Tmod2 Is a Regulator of Cocaine Responses through Control of Striatal and Cortical Excitability and Drug-Induced Plasticity.

Drugs of abuse induce neuroadaptations, including synaptic plasticity, that are critical for transition to addiction, and genes and pathways that regulate these neuroadaptations are potential therapeutic targets. Tropomodulin 2 (Tmod2) is an actin-regulating gene that plays an important role in synapse maturation and dendritic arborization and has been implicated in substance abuse and intellectual disability in humans. Here, we mine the KOMP2 data and find that Tmod2 knock-out mice show emotionality phenotypes that are predictive of addiction vulnerability. Detailed addiction phenotyping shows that Tmod2 deletion does not affect the acute locomotor response to cocaine administration. However, sensitized locomotor responses are highly attenuated in these knock-outs, indicating perturbed drug-induced plasticity. In addition, Tmod2 mutant animals do not self-administer cocaine indicating lack of hedonic responses to cocaine. Whole-brain MR imaging shows differences in brain volume across multiple regions, although transcriptomic experiments did not reveal perturbations in gene coexpression networks. Detailed electrophysiological characterization of Tmod2 KO neurons showed increased spontaneous firing rate of early postnatal and adult cortical and striatal neurons. Cocaine-induced synaptic plasticity that is critical for sensitization is either missing or reciprocal in Tmod2 KO nucleus accumbens shell medium spiny neurons, providing a mechanistic explanation of the cocaine response phenotypes. Combined, these data, collected from both males and females, provide compelling evidence that Tmod2 is a major regulator of plasticity in the mesolimbic system and regulates the reinforcing and addictive properties of cocaine.

Effects of adolescent nicotine exposure and withdrawal on intravenous cocaine self-administration during adulthood in male C57BL/6J mice.

Studies of adolescent drug use show (1) a pattern in which the use of tobacco precedes the use of other drugs and (2) a positive relationship between adolescent tobacco use and later drug use. These observations have led to the hypothesis that a causal relationship exists between early exposure to nicotine and the later use of hard drugs such as cocaine. Using male C57BL/6J mice, we tested the hypothesis that nicotine exposure in adolescence leads to increased intravenous self-administration (IVSA) of cocaine in adulthood. Using miniature osmotic pumps, we exposed mice and their littermate controls to nicotine (24 mg/kg/day) or vehicle, respectively, over the entire course of adolescence [postnatal days (P) 28-56]. Nicotine exposure was terminated on P56 and mice were not exposed to nicotine again during the experiment. On P73, mice were allowed to acquire cocaine IVSA (1.0 mg/kg/infusion) and a dose-response curve was generated (0.18, 0.32, 0.56, 1.0, 1.8 mg/kg/infusion). Lever pressing during extinction conditions was also evaluated. All mice rapidly learned to lever press for the combination of cocaine infusions and non-drug stimuli. Analysis of the dose-response curve revealed that adolescent nicotine-exposed mice self-administered significantly more (P < 0.05) cocaine than controls at all but the highest dose. No significant differences were observed between adolescent nicotine-exposed and control mice during the acquisition or extinction stages. These results indicate that adolescent nicotine exposure can increase cocaine IVSA in mice, which suggests the possibility of a causal link between adolescent tobacco use and later cocaine use in humans.

Discovery and validation of genes driving drug-intake and related behavioral traits in mice.

