A Novel ENU-Induced Mfn2 Mutation Causes Motor Deficits in Mice without Causing Peripheral Neuropathy.

Mitochondrial fission and fusion are required for maintaining functional mitochondria. The mitofusins (MFN1 and MFN2) are known for their roles in mediating mitochondrial fusion. Recently, MFN2 has been implicated in other important cellular functions, such as mitophagy, mitochondrial motility, and coordinating endoplasmic reticulum-mitochondria communication. In humans, over 100 MFN2 mutations are associated with a form of inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 2A (CMT2A). Here we describe an ENU-induced mutant mouse line with a recessive neuromuscular phenotype. Behavioral screening showed progressive weight loss and rapid deterioration of motor function beginning at 8 weeks. Mapping and sequencing revealed a missense mutation in exon 18 of Mfn2 (T1928C; Leu643Pro), within the transmembrane domain. Compared to wild-type and heterozygous littermates, Mfn2L643P/L643P mice exhibited diminished rotarod performance and decreases in activity in the open field test, muscular endurance, mean mitochondrial diameter, sensory tests, mitochondrial DNA content, and MFN2 protein levels. However, tests of peripheral nerve physiology and histology were largely normal. Mutant leg bones had reduced cortical bone thickness and bone area fraction. Together, our data indicate that Mfn2L643P causes a recessive motor phenotype with mild bone and mitochondrial defects in mice. Lack of apparent nerve pathology notwithstanding, this is the first reported mouse model with a mutation in the transmembrane domain of the protein, which may be valuable for researchers studying MFN2 biology.

The microbial community dynamics of cocaine sensitization in two behaviorally divergent strains of collaborative cross mice.

The gut-brain axis is increasingly recognized as an important pathway involved in cocaine use disorder. Microbial products of the murine gut have been shown to affect striatal gene expression, and depletion of the microbiome by antibiotic treatment alters cocaine-induced behavioral sensitization in C57BL/6J male mice. Some reports suggest that cocaine-induced behavioral sensitization is correlated with drug self-administration behavior in mice. Here, we profile the composition of the naïve microbiome and its response to cocaine sensitization in two collaborative cross (CC) strains. These strains display extremely divergent behavioral responses to cocaine sensitization. A high-responding strain, CC004/TauUncJ (CC04), has a gut microbiome that contains a greater amount of Lactobacillus than the cocaine-nonresponsive strain CC041/TauUncJ (CC41). The gut microbiome of CC41 is characterized by an abundance of Eisenbergella, Robinsonella and Ruminococcus. In response to cocaine, CC04 has an increased Barnsiella population, while the gut microbiome of CC41 displays no significant changes. PICRUSt functional analysis of the functional potential of the gut microbiome in CC04 shows a significant number of potential gut-brain modules altered after exposure to cocaine, specifically those encoding for tryptophan synthesis, glutamine metabolism, and menaquinone synthesis (vitamin K2). Depletion of the microbiome by antibiotic treatment revealed an altered cocaine-sensitization response following antibiotics in female CC04 mice. Depleting the microbiome by antibiotic treatment in males revealed increased infusions for CC04 during a cocaine intravenous self-administration dose-response curve. Together these data suggest that genetic differences in cocaine-related behaviors may involve the microbiome.

Probing different paradigms of morphine withdrawal on sleep behavior in male and female C57BL/6J mice.

Opioid misuse has dramatically increased over the last few decades resulting in many people suffering from opioid use disorder (OUD). The prevalence of opioid overdose has been driven by the development of new synthetic opioids, increased availability of prescription opioids, and more recently, the COVID-19 pandemic. Coinciding with increases in exposure to opioids, the United States has also observed increases in multiple Narcan (naloxone) administrations as a life-saving measures for respiratory depression, and, thus, consequently, naloxone-precipitated withdrawal. Sleep dysregulation is a main symptom of OUD and opioid withdrawal syndrome, and therefore, should be a key facet of animal models of OUD. Here we examine the effect of precipitated and spontaneous morphine withdrawal on sleep behaviors in C57BL/6 J mice. We find that morphine administration and withdrawal dysregulate sleep, but not equally across morphine exposure paradigms. Furthermore, many environmental triggers promote relapse to drug-seeking/taking behavior, and the stress of disrupted sleep may fall into that category. We find that sleep deprivation dysregulates sleep in mice that had previous opioid withdrawal experience. Our data suggest that the 3-day precipitated withdrawal paradigm has the most profound effects on opioid-induced sleep dysregulation and further validates the construct of this model for opioid dependence and OUD.

