Substrain Differences Research Articles

Abstract Mo008: Sub-strain Dependent Vulnerability to Psychosocial Stress Exacerbates Doxorubicin-induced Cardiotoxicity in Adult Male Mice

Introduction: Psychosocial stress is a significant cardiovascular risk factor and an enormous burden on cancer patients. However, limited knowledge is available on the underlying mechanisms at least in part because of lack of translationally relevant animal models. Previous work from our lab established that adult exposure to social stress can exacerbate the cardiovascular effect of juvenile doxorubicin (DOX) exposure. However, the effect of concomitant DOX and stress exposure in adult mice is unknown. Methods: In this study, a ‘two-hit’ mouse model was established, in which chronic subordination stress (CSS) and DOX treatment were given simultaneously. Twelve-week-old male C57BL/6N (6N) and C57BL/6J (6J) mice were subjected to daily episodes of social defeat by dominant CD1 mice with whom they remained in continuous sensory contact with for 28 days, thus establishing social subordination. One week after the start of CSS, mice were administered DOX (8 mg/kg/week) for 3 weeks. Thereafter, cardiac function was assessed by echocardiography and gene expression of inflammatory and pro-fibrotic markers were determined by real-time PCR. Cardiac fibrosis and inflammation were assessed by histopathologic analysis. Results: Cardiac dysfunction and significant cardiac fibrosis were observed in 6N but not in 6J mice that were exposed to both DOX and CSS, as evidenced by reduced ejection fraction, cardiac output, and increased trichrome staining, respectively. Additionally, gene expression analysis demonstrated significant upregulation of atrial natriuretic peptide and interleukin-6 in 6N mice exposed to both DOX and CSS compared to 6J mice. Finally, DOX caused cardiac atrophy and abrogated CSS-induced cardiac hypertrophy in 6N but not in 6J mice, as evidenced by measuring left ventricular mass and heart weight/tibia length. Conclusion: Exposure to CSS exacerbated the cardiotoxic effects of DOX in a sub-strain dependent manner. These findings highlight psychosocial stress as a risk factor for worse cardiovascular outcome in cancer patients receiving cardiotoxic regimens. The study underscores sub-strain differences in the cardiac responses of 6N and 6J mouse to CSS alone and CSS/DOX treatment that may serve as a discovery platform for the identification of genetic biomarkers of resilience.

Cyfip2 allelic variation in C57BL/6J and C57BL/6NJ mice alters free-choice ethanol drinking but not binge-like drinking or wheel-running activity.

Since the origin of the C57BL/6 (B6) mouse strain, several phenotypically and genetically distinct B6 substrains have emerged. For example, C57BL/6J mice (B6J) display greater voluntary ethanol consumption and locomotor response to psychostimulants and differences in nucleus accumbens synaptic physiology relative to C57BL/6N (B6N) mice. A non-synonymous serine to phenylalanine point mutation (S968F) in the cytoplasmic FMR1-interacting protein 2 (Cyfip2) gene underlies both the differential locomotor response to cocaine and the accumbal physiology exhibited by these substrains. We examined whether Cyfip2 allelic variation underlies B6 substrain differences in other reward-related phenotypes, such as ethanol intake and wheel-running activity. We compared voluntary ethanol consumption, wheel-running, and binge-like ethanol drinking in male and female B6J and B6NJ mice. When substrain differences were observed, additional experiments were performed in two novel mouse models in which the B6N Cyfip2 mutation was either introduced (S968F) into the B6J background or corrected (F968S) via CRISPR/Cas9 technology. B6J consumed significantly more ethanol than B6NJ and allelic variation in Cyfip2 contributed substantially to this substrain difference. In contrast, B6NJ displayed significantly more daily wheel-running than B6J, with Cyfip2 allelic variation playing only a minor role in this substrain difference. Lastly, no substrain differences were observed in binge-like ethanol drinking. These results contribute to the characterization of behavior-genetic differences between B6 substrains, support previous work indicating that free-choice and binge-like ethanol drinking are dependent on partially distinct genetic networks, and identify a novel phenotypic difference between B6 substrains in wheel-running activity.