Substance use disorders are heritable disorders characterized by compulsive drug use, the biological mechanisms for which remain largely unknown. Genetic correlations reveal that predisposing drug-naïve phenotypes, including anxiety, depression, novelty preference and sensation seeking, are predictive of drug-use phenotypes, thereby implicating shared genetic mechanisms. High-throughput behavioral screening in knockout (KO) mice allows efficient discovery of the function of genes. We used this strategy in two rounds of candidate prioritization in which we identified 33 drug-use candidate genes based upon predisposing drug-naïve phenotypes and ultimately validated the perturbation of 22 genes as causal drivers of substance intake. We selected 19/221 KO strains (8.5%) that had a difference from control on at least one drug-naïve predictive behavioral phenotype and determined that 15/19 (~80%) affected the consumption or preference for alcohol, methamphetamine or both. No mutant exhibited a difference in nicotine consumption or preference which was possibly confounded with saccharin. In the second round of prioritization, we employed a multivariate approach to identify outliers and performed validation using methamphetamine two-bottle choice and ethanol drinking-in-the-dark protocols. We identified 15/401 KO strains (3.7%, which included one gene from the first cohort) that differed most from controls for the predisposing phenotypes. 8 of 15 gene deletions (53%) affected intake or preference for alcohol, methamphetamine or both. Using multivariate and bioinformatic analyses, we observed multiple relations between predisposing behaviors and drug intake, revealing many distinct biobehavioral processes underlying these relationships. The set of mouse models identified in this study can be used to characterize these addiction-related processes further.

Reorganization of circuits underlying cerebellar modulation of prefrontal cortical dopamine in mouse models of autism spectrum disorder.

Imaging, clinical, and pre-clinical studies have provided ample evidence for a cerebellar involvement in cognitive brain function including cognitive brain disorders, such as autism and schizophrenia. We previously reported that cerebellar activity modulates dopamine release in the mouse medial prefrontal cortex (mPFC) via two distinct pathways: (1) cerebellum to mPFC via dopaminergic projections from the ventral tegmental area (VTA) and (2) cerebellum to mPFC via glutamatergic projections from the mediodorsal and ventrolateral thalamus (ThN md and vl). The present study compared functional adaptations of cerebello-cortical circuitry following developmental cerebellar pathology in a mouse model of developmental loss of Purkinje cells (Lurcher) and a mouse model of fragile X syndrome (Fmr1 KO mice). Fixed potential amperometry was used to measure mPFC dopamine release in response to cerebellar electrical stimulation. Mutant mice of both strains showed an attenuation in cerebellar-evoked mPFC dopamine release compared to respective wildtype mice. This was accompanied by a functional reorganization of the VTA and thalamic pathways mediating cerebellar modulation of mPFC dopamine release. Inactivation of the VTA pathway by intra-VTA lidocaine or kynurenate infusions decreased dopamine release by 50 % in wildtype and 20-30 % in mutant mice of both strains. Intra-ThN vl infusions of either drug decreased dopamine release by 15 % in wildtype and 40 % in mutant mice of both strains, while dopamine release remained relatively unchanged following intra-ThN md drug infusions. These results indicate a shift in strength towards the thalamic vl projection, away from the VTA. Thus, cerebellar neuropathologies associated with autism spectrum disorders may cause a reduction in cerebellar modulation of mPFC dopamine release that is related to a reorganization of the mediating neuronal pathways.

Intravenous fentanyl self-administration in male and female C57BL/6J and DBA/2J mice.

The genetic mechanisms underlying fentanyl addiction, a highly heritable disease, are unknown. Identifying these mechanisms will lead to better risk assessment, early diagnosis, and improved intervention. To this end, we used intravenous fentanyl self-administration to quantify classical self-administration phenotypes and addiction-like fentanyl seeking in male and female mice from the two founder strains of the BXD recombinant inbred mouse panel (C57BL/6J and DBA/2J). We reached three primary conclusions from these experiments. First, mice from all groups rapidly acquired intravenous fentanyl self-administration and exhibited a dose-response curve, extinction burst, and extinction of the learned self-administration response. Second, fentanyl intake (during acquisition and dose response) and fentanyl seeking (during extinction) were equivalent among groups. Third, strain effects, sex effects, or both were identified for several addiction-like behaviors (cue-induced reinstatement, stress-induced reinstatement, escalation of intravenous fentanyl self-administration). Collectively, these data indicate that C57BL/6J and DBA/2J mice of both sexes were able to acquire, regulate, and extinguish intravenous fentanyl self-administration. Moreover, these data reveal novel strain and sex effects on addiction-like behaviors in the context of intravenous fentanyl self-administration in mice and indicate that the full BXD panel can be used to identify and dissect the genetic mechanisms underlying these effects.

Effects of isolation housing stress and mouse strain on intravenous cocaine self-administration, sensory stimulus self-administration, and reward preference.