Repeated dosing with cocaine produces strain-dependent effects on responding for conditioned reinforcement in Collaborative Cross mice.

RATIONALE: Cocaine use disorder (CUD) is a highly heritable form of substance use disorder, with genetic variation accounting for a substantial proportion of the risk for transitioning from recreational use to a clinically impairing addiction. With repeated exposures to cocaine, psychomotor and incentive sensitization are observed in rodents. These phenomena are thought to model behavioral changes elicited by the drug that contribute to the progression into addiction, but little is known about how genetic variation may moderate these consequences. OBJECTIVES: Here, we describe the use of two Collaborative Cross (CC) recombinant inbred mouse strains that either exhibit high (CC018/UncJ) or no (CC027/GeniUncJ) psychomotor sensitization in response to cocaine to measure phenotypes related to incentive sensitization after repeated cocaine exposures; given the relationship of incentive motivation to nucleus accumbens core (NAc) dopamine release and reuptake, we also assessed these neurochemical mechanisms. METHODS: Adult male and female CC018/UncJ and CC027/GeniUncJ mice underwent Pavlovian conditioning to associate a visual cue with presentation of a palatable food reward, then received five, every-other-day injections of cocaine or vehicle. Following Pavlovian re-training, they underwent testing acquisition of a new operant response for the visual cue, now serving as a conditioned reinforcer. Subsequently, electrically evoked dopamine release was assessed using fast-scan cyclic voltammetry from acute brain slices containing the NAc. RESULTS: While both strains acquired the Pavlovian association, only CC018/UncJ mice showed conditioned reinforcement and incentive sensitization in response to cocaine, while CC027/GeniUncJ mice did not. Voltammetry data revealed that CC018/UncJ, compared to CC027/GeniUnc, mice exhibited higher baseline dopamine release and uptake. Moreover, chronic cocaine exposure blunted tonic and phasic dopamine release in CC018/UncJ, but not CC027/GeniUncJ, mice. CONCLUSIONS: Genetic background is a moderator of cocaine-induced neuroadaptations in mesolimbic dopamine signaling, which may contribute to both psychomotor and incentive sensitization and indicate a shared biological mechanism of variation.

Reproducible changes in the anorexia nervosa gut microbiota following inpatient therapy remain distinct from non-eating disorder controls.

The composition of the gut microbiota in patients with anorexia nervosa (AN), and the ability of this microbial community to influence the host, remains uncertain. To achieve a broader understanding of the role of the intestinal microbiota in patients with AN, we collected fecal samples before and following clinical treatment at two geographically distinct eating disorder units (Center of Excellence for Eating Disorders [UNC-CH] and ACUTE Center for Eating Disorders [Denver Health]). Gut microbiotas were characterized in patients with AN, before and after inpatient treatment, and in non-eating disorder (non-ED) controls using shotgun metagenomic sequencing. The impact of inpatient treatment on the AN gut microbiota was remarkably consistent between eating disorder units. Although weight in patients with AN showed improvements, AN microbiotas post-treatment remained distinct from non-ED controls. Additionally, AN gut microbiotas prior to treatment exhibited more fermentation pathways and a lower ability to degrade carbohydrates than non-ED controls. As the intestinal microbiota can influence nutrient metabolism, our data highlight the complex microbial communities in patients with AN as an element needing further attention post inpatient treatment. Additionally, this study defines the effects of renourishment on the AN gut microbiota and serves as a platform to develop precision nutrition approaches to potentially mitigate impediments to recovery.

The collaborative cross strains and their founders vary widely in cocaine-induced behavioral sensitization.