Substrain differences in Sry expression at the stage of sex determination in C57BL/6 mouse strains.

The expression of sex determining region of the Y chromosome (Sry) in the fetal gonads is important for male development. In a mouse model of disorders of sex development (C57BL/6 (B6)-XYPOS), the gonadal phenotype and the timing of Sry expression differ due to differences among B6 substrains as the genetic background. Since differences in Sry expression among B6 substrains have been speculated, the present study examined Sry expression in B6J, B6JJmsSlc, and B6NCrl mice. These substrains differed in the number of Sry-expressing cells in the gonads of embryonic mice at each developmental stage, with B6NCrl having more than the other strains. The substrains differed also in the number of Sry-expressing cells between the left and right gonads, with B6J and B6NCrl, but not B6JJmsSlc, showing left gonad-dominant Sry expression. Substrain differences existed also in the distribution of Sry-expressing cells in the medial and lateral directions of gonads. In addition, in the left gonad-dominant Sry-expressing substrains B6J and B6NCrl, the medial and central regions of the left gonad had more Sry-expressing cells than those of the right gonad. Substrains of B6 mice have not always been considered in sex differentiation studies. In the present study, however, we observed substrain differences in the number of Sry-expressing cells, left-right distribution, and medial/lateral distribution during the early stages of gonadal development in B6 mice. Therefore, future studies on sex differentiation in B6 mice should consider substrain differences.

Adriamycin-Induced Nephropathy is Robust in N and Modest in J Substrain of C57BL/6.

Adriamycin (ADR)-induced nephropathy remains the leading model to study human primary focal segmental glomerulosclerosis (FSGS), a common pathway for podocyte damage and glomerular loss of function that leads to chronic kidney disease. However, the use of this model for reverse genetics is limited by historical categorization of C57BL/6 mice as an ADR-resistant strain, which is also the most common genetically modified strain. Additionally, conflicting reports exist utilizing C57BL/6 for ADR-nephrosis due to lack of understanding of substrain differences (J/N) and variability in ADR dosage, timing, and frequency to induce damage. We have undertaken a systematic approach to elucidate the specifics of ADR-nephrosis in C57BL/6 N and J substrains. We induced nephropathy with 2 doses of ADR, and measured albuminuria for 6 weeks and performed histological evaluations. Our findings revealed induction of robust and modest proteinuria in N and J substrains, respectively. The serum creatinine levels were elevated in N, but not J substrain. Both the substrains showed reduction in body weight with N greater than J, although mortality remained at 0% in both substrains. Histological analysis showed worse renal lesions in the N than the J substrain. Podocyte markers synaptopodin, nephrin, podocin, and WT1 were reduced to a greater extent in the N than the J substrain. In summary, we provide the nephrology community with a reproducible mouse model for FSGS, in a strain otherwise assumed to be ADR-resistant and highlight the differences between J and N substrains. This enables future studies, especially concerning genetically manipulated animal models in C57BL/6.

Zhx2 Is a Candidate Gene Underlying Oxymorphone Metabolite Brain Concentration Associated with State-Dependent Oxycodone Reward.