Sensory stimuli are natural rewards in mice and humans. Consequently, preference for a drug reward relative to a sensory reward may be an endophenotype of addiction vulnerability. In this study, we developed a novel behavioral assay to quantify the preference for intravenous drug self-administration relative to sensory stimulus self-administration. We used founder strains of the BXD recombinant inbred mouse panel (C57BL/6J, DBA/2J) and a model of stress (isolation vs enriched housing) to assess genetic and epigenetic effects. Following 10 weeks of differential housing, all mice were tested under three reward conditions: sensory rewards available, cocaine rewards available, both rewards available. When a single reward was available (sensory stimuli or cocaine; delivered using distinct levers), DBA/2J mice self-administered significantly more rewards than C57BL/6J mice. When both rewards were available, DBA/2J mice exhibited a significant preference for cocaine relative to sensory stimuli; in contrast, C57BL/6J mice exhibited no preference. Housing condition influenced sensory stimulus self-administration and strain-dependently influenced inactive lever pressing when both rewards were available. Collectively, these data reveal strain effects, housing effects, or both on reward self-administration and preference. Most importantly, this study reveals that genetic mechanisms underlying preference for a drug reward relative to a nondrug reward can be dissected using the full BXD panel.

Gene expression genetics of the striatum of Diversity Outbred mice.

Brain transcriptional variation is a heritable trait that mediates complex behaviors, including addiction. Expression quantitative trait locus (eQTL) mapping reveals genomic regions harboring genetic variants that influence transcript abundance. In this study, we profiled transcript abundance in the striatum of 386 Diversity Outbred (J:DO) mice of both sexes using RNA-Seq. All mice were characterized using a behavioral battery of widely-used exploratory and risk-taking assays prior to transcriptional profiling. We performed eQTL mapping, incorporated the results into a browser-based eQTL viewer, and deposited co-expression network members in GeneWeaver. The eQTL viewer allows researchers to query specific genes to obtain allelic effect plots, analyze SNP associations, assess gene expression correlations, and apply mediation analysis to evaluate whether the regulatory variant is acting through the expression of another gene. GeneWeaver allows multi-species comparison of gene sets using statistical and combinatorial tools. This data resource allows users to find genetic variants that regulate differentially expressed transcripts and place them in the context of other studies of striatal gene expression and function in addiction-related behavior.

Gene expression genetics of the striatum of Diversity Outbred mice.

Brain transcriptional variation is a heritable trait that mediates complex behaviors, including addiction. Expression quantitative trait locus (eQTL) mapping reveals genomic regions harboring genetic variants that influence transcript abundance. In this study, we profiled transcript abundance in the striatum of 386 Diversity Outbred (J:DO) mice of both sexes using RNA-Seq. All mice were characterized using a behavioral battery of widely-used exploratory and risk-taking assays prior to transcriptional profiling. We performed eQTL mapping, incorporated the results into a browser-based eQTL viewer, and deposited co-expression network members in GeneWeaver. The eQTL viewer allows researchers to query specific genes to obtain allelic effect plots, analyze SNP associations, assess gene expression correlations, and apply mediation analysis to evaluate whether the regulatory variant is acting through the expression of another gene. GeneWeaver allows multi-species comparison of gene sets using statistical and combinatorial tools. This data resource allows users to find genetic variants that regulate differentially expressed transcripts and place them in the context of other studies of striatal gene expression and function in addiction-related behavior.

Microbial glutamate metabolism predicts intravenous cocaine self-administration in diversity outbred mice.