Cocaine use and overdose deaths attributed to cocaine have increased significantly in the United States in the last 10 years. Despite the prevalence of cocaine use disorder (CUD) and the personal and societal problems it presents, there are currently no approved pharmaceutical treatments. The absence of treatment options is due, in part, to our lack of knowledge about the etiology of CUDs. There is ample evidence that genetics plays a role in increasing CUD risk but thus far, very few risk genes have been identified in human studies. Genetic studies in mice have been extremely useful for identifying genetic loci and genes, but have been limited to very few genetic backgrounds, leaving substantial phenotypic, and genetic diversity unexplored. Herein we report the measurement of cocaine-induced behavioral sensitization using a 19-day protocol that captures baseline locomotor activity, initial locomotor response to an acute exposure to cocaine and locomotor sensitization across 5 exposures to the drug. These behaviors were measured in 51 genetically diverse Collaborative Cross (CC) strains along with their inbred founder strains. The CC was generated by crossing eight genetically diverse inbred strains such that each inbred CC strain has genetic contributions from each of the founder strains. Inbred CC mice are infinitely reproducible and provide a stable, yet diverse genetic platform on which to study the genetic architecture and genetic correlations among phenotypes. We have identified significant differences in cocaine locomotor sensitivity and behavioral sensitization across the panel of CC strains and their founders. We have established relationships among cocaine sensitization behaviors and identified extreme responding strains that can be used in future studies aimed at understanding the genetic, biological, and pharmacological mechanisms that drive addiction-related behaviors. Finally, we have determined that these behaviors exhibit relatively robust heritability making them amenable to future genetic mapping studies to identify addiction risk genes and genetic pathways that can be studied as potential targets for the development of novel therapeutics.

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.

Behavioral phenotypes revealed during reversal learning are linked with novel genetic loci in diversity outbred mice.

Impulsive behavior and impulsivity are heritable phenotypes that are strongly associated with risk for substance use disorders. Identifying the neurogenetic mechanisms that influence impulsivity may also reveal novel biological insights into addiction vulnerability. Our past studies using the BXD and Collaborative Cross (CC) recombinant inbred mouse panels have revealed that behavioral indicators of impulsivity measured in a reversal-learning task are heritable and are genetically correlated with aspects of intravenous cocaine self-administration. Genome-wide linkage studies in the BXD panel revealed a quantitative trait locus (QTL) on chromosome 10, but we expect to identify additional QTL by testing in a population with more genetic diversity. To this end, we turned to Diversity Outbred (DO) mice; 392 DO mice (156 males, 236 females) were phenotyped using the same reversal learning test utilized previously. Our primary indicator of impulsive responding, a measure that isolates the relative difficulty mice have with reaching performance criteria under reversal conditions, revealed a genome-wide significant QTL on chromosome 7 (max LOD score = 8.73, genome-wide corrected p<0.05). A measure of premature responding akin to that implemented in the 5-choice serial reaction time task yielded a suggestive QTL on chromosome 17 (max LOD score = 9.14, genome-wide corrected <0.1). Candidate genes were prioritized (2900076A07Rik, Wdr73 and Zscan2) based upon expression QTL data we collected in DO and CC mice and analyses using publicly available gene expression and phenotype databases. These findings may advance understanding of the genetics that drive impulsive behavior and enhance risk for substance use disorders.

Gut microbial communities from patients with anorexia nervosa do not influence body weight in recipient germ-free mice.

Anorexia nervosa (AN) is a psychiatric disorder that presents with profound weight dysregulation, metabolic disturbances, and an abnormal composition of gut microbial communities. As the intestinal microbiota can influence host metabolism, the impact of enteric microbial communities from patients with AN on host weight and adiposity was investigated. Germ-free (GF) mice were colonized with fecal microbiotas from either patients with AN (n = 4) prior to inpatient treatment (AN T1, n = 50 recipient mice), the same 4 patients following clinical renourishment (AN T2, n = 53 recipient mice), or age- and sex-matched non-AN controls (n = 4 human donors; non-AN, n = 50 recipient mice). Biological and fecal microbiota data were analyzed with linear mixed-effects models. Body weight did not differ significantly between AN recipient mice (T1 and T2) and non-AN recipient mice following 4 weeks of colonization. Enteric microbiotas from recipient mice colonized with AN T1 and AN T2 fecal microbiotas were more similar to each other compared with enteric microbiotas from non-AN recipient mice. Specific bacterial families in the Actinobacteria, Bacteroidetes, and Firmicutes phyla were significantly associated with body weight, fat mass, and cecum weight irrespective of the donor group. These data suggest that body weight, fat mass, and cecum weight of colonized GF mice are associated with human fecal microbes and independent of donor AN status, although additional analyses with larger cohorts are warranted.