Understanding the pharmacogenomics of opioid metabolism and behavior is vital to therapeutic success, as mutations can dramatically alter therapeutic efficacy and addiction liability. We found robust, sex-dependent BALB/c substrain differences in oxycodone behaviors and whole brain concentration of oxycodone metabolites. BALB/cJ females showed robust state-dependent oxycodone reward learning as measured via conditioned place preference when compared with the closely related BALB/cByJ substrain. Accordingly, BALB/cJ females also showed a robust increase in brain concentration of the inactive metabolite noroxycodone and the active metabolite oxymorphone compared with BALB/cByJ mice. Oxymorphone is a highly potent, full agonist at the mu opioid receptor that could enhance drug-induced interoception and state-dependent oxycodone reward learning. Quantitative trait locus (QTL) mapping in a BALB/c F2 reduced complexity cross revealed one major QTL on chromosome 15 underlying brain oxymorphone concentration that explained 32% of the female variance. BALB/cJ and BALB/cByJ differ by fewer than 10,000 variants, which can greatly facilitate candidate gene/variant identification. Hippocampal and striatal cis-expression QTL (eQTL) and exon-level eQTL analysis identified Zhx2, a candidate gene coding for a transcriptional repressor with a private BALB/cJ retroviral insertion that reduces Zhx2 expression and sex-dependent dysregulation of cytochrome P450 enzymes. Whole brain proteomics corroborated the Zhx2 eQTL and identified upregulated CYP2D11 that could increase brain oxymorphone in BALB/cJ females. To summarize, Zhx2 is a highly promising candidate gene underlying brain oxycodone metabolite levels. Future studies will validate Zhx2 and its site of action using reciprocal gene editing and tissue-specific viral manipulations in BALB/c substrains. SIGNIFICANCE STATEMENT: Our findings show that genetic variation can result in sex-specific alterations in whole brain concentration of a bioactive opioid metabolite after oxycodone administration, reinforcing the need for sex as a biological factor in pharmacogenomic studies. The cooccurrence of female-specific increased oxymorphone and state-dependent reward learning suggests that this minor yet potent and efficacious metabolite of oxycodone could increase opioid interoception and drug-cue associative learning of opioid reward, which has implications for cue-induced relapse of drug-seeking behavior and for precision pharmacogenetics.

Cocaine-Induced Locomotor Activation Differs Across Inbred Mouse Substrains.

Cocaine use disorders (CUD) are devastating for affected individuals and impose a significant societal burden, but there are currently no FDA-approved therapies. The development of novel and effective treatments has been hindered by substantial gaps in our knowledge about the etiology of these disorders. The risk for developing a CUD is influenced by genetics, the environment and complex interactions between the two. Identifying specific genes and environmental risk factors that increase CUD risk would provide an avenue for the development of novel treatments. Rodent models of addiction-relevant behaviors have been a valuable tool for studying the genetics of behavioral responses to drugs of abuse. Traditional genetic mapping using genetically and phenotypically divergent inbred mice has been successful in identifying numerous chromosomal regions that influence addiction-relevant behaviors, but these strategies rarely result in identification of the causal gene or genetic variant. To overcome this challenge, reduced complexity crosses (RCC) between closely related inbred mouse strains have been proposed as a method for rapidly identifying and validating functional variants. The RCC approach is dependent on identifying phenotypic differences between substrains. To date, however, the study of addiction-relevant behaviors has been limited to very few sets of substrains, mostly comprising the C57BL/6 lineage. The present study expands upon the current literature to assess cocaine-induced locomotor activation in 20 inbred mouse substrains representing six inbred strain lineages (A/J, BALB/c, FVB/N, C3H/He, DBA/2 and NOD) that were either bred in-house or supplied directly by a commercial vendor. To our knowledge, we are the first to identify significant differences in cocaine-induced locomotor response in several of these inbred substrains. The identification of substrain differences allows for the initiation of RCC populations to more rapidly identify specific genetic variants associated with acute cocaine response. The observation of behavioral profiles that differ between mice generated in-house and those that are vendor-supplied also presents an opportunity to investigate the influence of environmental factors on cocaine-induced locomotor activity.

Distinct Age-Specific Effects on Olfactory Associative Learning in C57BL/6 Substrains.

