relation. The ED50 was 109 mg/kg orally against tonic extension. Higher doses were necessary to abolish clonic convulsion and running response. Because impaired cerebral (Na+, K+)-ATPase activity is supposed to play a role in epileptogenesis, we tested MLM on in vitro cortical enzymatic activity of DBA/2J mice. Basal (Na+, K+)-ATPase activity was unchanged by several concentrations of MLM in normal C57BL/6J and audiogenic DBA/2J mice. K+ activation (from 3 to 18 mM) of (Na+, K+)-ATPase is abolished in DBA/2J mice as compared with C57BL/6J mice, suggesting impaired glial (Na+, K+)-ATPase. In the presence of MLM (from 30 to 1000 mg/L), cortical (Na+, K+)-ATPase of DBA/2J mice is activated by high concentrations of K+, as in C57BL/6J mice. Results suggest that the antiepileptic activity of MLM in audiogenic mice may be secondary to an activation of a deficient glial (Na+, K+)-ATPase.     

Prenatal development of the hippocampus in two strains of inbred mice

In this paper we present some morphological observations on the morphogenesis of the hippocampus in two inbred strains of mice, C57BL/6J (C57) and DBA/2J (DBA), from day 16 (E16) to day 17 (E17) of prenatal life. At E16 some differences in the histoarchitecture of the hippocampal anlage begin to be detectable. At E17 the differences become more relevant, particularly those concerning the lamination pattern. The more relevant differences concern the differentiation of the pyramidal layer, which appears well defined in C57 mice, while in DBA mice there is no clearcut delimitation between the pyramidal layer and the intermediate zone, where numerous migrating neurons are present. These observations may be interpreted in terms of different migration rates for the neurons in the two strains.     

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.     

Establishment of chronic intravenous drug self-administration in the C57BL/6J mouse

A wide variety of drugs that have significant human abuse potential have been demonstrated to function as positive reinforcers in animals. The present study was designed to characterize a new mouse model of chronic intravenous drug self-administration. Adult male C57BL/6J mice, implanted with external jugular infusion catheters, were given access to response-contingent injections. They did not initiate responding for saline delivery, whereas the C57BL/6J mice initiated morphine, cocaine, methamphetamine and pentobarbital self-administration. Drug-maintained responding was consistently and significantly higher for each compound than for saline responding. In contrast to C57BL/6J mice, DBA/2J mice failed to initiate cocaine self-administration. Thus, chronic intravenous drug self-administration procedures can be adapted to the inbred mouse.