The gut microbiome is thought to play a critical role in the onset and development of psychiatric disorders, including depression and substance use disorder (SUD). To test the hypothesis that the microbiome affects addiction predisposing behaviors and cocaine intravenous self-administration (IVSA) and to identify specific microbes involved in the relationship, we performed 16S rRNA gene sequencing on feces from 228 diversity outbred mice. Twelve open field measures, two light-dark assay measures, one hole board and novelty place preference measure significantly differed between mice that acquired cocaine IVSA (ACQ) and those that failed to acquire IVSA (FACQ). We found that ACQ mice are more active and exploratory and display decreased fear than FACQ mice. The microbial abundances that differentiated ACQ from FACQ mice were an increased abundance of Barnesiella, Ruminococcus, and Robinsoniella and decreased Clostridium IV in ACQ mice. There was a sex-specific correlation between ACQ and microbial abundance, a reduced Lactobacillus abundance in ACQ male mice, and a decreased Blautia abundance in female ACQ mice. The abundance of Robinsoniella was correlated, and Clostridium IV inversely correlated with the number of doses of cocaine self-administered during acquisition. Functional analysis of the microbiome composition of a subset of mice suggested that gut-brain modules encoding glutamate metabolism genes are associated with the propensity to self-administer cocaine. These findings establish associations between the microbiome composition and glutamate metabolic potential and the ability to acquire cocaine IVSA thus indicating the potential translational impact of targeting the gut microbiome or microbial metabolites for treatment of SUD. This article is part of the Special Issue on "Microbiome & the Brain: Mechanisms & Maladies".

DISCOVERY AND VALIDATION OF GENES DRIVING DRUG-INTAKE AND RELATED BEHAVIORAL TRAITS IN MICE.

Substance use disorders (SUDs) are heritable disorders characterized by compulsive drug use, but the biological mechanisms driving addiction remain largely unknown. Genetic correlations reveal that predisposing drug-naïve phenotypes, including anxiety, depression, novelty preference, and sensation seeking, are predictive of drug-use phenotypes, implicating shared genetic mechanisms. Because of this relationship, high-throughput behavioral screening of predictive phenotypes in knockout (KO) mice allows efficient discovery of genes likely to be involved in drug use. We used this strategy in two rounds of screening in which we identified 33 drug-use candidate genes and ultimately validated the perturbation of 22 of these genes as causal drivers of substance intake. In our initial round of screening, we employed the two-bottle-choice paradigms to assess alcohol, methamphetamine, and nicotine intake. We identified 19 KO strains that were extreme responders on at least one predictive phenotype. Thirteen of the 19 gene deletions (68%) significantly affected alcohol use three methamphetamine use, and two both. In the second round of screening, we employed a multivariate approach to identify outliers and performed validation using methamphetamine two-bottle choice and ethanol drinking-in-the-dark protocols. We identified 15 KO strains that were extreme responders across the predisposing drug-naïve phenotypes. Eight of the 15 gene deletions (53%) significantly affected intake or preference for three alcohol, eight methamphetamine or three both (3). We observed multiple relations between predisposing behaviors and drug intake, revealing many distinct biobehavioral processes underlying these relationships. The set of mouse models identified in this study can be used to characterize these addiction-related processes further.

Consensus paper: pathological role of the cerebellum in autism.

There has been significant advancement in various aspects of scientific knowledge concerning the role of cerebellum in the etiopathogenesis of autism. In the current consensus paper, we will observe the diversity of opinions regarding the involvement of this important site in the pathology of autism. Recent emergent findings in literature related to cerebellar involvement in autism are discussed, including: cerebellar pathology, cerebellar imaging and symptom expression in autism, cerebellar genetics, cerebellar immune function, oxidative stress and mitochondrial dysfunction, GABAergic and glutamatergic systems, cholinergic, dopaminergic, serotonergic, and oxytocin-related changes in autism, motor control and cognitive deficits, cerebellar coordination of movements and cognition, gene-environment interactions, therapeutics in autism, and relevant animal models of autism. Points of consensus include presence of abnormal cerebellar anatomy, abnormal neurotransmitter systems, oxidative stress, cerebellar motor and cognitive deficits, and neuroinflammation in subjects with autism. Undefined areas or areas requiring further investigation include lack of treatment options for core symptoms of autism, vermal hypoplasia, and other vermal abnormalities as a consistent feature of autism, mechanisms underlying cerebellar contributions to cognition, and unknown mechanisms underlying neuroinflammation.

Working memory and pattern separation in founder strains of the BXD recombinant inbred mouse panel.