C57BL/6 is the most widely used mouse strain in the laboratories. Two substrains of C57BL/6, C57BL/6J (B6J), and C57BL/6N (B6N) are well-known backgrounds for genetic modification and have been shown difference in quite a few tests, including open field test, rotarod test, and Morris water maze. However, difference between these two substrains in olfaction-dependent behaviors remains unknown. Here, we used olfactory two-alternative choice task, which is modified to have two training stages, to evaluate animals’ ability in instrumental learning and olfactory association. In the first (rule learning) stage, the mice were trained to use the operant chamber to collect water rewards. An odor cue was provided in the procedure, with no indication about reward locations. In the following (discrimination learning) stage, two odor cues were provided, with each indicating a specific water port. The animals were rewarded upon correct port choices following cue deliveries. We found that during young adulthood (7–10 weeks old), proportionally more B6J than B6N mice were able to pass rule learning (58.3% vs. 29.2%) and ultimately acquire this task (54.2% vs. 25%), with the two substrains showing similar pass rates in discrimination learning (92.9% vs. 85.7%). Surprisingly, at a more mature age (17 weeks old), this substrain difference disappeared. Mature B6N mice had a significant improvement in pass percentages of rule learning and overall task, whereas similar improvement was not observed in the B6J counterparts. Instead, mature B6J mice had an improved speed in rule learning and overall task. We further examined behavioral patterns of 8-week-old B6J and B6N mice in the olfactory habituation or dishabituation test. We observed normal olfactory habituation from subjects of both substrains, with the B6J mice exhibiting stronger investigative responses to newly presented odorants. These results reveal for the first time that B6J and B6N mice are different in acquisition processes of a behavioral task that requires instrumental learning and olfactory association, and that maturation appears to employ different effects on these two substrains during these processes. Furthermore, young adult B6J and B6N mice might be similar in olfactory habituation but different in the olfactory aspects of novelty seeking.

Differential effects of noise exposure between substrains of CBA mice

Noise trauma involves a plethora of mechanisms including reactive oxygen species, apoptosis, tissue damage, and inflammation. Recently, circadian mechanisms were also found to contribute to the vulnerability to noise trauma in mice, with greater damage occurring during their active phase (nighttime), when compared to similar noise exposures during their inactive phase (daytime). These effects seem to be regulated by mechanisms involving Bdnf responses to noise trauma and circulating levels of corticosterone (CORT). However, recent studies using different noise paradigms show contradicting results and it remains unclear how universal these findings are. Here we show that these findings differ even between substrains of mice and are restricted to a narrow window of noise intensity. We found that CBA/Sca mice exposed to 103 dB SPL display differential day/night noise sensitivity as measured by auditory brainstem responses (ABRs), but not at 100 (where full recovery is observed in day or night exposed mice) or 105 dB SPL (where permanent damage is found in both groups). In contrast, neither CBA/CaJ or CBA/JRj displayed such differences in day/night noise sensitivity, whatever noise intensity used. These effects appeared to be independent from outer hair cell function, as distortion product otoacoustic emissions appeared equally affected by day or night noise exposure, in all strains and in all noise conditions. Minor differences in ribbon counts or synaptic pairing were found in CBA/Sca mice, which were inconsistent with ABR wave 1 amplitude changes. Interestingly, CORT levels peaked in CBA/Sca mice at the onset of darkness at zeitgeber time 12 reaching levels of 43.8 ng/ml, while in the CBA/CaJ and the CBA/JRj, levels were 11.9 and 15.6 ng/ml respectively and peaking 4 h earlier (zeitgeber time 8). These findings were consistent with higher period of daily rhythm in CBA/Sca mice when measured in complete darkness using running wheels (23.7 h), than in CBA/CaJ (23.45 h) or CBA/JRj (23.13 h). In conclusion, our study suggests that the differential vulnerability to noise trauma between inactive and active phase is not universal and is as sensitive as substrain differences that might be governed by the circadian amplitude of the circulating CORT profiles.

Strain specific behavioral and physiological responses to constant light in male CBA/J and CBA/CaJ mice.

Objective:Being visually impaired increases the likelihood of sleep disorders and altered behavior. This study investigated physiological and behavioral differences in two similar mice substrains when exposed to constant light (LL) - CBA/J with retinal degeneration and CBA/CaJ mice (no retinal degeneration).Material and Methods:Male CBA/J and CBA/CaJ mice were placed into a 12:12 light:dark cycle or constant light (LL). Open field behavior, metabolic markers, and home-cage circadian activity were observed.Results:CBA/CaJ mice have greater circadian period lengthening, increased weight gain, reduced glucose, and increased novelty-induced locomotor activity in LL, compared to CBA/J mice. LL reduced thyroid hormone and insulin in both substrains.Discussion:While several baseline substrain differences were elucidated, CBA/CaJ mice were more effected by the exposure to LL than the blind CBA/J mice. These results illustrate that LL causes alterations in physiology and behavior and that circadian photoreceptivity might contribute to these effects.

