A quantitative trait variant in Gabra2 underlies increased methamphetamine stimulant sensitivity.

Psychostimulant (methamphetamine, cocaine) use disorders have a genetic component that remains mostly unknown. We conducted genome-wide quantitative trait locus (QTL) analysis of methamphetamine stimulant sensitivity. To facilitate gene identification, we employed a Reduced Complexity Cross between closely related C57BL/6 mouse substrains and examined maximum speed and distance traveled over 30 min following methamphetamine (2 mg/kg, i.p.). For maximum methamphetamine-induced speed following the second and third administration, we identified a single genome-wide significant QTL on chromosome 11 that peaked near the Cyfip2 locus (LOD = 3.5, 4.2; peak = 21 cM [36 Mb]). For methamphetamine-induced distance traveled following the first and second administration, we identified a genome-wide significant QTL on chromosome 5 that peaked near a functional intronic indel in Gabra2 coding for the alpha-2 subunit of the GABA-A receptor (LOD = 3.6-5.2; peak = 34-35 cM [66-67 Mb]). Striatal cis-expression QTL mapping corroborated Gabra2 as a functional candidate gene underlying methamphetamine-induced distance traveled. CRISPR/Cas9-mediated correction of the mutant intronic deletion on the C57BL/6J background to the wild-type C57BL/6NJ allele was sufficient to reduce methamphetamine-induced locomotor activity toward the wild-type C57BL/6NJ-like level, thus validating the quantitative trait variant (QTV). These studies show the power and efficiency of Reduced Complexity Crosses in identifying causal variants underlying complex traits. Functionally restoring Gabra2 expression decreased methamphetamine stimulant sensitivity and supports preclinical and human genetic studies implicating the GABA-A receptor in psychostimulant addiction-relevant traits. Importantly, our findings have major implications for studying psychostimulants in the C57BL/6J strain-the gold standard strain in biomedical research.

Genetic Modulation of Initial Sensitivity to Δ9-Tetrahydrocannabinol (THC) Among the BXD Family of Mice.

Cannabinoid receptor 1 activation by the major psychoactive component in cannabis, Δ9-tetrahydrocannabinol (THC), produces motor impairments, hypothermia, and analgesia upon acute exposure. In previous work, we demonstrated significant sex and strain differences in acute responses to THC following administration of a single dose (10 mg/kg, i.p.) in C57BL/6J (B6) and DBA/2J (D2) inbred mice. To determine the extent to which these differences are heritable, we quantified acute responses to a single dose of THC (10 mg/kg, i.p.) in males and females from 20 members of the BXD family of inbred strains derived by crossing and inbreeding B6 and D2 mice. Acute THC responses (initial sensitivity) were quantified as changes from baseline for: 1. spontaneous activity in the open field (mobility), 2. body temperature (hypothermia), and 3. tail withdrawal latency to a thermal stimulus (antinociception). Initial sensitivity to the immobilizing, hypothermic, and antinociceptive effects of THC varied substantially across the BXD family. Heritability was highest for mobility and hypothermia traits, indicating that segregating genetic variants modulate initial sensitivity to THC. We identified genomic loci and candidate genes, including Ndufs2, Scp2, Rps6kb1 or P70S6K, Pde4d, and Pten, that may control variation in THC initial sensitivity. We also detected strong correlations between initial responses to THC and legacy phenotypes related to intake or response to other drugs of abuse (cocaine, ethanol, and morphine). Our study demonstrates the feasibility of mapping genes and variants modulating THC responses in the BXDs to systematically define biological processes and liabilities associated with drug use and abuse.

Sex and Strain Variation in Initial Sensitivity and Rapid Tolerance to Δ9-Tetrahydrocannabinol.