Working memory and pattern separation are fundamental cognitive abilities which, when impaired, significantly diminish quality of life. Discovering genetic mechanisms underlying innate and disease-induced variation in these cognitive abilities is a critical step towards treatments for common and devastating neurodegenerative conditions such as Alzheimer's disease. In this regard, the trial-unique nonmatching-to-location assay (TUNL) is a touchscreen operant conditioning procedure allowing simultaneous quantification of working memory and pattern separation in mice and rats. In the present study, we used the TUNL assay to quantify these cognitive abilities in C57BL/6J and DBA/2J mice. These strains are the founders of the BXD recombinant inbred mouse panel which enables discovery of genetic mechanisms underlying phenotypic variation. TUNL testing revealed that pattern separation was significantly influenced by mouse strain, whereas working memory was not. Moreover, horizontal distance and vertical distance between choice-phase stimuli had dissociable effects on TUNL performance. These findings provide novel data on mouse strain differences in pattern separation and support previous findings of equivalent working memory performance in C57BL/6J and DBA/2J mice. Although working memory of the BXD founder strains was equivalent in this study, working memory of BXD strains may be divergent because of transgressive segregation. Collectively, data presented here indicate that pattern separation is heritable in the mouse and that the BXD panel can be used to identify mechanisms underlying variation in pattern separation.

Connecting the dots of the cerebro-cerebellar role in cognitive function: neuronal pathways for cerebellar modulation of dopamine release in the prefrontal cortex.

Cerebellar involvement in autism, schizophrenia, and other cognitive disorders is typically associated with prefrontal cortical pathology. However, the underlying neuronal mechanisms are largely unknown. It has previously been shown in mice that stimulation of the dentate nucleus (DN) of the cerebellum evokes dopamine (DA) release in the medial prefrontal cortex (mPFC). Here, we investigated the neuronal circuitry by which the cerebellum modulates mPFC DA release. Fixed potential amperometry was used to determine the contribution of two candidate pathways by which the cerebellum may modulate mPFC DA release. In urethane anesthetized mice, DA release evoked by DN stimulation (50 Hz) was recorded in mPFC following local anesthetic lidocaine (0.02 µg) or ionotropic glutamate receptor antagonist kynurenate (0.5 µg) infusions into the mediodorsal or ventrolateral thalamic nucleus (ThN md; ThN vl), or the ventral tegmental area (VTA). Following intra-VTA lidocaine or kynurenate infusions, DA release was decreased by ∼50%. Following intra-ThN md and ThN vl infusions of either drug, DA release was decreased by ∼35% and 15%, respectively. Reductions in DA release following lidocaine or kynurenate infusions were not significantly different indicating that neuronal cells in the VTA and ThN were activated primarily if not entirely by glutamatergic inputs. The present study suggests that neuropathological changes in the cerebellum commonly observed in autism, schizophrenia, and other cognitive disorders could result in a loss of functionality of cerebellar-mPFC circuitry that is manifested as aberrant dopaminergic activity in the mPFC. Additionally, these results specifically implicate glutamate as a modulator of mPFC dopaminergic activity.

Strain and sex dependent effects of isolation housing relative to environmental enrichment on operant sensation seeking in mice.

Sensation seeking is a multidimensional phenotype that predicts the development of drug addiction in humans and addiction-like drug seeking in rodents. Several lines of evidence suggest that chronic stress increases sensation seeking and addiction-like drug seeking through common genetic mechanisms. Discovery and characterization of these mechanisms would reveal how chronic stress interacts with the genome to influence sensation seeking and how drugs of abuse hijack these fundamental reward mechanisms to drive addiction. To this end, we tested the hypothesis that chronic isolation housing stress (relative to environmental enrichment) influences operant sensation seeking as a function of strain, sex, or their interaction. To determine if the BXD recombinant inbred panel could be used to identify genetic and epigenetic mechanisms underlying any identified gene-by-environment interactions, we used mice from the two BXD founder strains. Following 10 weeks of differential housing, we assessed operant sensation seeking using several reinforcement schedules. The primary finding from this study was that DBA/2J but not C57BL/6J mice were significantly vulnerable to an isolation-induced increase (relative to environmental enrichment) in sensation seeking during extinction when the sensory reward was no longer available; this effect was significantly more robust in females. These data reveal a previously unknown isolation-induced effect on extinction of operant sensation seeking that is sex-dependent, addiction-relevant, and that can be dissected using the BXD recombinant inbred panel.