Involvement of Neuropeptide Galanin Receptors 2 and 3 in Learning, Memory and Anxiety in Aging Mice.

The neuropeptide galanin (GAL), which is expressed in limbic brain structures, has a strong impact on the regulation of mood and behavior. GAL exerts its effects via three G protein-coupled receptors (GAL1–3-R). Little is known about the effects of aging and loss of GAL-Rs on hippocampal-mediated processes connected to neurogenesis, such as learning, memory recall and anxiety, and cell proliferation and survival in the dorsal dentate gyrus (dDG) in mice. Our results demonstrate that loss of GAL3-R, but not GAL2-R, slowed learning and induced anxiety in older (12–14-month-old) mice. Lack of GAL2-R increased cell survival (BrdU incorporation) in the dDG of young mice. However, normal neurogenesis was observed in vitro using neural stem and precursor cells obtained from GAL2-R and GAL3-R knockouts upon GAL treatment. Interestingly, we found sub-strain differences between C57BL/6J and C57BL/6N mice, the latter showing faster learning, less anxiety and lower cell survival in the dDG. We conclude that GAL-R signaling is involved in cognitive functions and can modulate the survival of cells in the neurogenic niche, which might lead to new therapeutic applications. Furthermore, we observed that the mouse sub-strain had a profound impact on the behavioral parameters analyzed and should therefore be carefully considered in future studies.

Affective Disruption During Forced Ethanol Abstinence in C57BL/6J and C57BL/6NJ Mice.

In alcohol-dependent individuals, acute alcohol withdrawal results in severe physiological disruption, including potentially lethal central nervous system hyperexcitability. Although benzodiazepines successfully mitigate such symptoms, this treatment does not significantly reduce recidivism rates in postdependent individuals. Instead, persistent affective disturbances that often emerge weeks to months after initial detoxification appear to play a significant role in relapse risk; however, it remains unclear whether genetic predispositions contribute to their emergence, severity, and/or duration. Interestingly, significant genotypic and phenotypic differences have been observed among distinct C57BL/6 (B6) substrains, and, in particular, C57BL/6J (B6J) mice have been found to reliably exhibit higher voluntary ethanol (EtOH) intake and EtOH preference compared to several C57BL/6N (B6N)-derived substrains. To date, however, B6 substrains have not been directly compared on measures of acute withdrawal severity or affective-behavioral disruption during extended abstinence. Male and female B6J and B6NJ mice were exposed to either a 7-day chronic intermittent EtOH vapor (CIE) protocol or to ordinary room air in inhalation chambers. Subsequently, blood EtOH concentrations and handling-induced convulsions were evaluated during acute withdrawal, and mice were then tested weekly for affective behavior on the sucrose preference test, light-dark box test, and forced swim test throughout 4weeks of (forced) abstinence. Despite documented differences in voluntary EtOH intake between these substrains, we found little evidence for substrain differences in either acute withdrawal or long-term abstinence between B6J and B6NJ mice. In B6J and B6NJ mice, both the acute and long-term sequelae of EtOH withdrawal are dependent on largely nonoverlapping gene networks relative to those underlying voluntary EtOH drinking.