Background and Objectives: For cannabis and other drugs of abuse, initial response and/or tolerance to drug effects can predict later dependence and problematic use. Our objective is to identify sex and genetic (strain) differences in initial response and rapid tolerance to Δ9-tetrahydrocannabinol (THC), the main psychoactive ingredient in cannabis, between highly genetically divergent inbred mouse strains-C57BL/6J (B6) and DBA/2J (D2). Experimental Approach: Sex and strain responses relative to baseline were quantified following daily exposure (i.p.) to 10 mg/kg THC or vehicle (VEH) over the course of 5 days. Dependent measures included hypothermia (decreased body temperature) and ataxia (decreased spontaneous activity in the open field), and antinociception (increase in tail withdrawal latency to a thermal stimulus). Initial sensitivity to THC was defined as the difference in response between baseline and day 1. Rapid tolerance to THC was defined as the difference in response between days 1 and 2. Results: B6 exhibited greater THC-induced motor activity suppression and initial sensitivity to ataxia relative to the D2 strain. Females demonstrated greater levels of THC-induced hypothermia and initial sensitivity relative to males. Higher levels of THC-induced antinociception and initial sensitivity were observed for D2 relative to B6. Rapid tolerance to THC was observed for hypothermia and antinociception. Much less tolerance was observed for THC-induced ataxia. D2 exhibited rapid tolerance to THC-induced hypothermia and antinociception at time points associated with peak THC initial response. Likewise, at the peak initial THC response time point, females demonstrated greater levels of rapid tolerance to hypothermic effects relative to males. Conclusions: Both sex and genetic factors drive variation in initial response and rapid tolerance to the ataxic, antinociceptive, and hypothermic effects of THC. As these traits directly result from THC activation of the cannabinoid receptor 1, gene variants between B6 and D2 in cannabinoid signaling pathways are likely to mediate strain differences in response to THC.

Genetic Factors Mediate the Impact of Chronic Stress and Subsequent Response to Novel Acute Stress.

Individual differences in physiological and biobehavioral adaptation to chronic stress are important predictors of health and fitness; genetic differences play an important role in this adaptation. To identify these differences we measured the biometric, neuroendocrine, and transcriptional response to stress among inbred mouse strains with varying degrees of genetic similarity, C57BL/6J (B), C57BL/6NJ (N), and DBA/2J (D). The B and D strains are highly genetically diverse whereas the B and N substrains are highly similar. Strain differences in hypothalamic-pituitary-adrenal (HPA) axis cross-sensitization were determined by plasma corticosterone (CORT) levels and hippocampal gene expression following 7-weeks of chronic mild stress (CMS) or normal housing (NH) and subsequent exposure to novel acute restraint. Fecal CORT metabolites and body and organ weights were also measured. All strains exposed to CMS had reduced heart weights, whereas body weight gain was attenuated only in B and N strains. Acute stress alone produced larger plasma CORT responses in the D and N strains compared to the B strain. CMS paired with acute stress produced cross-sensitization of the CORT response in the N strain. The N strain also had the largest number of hippocampal transcripts with up-regulated expression in response to stress. In contrast, the D strain had the largest number of transcripts with down-regulated expression following CMS and acute stress. In summary, we observed differential responses to CMS at both the physiological and molecular level among genetically diverse strains, indicating that genetic factors drive individual differences in experience-dependent regulation of the stress response.

Comparison and Functional Genetic Analysis of Striatal Protein Expression Among Diverse Inbred Mouse Strains.

C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains are highly variable genetically and differ in a large number of behavioral traits related to striatal function, including depression, anxiety, stress response, and response to drugs of abuse. The genetic basis of these phenotypic differences are, however, unknown. Here, we present a comparison of the striatal proteome between B6 and D2 and relate differences at the protein level to strain differences at the mRNA level. We also leverage a recombinant inbred BXD population derived from B6 and D2 strains to investigate the role of genetic variation on the regulation of mRNA and protein levels. Finally, we test the hypothesis that differential protein expression contributes to differential behavioral responses between the B6 and D2 strain. We detected the expression of over 2,500 proteins in membrane-enriched protein fractions from B6 and D2 striatum. Of these, 160 proteins demonstrated significant differential expression between B6 and D2 strains at a 10% false discovery level, including COMT, GABRA2, and cannabinoid receptor 1 (CNR1)-key proteins involved in synaptic transmission and behavioral response. Similar to previous reports, there was little overlap between protein and transcript levels (25%). However, the overlap was greater (51%) for proteins demonstrating genetic regulation of cognate gene expression. We also found that striatal proteins with significantly higher or lower relative expression in B6 and D2 were enriched for dopaminergic and glutamatergic synapses and processes involved in synaptic plasticity [e.g., long-term potentiation (LTP) and long-term depression (LTD)]. Finally, we validated higher expression of CNR1 in B6 striatum and demonstrated greater sensitivity of this strain to the locomotor inhibiting effects of the CNR1 agonist, Δ9-tetrahydrocannabinol (THC). Our study is the first comparison of differences in striatal proteins between the B6 and D2 strains and suggests that alterations in the striatal proteome may underlie strain differences in related behaviors, such as drug response. Furthermore, we propose that genetic variants that impact transcript levels are more likely to also exhibit differential expression at the protein level.