Bayesian modeling of skewed X inactivation in genetically diverse mice identifies a novel Xce allele associated with copy number changes.

Female mammals are functional mosaics of their parental X-linked gene expression due to X chromosome inactivation (XCI). This process inactivates one copy of the X chromosome in each cell during embryogenesis and that state is maintained clonally through mitosis. In mice, the choice of which parental X chromosome remains active is determined by the X chromosome controlling element (Xce), which has been mapped to a 176-kb candidate interval. A series of functional Xce alleles has been characterized or inferred for classical inbred strains based on biased, or skewed, inactivation of the parental X chromosomes in crosses between strains. To further explore the function structure basis and location of the Xce, we measured allele-specific expression of X-linked genes in a large population of F1 females generated from Collaborative Cross (CC) strains. Using published sequence data and applying a Bayesian "Pólya urn" model of XCI skew, we report two major findings. First, inter-individual variability in XCI suggests mouse epiblasts contain on average 20-30 cells contributing to brain. Second, CC founder strain NOD/ShiLtJ has a novel and unique functional allele, Xceg, that is the weakest in the Xce allelic series. Despite phylogenetic analysis confirming that NOD/ShiLtJ carries a haplotype almost identical to the well-characterized C57BL/6J (Xceb), we observed unexpected patterns of XCI skewing in females carrying the NOD/ShiLtJ haplotype within the Xce. Copy number variation is common at the Xce locus and we conclude that the observed allelic series is a product of independent and recurring duplications shared between weak Xce alleles.

Behavioral characterization of a novel Cisd2 mutant mouse.

Wolfram syndrome (WFS) is a rare autosomal recessive disorder characterized by diabetes mellitus and insipidus, progressive optic atrophy and sensorineural deafness. An increased incidence of psychiatric disorders has also been reported in WFS patients. There are two subtypes of WFS. Type 1 (WFS1) is caused by mutations in the WFS1 gene and type 2 (WFS2) results from mutations in the CISD2 gene. Existing Wfs1 knockout mice exhibit many WFS1 cardinal symptoms including diabetic nephropathy, metabolic disruptions and optic atrophy. Far fewer studies have examined loss of Cisd2 function in mice. We identified B6.DDY-Cisd2m1Lmt, a mouse model with a spontaneous mutation in the Cisd2 gene. B6.DDY-Cisd2m1Lmt mice were initially identified based on the presence of audible sonic vocalizations as well as decreased body size and weight compared to unaffected wildtype littermates. Although Wfs1 knockout mice have been characterized for numerous behavioral phenotypes, similar studies have been lacking for Cisd2 mutant mice. We tested B6.DDY-Cisd2m1Lmt mice in a battery of behavioral assays that model phenotypes related to neurological and psychiatric disorders including anxiety, sensorimotor gating, stress response, social interaction and learning and memory. B6.DDY-Cisd2m1Lmt mice displayed hypoactivity across several behavioral tests, exhibited increased stress response and had deficits in spatial learning and memory and sensorimotor gating compared to wildtype littermates. Our data indicate that the B6.DDY-Cisd2m1Lmt mouse strain is a useful model to investigate potential mechanisms underlying the neurological and psychiatric symptoms observed in WFS.

Pharmacokinetic and pharmacodynamic analyses of cocaine and its metabolites in behaviorally divergent inbred mouse strains.