C57BL/6J substrain differences in response to high-fat diet intervention

C57BL/6J-related mouse strains are widely used animal models for diet-induced obesity (DIO). Multiple vendors breed C57BL/6J-related substrains which may introduce genetic drift and environmental confounders such as microbiome differences. To address potential vendor/substrain specific effects, we compared DIO of C57BL/6J-related substrains from three different vendors: C57BL/6J (Charles Rivers), C57BL/6JBomTac (Taconic Bioscience) and C57BL/6JRj (Janvier). After local acclimatization, DIO was induced by either a high-fat diet (HFD, 60% energy from fat) or western diet (WD, 42% energy from fat supplemented with fructose in the drinking water). All three groups on HFD gained a similar amount of total body weight, yet the relative amount of fat percentage and mass of inguinal- and epididymal white adipose tissue (iWAT and eWAT) was lower in C57BL/6JBomTac compared to the two other C57BL/6J-releated substrains. In contrast to HFD, the three groups on WD responded differently in terms of body weight gain, where C57BL/6J was particularly prone to WD. This was associated with a relative higher amount of eWAT, iWAT, and liver triglycerides. Although the HFD and WD had significant impact on the microbiota, we did not observe any major differences between the three groups of mice. Together, these data demonstrate significant differences in HFD- and WD-induced adiposity in C57BL/6J-related substrains, which should be considered in the design of animal DIO studies.

Inbred Substrain Differences Influence Neuroimmune Response and Drinking Behavior.

The inbred mouse strain C57BL/6 is widely used in both models of addiction and immunological disease. However, there are pronounced phenotypic differences in ethanol (EtOH) consumption and innate immune response between C57BL/6 substrains. The focus of this study was to examine the effects of substrain on innate immune response and neuroimmune-induced escalation of voluntary EtOH consumption. The main goal was to identify whether substrain differences in immune response can account for differences in EtOH behavior. We compared acute innate immune response with a viral dsRNA mimic, polyinosinic:polycytidylic acid (poly(I:C)), in brain using qRT-PCR in both C57BL/6N and C57BL/6J mice. Next, we used a neuroimmune model of escalation using poly(I:C) to compare drinking behavior between substrains. Finally, we compared brain neuroimmune response with both EtOH and repeated poly(I:C) in both substrains as a way to account for differences in EtOH behavior. We found that C57BL/6 substrains have differing immune response and drinking behaviors. C57BL/6N mice have a shorter but more robust inflammatory response to acute poly(I:C). In contrast, C57BL/6J mice have a smaller but longer-lasting acute immune response to poly(I:C). In our neuroimmune-induced escalation model, C57BL/6J mice but not C57BL/6N mice escalate EtOH intake after poly(I:C). Finally, only C57BL/6J mice show enhanced proinflammatory transcript abundance after poly(I:C) and EtOH, suggesting that longer-lasting immune responses are critical to neuroimmune drinking phenotypes. Altogether, this work has elucidated additional influences that substrain has on both innate immune response and drinking phenotypes. Our observations highlight the importance of considering and reporting the source and background used for production of transgenic and knockout mice. These data provide further evidence that genetic background must be carefully considered when investigating the role of neuroimmune signaling in EtOH abuse.

C57BL/6 substrain differences in formalin-induced pain-like behavioral responses

Substantial evidence from preclinical models of pain suggests that basal and noxious nociceptive sensitivity, as well as antinociceptive responses to drugs, show significant heritability. Individual differences to these responses have been observed across species from rodents to humans. The use of closely related C57BL/6 inbred mouse substrains can facilitate gene mapping of acute nociceptive behaviors in preclinical pain models. In this study, we investigated behavioral differences between C57BL/6 J (B6 J) and C57BL/6 N (B6 N) substrains in the formalin test, a widely used tonic inflammatory pain model, using a battery of pain-related phenotypes, including reflexive tests, nesting, voluntary wheel running, sucrose preference and anxiety-like behavior in the light/dark test at two different time points (1-h and 24-h). Our results show that these substrains did not differ in reflexive thermal and mechanical responses at the 1-h time point. However, B6 N substrain mice showed increased sensitivity to spontaneous pain-like behaviors. In addition, B6 N substrain continued to show higher levels of mechanical hypersensitivity compared to controls at 24-h. indicating that mechanical hypersensitivity is a more persistent pain-related phenotype induced by formalin. Finally, no sex differences were observed in our outcome measures. Our results provide a comprehensive behavioral testing paradigm in response to an inflammatory agent for future mouse genetic studies in pain.

C57BL/6 Substrain Differences in Pharmacological Effects after Acute and Repeated Nicotine Administration.