Environmental enrichment influences novelty reactivity, novelty preference, and anxiety via distinct genetic mechanisms in C57BL/6J and DBA/2J mice.

Environmental factors such as stress drive the development of drug addiction in genetically vulnerable individuals; the genes underlying this vulnerability are unknown. One strategy for uncovering these genes is to study the impact of environmental manipulation on high-throughput phenotypes that predict drug use and addiction-like behaviors. In the present study, we assessed the viability of this approach by evaluating the relative effects of environmental enrichment and isolation housing on three high-throughput phenotypes known to predict variation on distinct aspects of intravenous drug self-administration. Prior to behavioral testing, male and female C57BL/6J and DBA/2J mice (BXD founders) were housed in enrichment or isolation for ten weeks beginning at weaning. Enrichment significantly reduced novelty reactivity; this effect was significantly more robust in C57BL/6J mice relative to DBA/2J mice. Enrichment significantly reduced novelty preference; this effect was significantly dependent on novel environment characteristics and was significantly more robust in DBA/2J mice relative to C57BL/6J mice. Enrichment significantly increased anxiety; this effect was not strain-dependent. Collectively, these data indicate that (1) environmental enrichment influences novelty reactivity, novelty preference, and anxiety via distinct genetic mechanisms in mice, and (2) the BXD panel can be used to discover the genetic and epigenetic mechanisms underlying this phenomenon.

Behavioral flexibility in a mouse model of developmental cerebellar Purkinje cell loss.

Although behavioral inflexibility and Purkinje cell loss are both well established in autism, it is unknown if these phenomena are causally related. Using a mouse model, we tested the hypothesis that developmental abnormalities of the cerebellum, including Purkinje cell loss, result in behavioral inflexibility. Specifically, we made aggregation chimeras (Lc/+<-->+/+) between lurcher (Lc/+) mutant embryos and wildtype (+/+) control embryos. Lurcher mice lose 100% of their Purkinje cells postnatally, while chimeric mice lose varying numbers of Purkinje cells. We tested these mice on the acquisition and serial reversals of an operant conditional visual discrimination, a test of behavioral flexibility in rodents. During reversals 1 and 2, all groups of mice committed similar numbers of "perseverative" errors (those committed while session performance was <= 40% correct). Lurchers, however, committed a significantly greater number of "learning" errors (those committed while session performance was between 41% and 85% correct) than both controls and chimeras, and most were unable to advance past reversal 3. During reversals 3 and 4, chimeras, as a group, committed more "perseverative", but not "learning" errors than controls, although a comparison of Purkinje cell number and performance in individual mice revealed that chimeras with fewer Purkinje cells made more "learning" errors and had shorter response latencies than chimeras with more Purkinje cells. These data suggest that developmental cerebellar Purkinje cell loss may affect higher level cognitive processes which have previously been shown to be mediated by the prefrontal cortex, and are commonly deficient in autism spectrum disorders.

Discovery of a Role for Rab3b in Habituation and Cocaine Induced Locomotor Activation in Mice Using Heterogeneous Functional Genomic Analysis.