Cocaine (COC) is a psychostimulant with a high potential for abuse and addiction. Risk for COC use disorder is driven, in part, by genetic factors. Animal models of addiction-relevant behaviors have proven useful for studying both genetic and nongenetic contributions to drug response. In a previous study, we examined initial locomotor sensitivity to COC in genetically diverse inbred mouse strains. That work highlighted the relevance of pharmacokinetics (PK) in initial locomotor response to COC but was limited by a single dose and two sampling points. The objective of the present study was to characterize the PK and pharmacodynamics of COC and its metabolites (norcocaine and benzoylecgonine) in six inbred mouse strains (I/LnJ, C57BL/6J, FVB/NJ, BTBR T+ tf/J, LG/J and LP/J) that exhibit extreme locomotor responses to cocaine. Mice were administered COC at one of four doses and concentrations of cocaine, norcocaine and benzoylecgonine were analyzed in both plasma and brain tissue at 5 different time points. Initial locomotor sensitivity to COC was used as a pharmacodynamic endpoint. We developed an empirical population PK model that simultaneously characterizes cocaine, norcocaine and benzoylecgonine in plasma and brain tissues. We observed interstrain variability occurring in the brain compartment that may contribute to pharmacodynamic differences among select strains. Our current work paves the way for future studies to explore strain-specific pharmacokinetic differences and identify factors other than PK that are responsible for the diverse behavioral response to COC across these inbred mouse strains.

Sequence variant analysis reveals poor correlations in microbial taxonomic abundance between humans and mice after gnotobiotic transfer.

Transplanting human gut microbiotas into germ-free (GF) mice is a popular approach to disentangle cause-and-effect relationships between enteric microbes and disease. Algorithm development has enabled sequence variant (SV) identification from 16S rRNA gene sequence data. SV analyses can identify which donor taxa colonize recipient GF mice, and how SV abundance in humans is replicated in these mice. Fecal microbiotas from 8 human subjects were used to generate 77 slurries, which were transplanted into 153 GF mice. Strong correlations between fecal and slurry microbial communities were observed; however, only 42.15 ± 9.95% of SVs successfully transferred from the donor to the corresponding recipient mouse. Firmicutes had a particularly low transfer rate and SV abundance was poorly correlated between donor and recipient pairs. Our study confirms human fecal microbiotas colonize formerly GF mice, but the engrafted community only partially resembles the input human communities. Our findings emphasize the importance of reporting a standardized transfer rate and merit the exploration of other animal models or in silico tools to understand the relationships between human gut microbiotas and disease.

Prospects for finding the mechanisms of sex differences in addiction with human and model organism genetic analysis.

Despite substantial evidence for sex differences in addiction epidemiology, addiction-relevant behaviors and associated neurobiological phenomena, the mechanisms and implications of these differences remain unknown. Genetic analysis in model organism is a potentially powerful and effective means of discovering the mechanisms that underlie sex differences in addiction. Human genetic studies are beginning to show precise risk variants that influence the mechanisms of addiction but typically lack sufficient power or neurobiological mechanistic access, particularly for the discovery of the mechanisms that underlie sex differences. Our thesis in this review is that genetic variation in model organisms are a promising approach that can complement these investigations to show the biological mechanisms that underlie sex differences in addiction.

Characterization of genetically complex Collaborative Cross mouse strains that model divergent locomotor activating and reinforcing properties of cocaine.

