Randal J. Nonneman

Remote Control of Neuronal Activity in Transgenic Mice Expressing Evolved G Protein-Coupled Receptors

Examining the behavioral consequences of selective CNS neuronal activation is a powerful tool for elucidating mammalian brain function in health and disease. Newly developed genetic, pharmacological, and optical tools allow activation of neurons with exquisite spatiotemporal resolution; however, the inaccessibility to light of widely distributed neuronal populations and the invasiveness required for activation by light or infused ligands limit the utility of these methods. To overcome these barriers, we created transgenic mice expressing an evolved G protein-coupled receptor (hM3Dq) selectively activated by the pharmacologically inert, orally bioavailable drug clozapine-N-oxide (CNO). Here, we expressed hM3Dq in forebrain principal neurons. Local field potential and single-neuron recordings revealed that peripheral administration of CNO activated hippocampal neurons selectively in hM3Dq-expressing mice. Behavioral correlates of neuronal activation included increased locomotion, stereotypy, and limbic seizures. These results demonstrate a powerful chemical-genetic tool for remotely controlling the activity of discrete populations of neurons in vivo.

Social Approach in Genetically-Engineered Mouse Lines Relevant to Autism

Profound impairment in social interaction is a core symptom of autism, a severe neurodevelopmental disorder. Deficits can include a lack of interest in social contact and low levels of approach and proximity to other children. In this study, a three‐chambered choice task was used to evaluate sociability and social novelty preference in five lines of mice with mutations in genes implicated in autism spectrum disorders. Fmr1tm1Cgr/Y(Fmr1−/y) mice represent a model for fragile X, a mental retardation syndrome that is partially comorbid with autism. We tested Fmr1−/ymice on two genetic backgrounds, C57BL/6J and FVB/N‐129/OlaHsd (FVB/129). Targeted disruption of Fmr1 resulted in low sociability on one measure, but only when the mutation was expressed on FVB/129. Autism has been associated with altered serotonin levels and polymorphisms in SLC6A4 (SERT), the serotonin transporter gene. Male mice with targeted disruption of Slc6a4 displayed significantly less sociability than wild‐type controls. Mice with conditional overexpression of Igf‐1 (insulin‐like growth factor‐1) offered a model for brain overgrowth associated with autism. Igf‐1 transgenic mice engaged in levels of social approach similar to wild‐type controls. Targeted disruption in other genes of interest, En2 (engrailed‐2) and Dhcr7, was carried on genetic backgrounds that showed low levels of exploration in the choice task, precluding meaningful interpretations of social behavior scores. Overall, results show that loss of Fmr1 or Slc6a4 gene function can lead to deficits in sociability. Findings from the fragile X model suggest that the FVB/129 background confers enhanced susceptibility to consequences of Fmr1 mutation on social approach.

Social Approach and Repetitive Behavior in Eleven Inbred Mouse Strains

Core symptoms of autism include deficits in social interaction, impaired communication, and restricted, repetitive behaviors. The repetitive behavior domain encompasses abnormal motoric stereotypy, an inflexible insistence on sameness, and resistance to change. In recent years, many genetic mouse models of autism and related disorders have been developed, based on candidate genes for disease susceptibility. The present studies are part of an ongoing initiative to develop appropriate behavioral tasks for the evaluation of mouse models relevant to autism. We have previously reported profiles for sociability, preference for social novelty, and resistance to changes in a learned pattern of behavior, as well as other functional domains, for 10 inbred mouse strains of divergent genetic backgrounds. The present studies extend this multi-component behavioral characterization to several additional strains: C58/J, NOD/LtJ, NZB/B1NJ, PL/J, SJL/J, SWR/J, and the wild-derived PERA/EiJ. C58/J, NOD/LtJ, NZB/B1NJ, SJL/J, and PERA/EiJ demonstrated low sociability, measured by time spent in proximity to an unfamiliar conspecific, with 30-60% of mice from these strains showing social avoidance. In the Morris water maze, NZB/B1NJ had a persistent bias for the quadrant where the hidden platform was located during acquisition, even after 9 days of reversal training. A particularly interesting profile was found for C58/J, which had low social preference, poor performance in the T-maze, and overt motoric stereotypy. Overall, this set of tasks and observational methods provides a strategy for evaluating novel mouse models in behavioral domains relevant to the autism phenotype.