Substance use disorders are prevalent and present a tremendous societal cost but the mechanisms underlying addiction behavior are poorly understood and few biological treatments exist. One strategy to identify novel molecular mechanisms of addiction is through functional genomic experimentation. However, results from individual experiments are often noisy. To address this problem, the convergent analysis of multiple genomic experiments can discern signal from these studies. In the present study, we examine genetic loci that modulate the locomotor response to cocaine identified in the recombinant inbred (BXD RI) genetic reference population. We then applied the GeneWeaver software system for heterogeneous functional genomic analysis to integrate and aggregate multiple studies of addiction genomics, resulting in the identification of Rab3b as a functional correlate of the locomotor response to cocaine in rodents. This gene encodes a member of the RAB family of Ras-like GTPases known to be involved in trafficking of secretory and endocytic vesicles in eukaryotic cells. The convergent evidence for a role of Rab3b includes co-occurrence in previously published genetic mapping studies of cocaine related behaviors; methamphetamine response and cocaine- and amphetamine-regulated transcript prepropeptide (Cartpt) transcript abundance; evidence related to other addictive substances; density of polymorphisms; and its expression pattern in reward pathways. To evaluate this finding, we examined the effect of RAB3 complex perturbation in cocaine response. B6;129-Rab3btm1Sud Rab3ctm1sud Rab3dtm1sud triple null mice (Rab3bcd -/-) exhibited significant deficits in habituation, and increased acute and repeated cocaine responses. This previously unidentified mechanism of the behavioral predisposition and response to cocaine is an example of many that can be identified and validated using aggregate genomic studies.

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.

Stimulus Complexity and Mouse Strain Drive Escalation of Operant Sensation Seeking Within and Across Sessions in C57BL/6J and DBA/2J Mice.

Sensation seeking is a heritable trait that is genetically correlated with substance use; the shared genetic mechanisms underlying these traits are largely unknown. The relationship of sensation seeking and substance use has practical importance because discovering genes that drive sensation seeking can reveal genes driving substance use, and quantification of sensation seeking in mice is higher throughput and less technically challenging than quantification of volitional drug use. In order to fully understand the genetic mechanisms driving sensation seeking, it is critical to first understand the nongenetic factors driving sensation seeking. In the present study, we used the operant sensation seeking paradigm to assess the effects of stimulus complexity on sensation seeking in C57BL/6J and DBA/2J mice. These strains are the founders of the BXD recombinant inbred mouse panel which enables the discovery of genes driving phenotypic variation. This study led to four principal conclusions. First, all sensory stimuli used in the study, regardless of complexity or number of stimulus modalities, served as reinforcers for C57BL/6J and DBA/2J mice. Second, for both C57BL/6J and DBA/2J mice, sensation seeking for a high complexity sensory stimulus was significantly greater than sensation seeking for a low complexity sensory stimulus. Third, for both C57BL/6J and DBA/2J mice, sensation seeking escalated significantly within-session when a multimodal sensory stimulus of medium or high complexity was used but not when a unimodal sensory stimulus of low complexity was used. Finally, both the magnitude of sensation seeking and the magnitude of within-session escalation of sensation seeking were significantly greater in mice from the DBA/2J strain relative to mice from the C57BL/6J strain. Collectively, these findings indicate that stimulus complexity and genetic background drive escalation of operant sensation seeking within and across sessions, and that the BXD recombinant inbred mouse panel can be used to discover the genetic mechanisms underlying these phenomena.

Visual Discrimination, Serial Reversal, and Extinction Learning in the mdx Mouse.

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy and the most common neuromuscular disorder. In addition to neuromuscular consequences, some individuals with DMD experience global intellectual dysfunction and executive dysfunction of unknown mechanistic origin. The cognitive profile of the mdx mouse, the most commonly used mouse model of DMD, has been incompletely characterized and has never been assessed using the touchscreen operant conditioning paradigm. The touchscreen paradigm allows the use of protocols that are virtually identical to those used in human cognitive testing and may, therefore, provide the most translational paradigm for quantifying mouse cognitive function. In the present study, we used the touchscreen paradigm to assess the effects of the mdx mutation on visual discrimination learning, serial reversal learning, and extinction learning. To enable measuring task-dependent learning and memory processes while holding demands on sensory-driven information processing constant, we developed equally salient visual stimuli and used them on all experimental stages. Acquisition of the initial pairwise visual discrimination was facilitated in mdx mice relative to wildtype littermates; this effect was not explained by genotypic differences in impulsivity, motivation, or motor deficits. The mdx mutation had no effect on serial reversal or extinction learning. Together, findings from this study and previous studies suggest that mdx effects on cognitive function are task-specific and may be influenced by discrimination type (spatial, visual), reward type (food, escape from a non-preferred environment), sex, and genetic background.