Identification of a Functional Non-coding Variant in the GABA A Receptor α2 Subunit of the C57BL/6J Mouse Reference Genome: Major Implications for Neuroscience Research.

GABA type-A (GABA-A) receptors containing the α2 subunit (GABRA2) are expressed in most brain regions and are critical in modulating inhibitory synaptic function. Genetic variation at the GABRA2 locus has been implicated in epilepsy, affective and psychiatric disorders, alcoholism and drug abuse. Gabra2 expression varies as a function of genotype and is modulated by sequence variants in several brain structures and populations, including F2 crosses originating from C57BL/6J (B6J) and the BXD recombinant inbred family derived from B6J and DBA/2J. Here we demonstrate a global reduction of GABRA2 brain protein and mRNA in the B6J strain relative to other inbred strains, and identify and validate the causal mutation in B6J. The mutation is a single base pair deletion located in an intron adjacent to a splice acceptor site that only occurs in the B6J reference genome. The deletion became fixed in B6J between 1976 and 1991 and is now pervasive in many engineered lines, BXD strains generated after 1991, the Collaborative Cross, and the majority of consomic lines. Repair of the deletion using CRISPR-Cas9-mediated gene editing on a B6J genetic background completely restored brain levels of GABRA2 protein and mRNA. Comparison of transcript expression in hippocampus, cortex, and striatum between B6J and repaired genotypes revealed alterations in GABA-A receptor subunit expression, especially in striatum. These results suggest that naturally occurring variation in GABRA2 levels between B6J and other substrains or inbred strains may also explain strain differences in anxiety-like or alcohol and drug response traits related to striatal function. Characterization of the B6J private mutation in the Gabra2 gene is of critical importance to molecular genetic studies in neurobiological research because this strain is widely used to generate genetically engineered mice and murine genetic populations, and is the most widely utilized strain for evaluation of anxiety-like, depression-like, pain, epilepsy, and drug response traits that may be partly modulated by GABRA2 function.

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry.

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

STIM1-dependent Ca(2+) microdomains are required for myofilament remodeling and signaling in the heart.

In non-excitable cells stromal interaction molecule 1 (STIM1) is a key element in the generation of Ca(2+) signals that lead to gene expression, migration and cell proliferation. A growing body of literature suggests that STIM1 plays a key role in the development of pathological cardiac hypertrophy. However, the precise mechanisms involving STIM-dependent Ca(2+) signaling in the heart are not clearly established. Here, we have investigated the STIM1-associated Ca(2+) signals in cardiomyocytes and their relevance to pathological cardiac remodeling. We show that mice with inducible, cardiac-restricted, ablation of STIM1 exhibited left ventricular reduced contractility, which was corroborated by impaired single cell contractility. The spatial properties of STIM1-dependent Ca(2+) signals determine restricted Ca(2+) microdomains that regulate myofilament remodeling and activate spatially segregated pro-hypertrophic factors. Indeed, mice lacking STIM1 showed less adverse structural remodeling in response to pressure overload-induced cardiac hypertrophy. These results highlight how STIM1-dependent Ca(2+) microdomains have a major impact on intracellular Ca(2+) homeostasis, cytoskeletal remodeling and cellular signaling, even when excitation-contraction coupling is present.