RATIONALE: Few effective treatments exist for cocaine use disorders due to gaps in knowledge about its complex etiology. Genetically defined animal models provide a useful tool for advancing our understanding of the biological and genetic underpinnings of addiction-related behavior and evaluating potential treatments. However, many attempts at developing mouse models of behavioral disorders were based on overly simplified single gene perturbations, often leading to inconsistent and misleading results in pre-clinical pharmacology studies. A genetically complex mouse model may better reflect disease-related behaviors. OBJECTIVES: Screening defined, yet genetically complex, intercrosses of the Collaborative Cross (CC) mice revealed two lines, RIX04/17 and RIX41/51, with extreme high and low behavioral responses to cocaine. We characterized these lines as well as their CC parents, CC004/TauUnc and CC041/TauUnc, to evaluate their utility as novel model systems for studying the biological and genetic mechanisms underlying behavioral responses to cocaine. METHODS: Behavioral responses to acute (initial locomotor sensitivity) and repeated (behavioral sensitization, conditioned place preference, intravenous self-administration) exposures to cocaine were assessed. We also examined the monoaminergic system (striatal tissue content and in vivo fast scan cyclic voltammetry), HPA axis reactivity, and circadian rhythms as potential mechanisms for the divergent phenotypic behaviors observed in the two strains, as these systems have a previously known role in mediating addiction-related behaviors. RESULTS: RIX04/17 and 41/51 show strikingly divergent initial locomotor sensitivity to cocaine with RIX04/17 exhibiting very high and RIX41/51 almost no response. The lines also differ in the emergence of behavioral sensitization with RIX41/51 requiring more exposures to exhibit a sensitized response. Both lines show conditioned place preference for cocaine. We determined that the cocaine sensitivity phenotype in each RIX line was largely driven by the genetic influence of one CC parental strain, CC004/TauUnc and CC041/TauUnc. CC004 demonstrates active operant cocaine self-administration and CC041 is unable to acquire under the same testing conditions, a deficit which is specific to cocaine as both strains show operant response for a natural food reward. Examination of potential mechanisms driving differential responses to cocaine show strain differences in molecular and behavioral circadian rhythms. Additionally, while there is no difference in striatal dopamine tissue content or dynamics, there are selective differences in striatal norepinephrine and serotonergic tissue content. CONCLUSIONS: These CC strains offer a complex polygenic model system to study underlying mechanisms of cocaine response. We propose that CC041/TauUnc and CC004/TauUnc will be useful for studying genetic and biological mechanisms underlying resistance or vulnerability to the stimulatory and reinforcing effects of cocaine.

Mean-Variance QTL Mapping Identifies Novel QTL for Circadian Activity and Exploratory Behavior in Mice.

We illustrate, through two case studies, that "mean-variance QTL mapping"-QTL mapping that models effects on the mean and the variance simultaneously-can discover QTL that traditional interval mapping cannot. Mean-variance QTL mapping is based on the double generalized linear model, which extends the standard linear model used in interval mapping by incorporating not only a set of genetic and covariate effects for mean but also set of such effects for the residual variance. Its potential for use in QTL mapping has been described previously, but it remains underutilized, with certain key advantages undemonstrated until now. In the first case study, a reduced complexity intercross of C57BL/6J and C57BL/6N mice examining circadian behavior, our reanalysis detected a mean-controlling QTL for circadian wheel running activity that interval mapping did not; mean-variance QTL mapping was more powerful than interval mapping at the QTL because it accounted for the fact that mice homozygous for the C57BL/6N allele had less residual variance than other mice. In the second case study, an intercross between C57BL/6J and C58/J mice examining anxiety-like behaviors, our reanalysis detected a variance-controlling QTL for rearing behavior; interval mapping did not identify this QTL because it does not target variance QTL. We believe that the results of these reanalyses, which in other respects largely replicated the original findings, support the use of mean-variance QTL mapping as standard practice.

Impact of vitamin D depletion during development on mouse sperm DNA methylation.

Suboptimal environmental conditions during development can substantially alter the epigenome. Stable environmentally-induced changes to the germline epigenome, in particular, have important implications for the health of the next generation. We showed previously that developmental vitamin D depletion (DVD) resulted in loss of DNA methylation at several imprinted loci over two generations. Here, we assessed the impact of DVD on genome-wide methylation in mouse sperm in order to characterize the number, extent and distribution of methylation changes in response to DVD and to find genes that may be susceptible to this prevalent environmental perturbation. We detected 15,827 loci that were differentially methylated in DVD mouse sperm vs. controls. Most epimutations (69%) were loss of methylation, and the extent of methylation change and number of CpGs affected in a region were associated with genic location and baseline methylation state. Methylation response to DVD at validated loci was only detected in offspring that exhibited a phenotypic response to DVD (increased body and testes weight) suggesting the two types of responses are linked, though a causal relationship is unclear. Epimutations localized to regions enriched for developmental and metabolic genes and pathway analyses showed enrichment for Cadherin, Wnt, PDGF and Integrin signaling pathways. These findings show for the first time that vitamin D status during development leads to substantial DNA methylation changes across the sperm genome and that locus susceptibility is linked to genomic and epigenomic context.

Reciprocal F1 Hybrids of Two Inbred Mouse Strains Reveal Parent-of-Origin and Perinatal Diet Effects on Behavior and Expression.