Tobacco smoking is the major cause of disability and death in the United States and around the world. In addition, tobacco dependence and addiction express themselves as complex behaviors involving an interplay of genetics, environment, and psychological state. Mouse genetic studies could potentially elucidate the novel genes and/or gene networks regulating various aspects of nicotine dependence. Using the closely related C57BL/6 (B6) mice substrains, recent reports have noted phenotypic differences within C57BL/6J (B6J) and C57BL/6N (B6N) mice for some drugs of abuse: alcohol, opiates, and cocaine. However, the differences in nicotine’s effects have not yet been described in these substrains. We examined the phenotypic differences in these substrains following the acute and repeated administration of nicotine in several pharmacological measures, including locomotion (after acute and repeated exposure), body temperature, nociception, and anxiety-like behaviors. We report substrain differences in the pharmacological effects of acute and repeated nicotine administration in the B6 substrains. Overall, we show enhanced nicotine sensitivity to locomotion, hypothermia, antinociception, and anxiety-like behaviors in the B6J mouse substrain compared to B6N. In the repeated administration paradigm, both the B6N and B6J substrains showed no sensitized locomotor responses after repeated exposure to nicotine at the two doses tested. This study thus provides evidence that the B6 mouse substrains may be useful for genetic studies to elucidate some of the genetic variants involved in tobacco dependence and addiction.

Experiments done in Black-6 mice: what does it mean?

Low replicability of animal experiments is perceived as a major hurdle in the field of biomedicine. Attempts to enhance the replicability and to reduce the variability in basic research has led to the recommendation to use isogenic mice. The C57BL/6 strain has evolved as a gold standard strain for this purpose. However, C57BL/6 mice are maintained as substrains by multiple vendors. Evidence exists that the subtle differences between these mouse lines have not been systematically investigated and are often ignored. In the present study, we characterized the female mice of two closely related substrains (C57BL/6J and C57BL/6N) from three vendors in Europe (Charles River Laboratories, Envigo, Janvier Labs) in a battery of behavioral tests. Our data show and confirm substantial behavioral differences between the C57BL/6J and C57BL/6N mice. Importantly, the substrain differences were largely affected by the origin of the animals, as a significant effect of vendor or interaction between the substrain and vendor occurred in all tests. This work highlights the importance of adhering to precise international nomenclature in all publications reporting animal experiments. Moreover, the generalization of research findings from a single mouse substrain can be seriously limited due to genetic drift and environmental variables occurring at different vendors. However, heterogenization of samples, by including animals of different substrains, can enhance generalizability. These issues need to be seriously addressed to improve reproducibility, replicability, and the translational potential of the mouse models.

Genetic differences between C57BL/6 substrains affect the process of testis differentiation in Y<sup>POS</sup> mice

C57BL/6J-XYPOS (B6J-XYPOS) mice, which have the Y chromosome derived from Mus musculus poschiavinus on a B6J genetic background, form ovotestes or ovaries. Previously, we replaced the genetic background of B6J-XYPOS mice with B6N and found that individuals with testes also appeared in addition to those with ovaries or ovotestes. To investigate the effect of the B6J genetic sequence on the testis differentiation, the genetic background of B6N-XYPOS mice was replaced with B6J again. The recovery of the B6J genetic background significantly decreased the incidence of testes; only ovaries developed. These results indicate that the testicular differentiation process tends to be perturbed especially in the B6J substrain. This shows the importance of substrain differences in mice usually treated as B6 collectively.

C57BL/6J and C57BL/6N substrains differentially influence phenotype severity in the Scn1a +/- mouse model of Dravet syndrome.