Development of a Mouse Test for Repetitive, Restricted Behaviors: Relevance to Autism

Repetitive behavior, a core symptom of autism, encompasses stereotyped responses, restricted interests, and resistance to change. These studies investigated whether different components of the repetitive behavior domain could be modeled in the exploratory hole-board task in mice. Four inbred mouse strains, C57BL/6J, BALB/cByJ, BTBR T+tf/J, and FVB/NJ, and mice with reduced expression of Grin1, leading to NMDA receptor hypofunction (NR1neo/neo mice), were tested for exploration and preference for olfactory stimuli in an activity chamber with a 16-hole floor-board. Reduced exploration and high preference for holes located in the corners of the chamber were observed in BALB/cByJ and BTBR T+tf/J mice. All inbred strains had initial high preference for a familiar olfactory stimulus (clean cage bedding). BTBR T+tf/J was the only strain that did not demonstrate a shift in hole preference towards an appetitive olfactory stimulus (cereal or a chocolate chip), following home cage exposure to the food. The NR1neo/neo mice showed lower hole selectivity and aberrant olfactory stimulus preference, in comparison to wildtype controls. The results indicate that NR1neo/neo mice have repetitive nose poke responses that are less modified by environmental contingencies than responses in wildtype mice. 25-30% of NMDA receptor hypomorphic mice also show self-injurious responses. Findings from the olfactory studies suggest that resistance to change and restricted interests might be modeled in mice by a failure to alter patterns of hole preference following familiarization with an appetitive stimulus, and by high preference persistently demonstrated for one particular olfactory stimulus. Further work is required to determine the characteristics of optimal mouse social stimuli in the olfactory hole-board test.

Analyses of allele-specific gene expression in highly divergent mouse crosses identifies pervasive allelic imbalance

Complex human traits are influenced by variation in regulatory DNA through mechanisms that are not fully understood. Because regulatory elements are conserved between humans and mice, a thorough annotation of cis regulatory variants in mice could aid in further characterizing these mechanisms. Here we provide a detailed portrait of mouse gene expression across multiple tissues in a three-way diallel. Greater than 80% of mouse genes have cis regulatory variation. Effects from these variants influence complex traits and usually extend to the human ortholog. Further, we estimate that at least one in every thousand SNPs creates a cis regulatory effect. We also observe two types of parent-of-origin effects, including classical imprinting and a new global allelic imbalance in expression favoring the paternal allele. We conclude that, as with humans, pervasive regulatory variation influences complex genetic traits in mice and provide a new resource toward understanding the genetic control of transcription in mammals.

A Gαs DREADD Mouse for Selective Modulation of cAMP Production in Striatopallidal Neurons

Here, we describe a newly generated transgenic mouse in which the Gs DREADD (rM3Ds), an engineered G protein-coupled receptor, is selectively expressed in striatopallidal medium spiny neurons (MSNs). We first show that in vitro, rM3Ds can couple to Gαolf and induce cAMP accumulation in cultured neurons and HEK-T cells. The rM3Ds was then selectively and stably expressed in striatopallidal neurons by creating a transgenic mouse in which an adenosine2A (adora2a) receptor-containing bacterial artificial chromosome was employed to drive rM3Ds expression. In the adora2A-rM3Ds mouse, activation of rM3Ds by clozapine-N-oxide (CNO) induces DARPP-32 phosphorylation, consistent with the known consequence of activation of endogenous striatal Gαs-coupled GPCRs. We then tested whether CNO administration would produce behavioral responses associated with striatopallidal Gs signaling and in this regard CNO dose-dependently decreases spontaneous locomotor activity and inhibits novelty induced locomotor activity. Last, we show that CNO prevented behavioral sensitization to amphetamine and increased AMPAR/NMDAR ratios in transgene-expressing neurons of the nucleus accumbens shell. These studies demonstrate the utility of adora2a-rM3Ds transgenic mice for the selective and noninvasive modulation of Gαs signaling in specific neuronal populations in vivo.This unique tool provides a new resource for elucidating the roles of striatopallidal MSN Gαs signaling in other neurobehavioral contexts.

Behavioral deficits in an Angelman syndrome model: Effects of genetic background and age

Angelman syndrome (AS) is a severe neurodevelopmental disorder associated with disruption of maternally inherited UBE3A (ubiquitin protein ligase E3A) expression. At the present time, there is no effective treatment for AS. Mouse lines with loss of maternal Ube3a (Ube3a(m-/p+)) recapitulate multiple aspects of the clinical AS profile, including impaired motor coordination, learning deficits, and seizures. Thus, these genetic mouse models could serve as behavioral screens for preclinical efficacy testing, a critical component of drug discovery for AS intervention. However, the severity and consistency of abnormal phenotypes reported in Ube3a(m-/p+) mice can vary, dependent upon age and background strain, which is problematic for the detection of beneficial drug effects. As part of an ongoing AS drug discovery initiative, we characterized Ube3a(m-/p+) mice on either a 129S7/SvEvBrd-Hprt(b-m2) (129) or C57BL/6J (B6) background across a range of functional domains and ages to identify reproducible and sufficiently large phenotypes suitable for screening therapeutic compounds. The results from the study showed that Ube3a(m-/p+) mice have significant deficits in acquisition and reversal learning in the Morris water maze. The findings also demonstrated that Ube3a(m-/p+) mice exhibit motor impairment in a rotarod task, hypoactivity, reduced rearing and marble-burying, and deficient fear conditioning. Overall, these profiles of abnormal phenotypes can provide behavioral targets for evaluating effects of novel therapeutic strategies relevant to AS.