Parent-of-origin effects (POE) in mammals typically arise from maternal effects or imprinting. In some instances, such POE have been associated with psychiatric disorders, as well as with changes in a handful of animal behaviors. However, POE on complex traits such as behavior remain largely uncharacterized. Moreover, although both behavior and epigenetic effects are known to be modified by perinatal environmental exposures such as nutrient deficiency, the architecture of such environment-by-POE is mostly unexplored. To study POE and environment-by-POE, we employ a relatively neglected but especially powerful experimental system for POE-detection: reciprocal F1 hybrids (RF1s). We exposed female NOD/ShiLtJ×C57Bl/6J and C57Bl/6J×NOD/ShiLtJ mice, perinatally, to one of four different diets, then after weaning recorded a set of behaviors that model psychiatric disease. Whole-brain microarray expression data revealed an imprinting-enriched set of 15 genes subject to POE. The most-significant expression POE, on the non-imprinted gene Carmil1 (a.k.a. Lrrc16a), was validated using qPCR in the same and in a new set of mice. Several behaviors, especially locomotor behaviors, also showed POE. Bayesian mediation analysis suggested Carmil1 expression suppresses behavioral POE, and that the imprinted gene Airn suppresses POE on Carmil1 expression. A suggestive diet-by-POE was observed on percent center time in the open field test, and a significant diet-by-POE was observed on one imprinted gene, Mir341, and on 16 non-imprinted genes. The relatively small, tractable set of POE and diet-by-POE detected on behavior and expression here motivates further studies examining such effects across RF1s on multiple genetic backgrounds.

Intravenous cocaine self-administration in a panel of inbred mouse strains differing in acute locomotor sensitivity to cocaine.

RATIONALE: Initial sensitivity to drugs of abuse often predicts subsequent use and abuse, but this relationship is not always observed in human studies. Moreover, studies examining the relationship between initial locomotor sensitivity and the rewarding and reinforcing effects of drugs in animal models have also been equivocal. Understanding the relationship between initial drug effects and propensity to continue use, potentially resulting in the development of a substance use disorder, may help to identify key targets for prevention and treatment. OBJECTIVES: We examined intravenous cocaine self-administration in a set of mouse strains that were previously identified to be at the phenotypic extremes for cocaine-induced locomotor activation to determine if initial locomotor sensitivity predicted acquisition, extinction, dose response, or progressive ratio (PR) breakpoint. METHODS: We selected eight inbred mouse strains based on locomotor sensitivity to 20 mg/kg cocaine. These strains, designated as low and high responders, were tested in an intravenous self-administration paradigm that included acquisition of 0.5 mg/(kg*inf) under a FR1 schedule, extinction, re-acquisition, dose response to 0.125, 0.25, 0.5, 1, and 2 mg/(kg*inf), and progressive ratio. RESULTS: We observed overall differences in self-administration behavior between high and low responders. Low responders self-administered less cocaine and had lower breakpoints under the PR schedule. However, we also observed strain differences within each group. Self-administration in the low responder, LG/J, more closely resembled the behavior of the high-responding group, and the high responder, P/J, had self-administration behavior that more closely resembled the low-responding group. CONCLUSIONS: We conclude that acute cocaine-induced locomotor activation does predict self-administration behavior, but in a strain-specific manner. These data support the idea that genetic background influences the relationship among addiction-related behaviors.

Reproducibility and replicability of rodent phenotyping in preclinical studies.

The scientific community is increasingly concerned with the proportion of published "discoveries" that are not replicated in subsequent studies. The field of rodent behavioral phenotyping was one of the first to raise this concern, and to relate it to other methodological issues: the complex interaction between genotype and environment; the definitions of behavioral constructs; and the use of laboratory mice and rats as model species for investigating human health and disease mechanisms. In January 2015, researchers from various disciplines gathered at Tel Aviv University to discuss these issues. The general consensus was that the issue is prevalent and of concern, and should be addressed at the statistical, methodological and policy levels, but is not so severe as to call into question the validity and the usefulness of model organisms as a whole. Well-organized community efforts, coupled with improved data and metadata sharing, have a key role in identifying specific problems and promoting effective solutions. Replicability is closely related to validity, may affect generalizability and translation of findings, and has important ethical implications.