SummaryMany disease‐relevant phenotypes modeled in inbred mice have been shown to be strain‐dependent, indicating the important influence of genetic background on disease phenotypes. Although C57BL/6 mice are one of the most commonly used inbred strains in laboratory research, there are multiple substrains (eg, B6J vs B6N) that have been separated for more than 50 years. Thus, understanding the substrain differences is important for scientific rigor and reproducibility. In this study, seizure susceptibility, spontaneous seizures, and survival were compared between Scn1a +/− mice on (C57BL/6J × 129S6/SvEvTac)F1 (F1J) vs (C57BL/6N × 129S6/SvEvTac)F1 (F1N) strain backgrounds. F1N.Scn1a +/− mice were more susceptible to hyperthermia‐induced seizures, yet had milder spontaneous seizures and improved survival relative to F1J.Scn1a +/− mice. Our results indicate that choice of C57BL/6 substrain may significantly alter disease phenotypes and should be considered carefully in experimental design using the Scn1a +/− Dravet mouse model, as well as other mouse models of epilepsy.

The time course of blood brain barrier leakage and its implications on the progression of methamphetamine-induced seizures

The initial goals of these experiments were to determine: 1) if blood-brain barrier (BBB) breakdown was a cause or an effect of METH-induced seizures; 2) all the brain regions where BBB is disrupted as seizures progress; and 3) the correlations between body temperature and vascular leakage and neurodegeneration. A fourth objective was added after initial experimentation to determine if sub-strain differences existed in adult male C57 B6 J (Jackson laboratories, JAX) versus C57 B6N (Charles River, CR) mice involving their susceptibility to BBB breakdown and seizure severity. With the 1st “maximal” intensity myoclonic-tonic seizure (MCT) varying degrees of IgG infiltration across the BBB (≤1 mm2) were prominent in olfactory system (OS) associated regions and in thalamus, hypothalamus and neocortex. IgG infiltration areas in the OS-associated regions of the bed nucleus of the stria terminalis, septum and more medial amygdala nuclei, and the hypothalamus were increased significantly by the time continuous behavioral seizures (CBS) developed. Mice receiving METH that had body temperatures of ≥40 °C had IgG infiltration along with MCT or CBS but peak body temperatures above 40 °C did not significantly increase IgG infiltration. Neurodegeneration seen at ≥6 h was restricted to the OS in both JAX and CR mice and was most prominent in the posteromedial cortical amygdaloid nucleus. Neurodegeneration in the anterior septum (tenia tecta) was seen only in the JAX mice. We hypothesize that METH-induced hypertension and hyperthermia lead to BBB breakdown and other vascular dysfunctions in the OS brain regions resulting in OS hyperexcitation. Excitation of the OS neural network then leads to the development of seizures. These seizures in turn exacerbate the energy depletions and the reactive oxygen stress produced by hyperthermia further damaging the BBB and vascular function. These events form a recurrent cycle that results in ever increasing seizure activity and neurotoxicity.

Variability in prescription opioid intake and reinforcement amongst 129 substrains.

Opioid abuse in the United States has reached epidemic proportions, with treatment admissions and deaths associated with prescription opioid abuse quadrupling over the past 10 years. Although genetics are theorized to contribute substantially to inter-individual variability in the development, severity and treatment outcomes of opioid abuse/addiction, little direct preclinical study has focused on the behavioral genetics of prescription opioid reinforcement and drug-taking. Herein, we employed different 129 substrains of mice currently available from The Jackson Laboratory (129S1/SvlmJ, 129X1/SvJ, 129S4/SvJaeJ and 129P3/J) as a model system of genetic variation and assayed mice for oral opioid intake and reinforcement, as well as behavioral and somatic signs of dependence. All substrains exhibited a dose-dependent increase in oral oxycodone and heroin preference and intake under limited-access procedures and all, but 129S1/SvlmJ mice, exhibited oxycodone reinforcement. Relative to the other substrains, 129P3/J mice exhibited higher heroin and oxycodone intake. While 129X1/SvJ exhibited the highest anxiety-like behavior during natural opioid withdrawal, somatic and behavior signs of precipitated withdrawal were most robust in 129P3/J mice. These results demonstrate the feasibility and relative sensitivity of our oral opioid self-administration procedures for detecting substrain differences in drug reinforcement/intake among 129 mice, of relevance to the identification of genetic variants contributing to high vs. low oxycodone reinforcement and intake.