Prosocial effects of oxytocin in two mouse models of autism spectrum disorders

Clinical evidence suggests that oxytocin treatment improves social deficits and repetitive behavior in autism spectrum disorders (ASDs). However, the neuropeptide has a short plasma half-life and poor ability to penetrate the blood-brain barrier. In order to facilitate the development of more bioavailable oxytocinergic compounds as therapeutics to treat core ASD symptoms, small animal models must be validated for preclinical screens. This study examined the preclinical utility of two inbred mouse strains, BALB/cByJ and C58/J, that exhibit phenotypes relevant to core ASD symptoms. Mice from both strains were intraperitoneally administered oxytocin, using either acute or sub-chronic regimens. Acute oxytocin did not increase sociability in BALB/cByJ; however, sub-chronic oxytocin had significant prosocial effects in both BALB/cByJ and C58/J. Increased sociability was observed 24 h following the final oxytocin dose in BALB/cByJ, while prosocial effects of oxytocin emerged 1-2 weeks post-treatment in C58/J. Furthermore, acute oxytocin decreased motor stereotypy in C58/J and did not induce hypoactivity or anxiolytic-like effects in an open field test. This study demonstrates that oxytocin administration can attenuate social deficits and repetitive behavior in mouse models of ASD, dependent on dose regimen and genotype. These findings provide validation of the BALB/cByJ and C58/J models as useful platforms for screening novel drugs for intervention in ASDs and for elucidating the mechanisms contributing to the prosocial effects of oxytocin.

Impaired Sociability and Cognitive Function in Nrcam-null Mice

NRCAM (Neuronal Cell Adhesion Molecule) has an important role in axonal guidance and the organization of neural circuitry during brain development. Association analyses in human populations have identified NRCAM as a candidate gene for autism susceptibility. In the present study, we evaluated Nrcam-null mice for sociability, social novelty preference, and reversal learning as a model for the social deficits, repetitive behavior, and cognitive rigidity characteristic of autism. Prepulse inhibition of acoustic startle responses was also measured, to reflect sensorimotor-gating deficits in autism spectrum disorders. Assays for anxiety-like behavior in an elevated plus maze and open field, motor coordination, and olfactory ability in a buried food test were conducted to provide control measures for the interpretation of results. Overall, the loss of Nrcam led to behavioral alterations in sociability, acquisition of a spatial task, and reversal learning, dependent on sex. In comparison to male wild type mice, male Nrcam-null mutants had significantly decreased sociability in a three-chambered choice task. Low sociability in the male null mutants was not associated with changes in anxiety-like behavior, activity, or motor coordination. Male, but not female, Nrcam-null mice had small decreases in prepulse inhibition. Nrcam deficiency in female mice led to impaired acquisition of spatial learning in the Morris water maze task. Reversal learning deficits were observed in both male and female Nrcam-null mice. These results provide evidence that NRCAM mediates domains of function relevant to symptoms observed in autism.

The Mouse Universal Genotyping Array: From Substrains to Subspecies

Genotyping microarrays are an important resource for genetic mapping, population genetics, and monitoring of the genetic integrity of laboratory stocks. We have developed the third generation of the Mouse Universal Genotyping Array (MUGA) series, GigaMUGA, a 143,259-probe Illumina Infinium II array for the house mouse (Mus musculus). The bulk of the content of GigaMUGA is optimized for genetic mapping in the Collaborative Cross and Diversity Outbred populations, and for substrain-level identification of laboratory mice. In addition to 141,090 single nucleotide polymorphism probes, GigaMUGA contains 2006 probes for copy number concentrated in structurally polymorphic regions of the mouse genome. The performance of the array is characterized in a set of 500 high-quality reference samples spanning laboratory inbred strains, recombinant inbred lines, outbred stocks, and wild-caught mice. GigaMUGA is highly informative across a wide range of genetically diverse samples, from laboratory substrains to other Mus species. In addition to describing the content and performance of the array, we provide detailed probe-level annotation and recommendations for quality control.