Autism is a neurodevelopmental disorder that affects approximately 0.6% of the U.S. population. The cause remains unknown. Lifetime costs of caring for autistic individuals are high, both in terms of a) financial expenses to the families, educational systems, and health care agencies;and b) quality of life for the affected individuals and their families. Strong evidence for genetic causes has spurred the search for genes mediating each of the three diagnostic symptoms of autism. Genetic mouse models offer an ideal experimental strategy to evaluate the consequences of candidate gene mutations. Treatments for the core symptoms of autism are currently limited to early behavioral interventions. Discovery of effective pharmacological treatments requires a greater understanding of the genes causing autism, and appropriate animal models to test the efficacy and safety of proposed treatments. Our Laboratory of Behavioral Neuroscience (LBN) is an international leader in behavioral assays for transgenic and knockout mice with mutations in genes expressed in brain pathways involved in neuropsychiatric disorders. Current focus is on genetic mouse models of autism. We collaborate with a large number of molecular genetics laboratories that contribute mutant lines of mice to our research program. Collaborative experiments in FY2009 included behavioral testing of shank1 knockout mice generated by Morgan Sheng at the Massachusetts Institute of Technology, of neuroligin-4 knockout mice generated by Nils Brose at the Max Planck Institute, and of COMT and dysbindin knockout mice with Danny Weinberger's CBDB group, IRP, NIMH. This project began in 2002 when Dr. Crawley initiated approaches to develop mouse behavioral tasks relevant to the symptoms of autism. She started by learning about the defining behavioral features of autism through a collaboration with Dr. Joseph Piven at the University of North Carolina Center for Autism Research, and through interactions with Dr. Susan Swedo's autism research program at NIMH and other clinical experts, as well as through ongoing participation in international autism conferences and workshops. Opportunities for members of LBN to observe autistic children in classrooms and in videotaped interviews have greatly enhanced our understanding of the diagnostic symptoms of autism to be modeled in mice. During 2006-2009, we discovered autism-relevant phenotypes of the inbred mouse strain BTBR T+tf/J (BTBR), and investigated the biological mechanisms underlying these phenotypes. Lack of sociability was confirmed in multiple independently bred cohorts, on multiple social tasks including juvenile play, adult social approach, adult reciprocal social interactions, and social transmission of food preference (McFarlane et al., 2008), relevant to the first diagnostic symptom of autism, abnormal social interactions. High levels of repetitive self-grooming were routinely detected in cohorts of BTBR (Yang et al., 2007a,b, 2009;McFarlane et al., 2008), relevant to the third diagnostic symptom of autism, repetitive behaviors. These phenotypes were identical when mice were raised on a conventional circadian cycle and tested in the light, versus on a reverse light cycle and tested in the dark phase of their circadian cycle, when mouse social behaviors are maximal (Moy et al., 2007;Yang et al., 2007a;McFarlane et al., 2008). The absence of a corpus callosum in BTBR is unlikely to explain their phenotypes, as corpus callosum lesions did not affect social behaviors in C57BL/6J (B6), a standard control strain of mice with high sociability (Yang et al., 2009). Social deficits and repetitive self-grooming in BTBR mice appear to be unrelated to maternal care factors, as confirmed by postnatal cross-fostering between BTBR and B6 (Yang et al., 2007b). Unusual pattterns of vocalizations were detected in BTBR as compared to control inbred strains B6, FVB/NJ, and 129X1/SvJ (Scattoni et al., 2008). Reduced social olfactory scent-marking and reduced vocalizations to pheromonal scent-marks were detected in BTBR as compared to B6 (Roullet et al., in preparation;Wohr et al., in preparation). FY2009 experiments addressed genetic and neurochemical mechanisms responsible for the autism-like phenotypes in BTBR. In collaboration with Dr. Elliott Sherr, University of California San Francisco, we began a quantitative trait loci linkage analysis (QTL) of the BTBR x B6 cross, designed to discover genes in the BTBR background that correlate with autism-like phenotypes. The first 100 F2 mice were tested behaviorally by LBN postdoctoral fellow Mu Yang, postbaccalaureate Mike Weber, and HHMI student intern Kayla Perry. Preliminary findings have encouraged our pursuit of the next 300 mice to complete this QTL. Drs. Mike Tyszka and Ralph Adolphs at the California Institute of Technology have joined this collaboration to conduct tractography of neuroanatomical pathways in BTBR versus B6, and in the QTL F2 mice selected from the extremes of the behavioral distributions. Stress-related hormones that might cause repetitive self-grooming are being assayed in BTBR by Dr. Jim Koenig at the University of Maryland Psychiatric Research Center. Oxytocin receptors and peptide levels are being assayed by Dr. Scott Young and his postdoctoral felllow Abbe Macbeth, IRP, NIMH, and by Dr. Koenig's group. Our Senior Laboratory Manager Dr. Jill Silverman is conducting parts of these experiments and coordinating the collaborations with Drs. Koenig and Young. A second approach toward identifying the genes mediating the diagnostic symptoms of autism is to evaluate the behaviors of mice with experimentally targeted mutations in candidate genes for autism. In FY2009, we began phenotyping experiments in mice with mutations in four candidate genes for autism: neuroligin-2, neuroligin-4, shank1, and shank3. Neuroligins and shanks are synaptic development genes for which mutations have been detected, each in a small number of autistic individuals. Social and repetitive behavioral assays, and control measures of general health, are being conducted by Drs. Yang and Silverman, postbaccalaureates Adam Katz and Sarah Turner, and HHMI student interns Leuk Woldeyohannes and Dieynaba Diagne. Postdoctoral fellow Markus Wohr and Postbaccalaureate Mark Harris are analyzing vocalizations in social settings. Postdoctoral fellow Florence Roullet and Postbaccalaureate Roheeni Saxena are analyzing social olfactory cues and responses. The third component of our mouse models of autism project is the translational evaluation of proposed treatments. The robust and highly replicated social deficits and repetitive self-grooming in BTBR provide a good model system for testing the ability of drugs and behavioral treatments to reverse and prevent autism-like symptoms. During FY2009, Dr. Silverman, Postbaccalaureate Charlotte Barkan, and HHMI student intern Seda Tolu completed dose-response curves for risperidone and MPEP. Risperidone, the first drug approved by the FDA for autism, primarily for the irritability symptom, blocked repetitive self-grooming in BTBR, but only at sedative doses. MPEP, an mGluR5 antagonist reported to reverse phenotypes in Fragile X mice, blocked repetitive self-grooming in BTBR at doses that were not sedative. Dr. Yang, Postbaccalaureate Mike Weber, and HHMI student intern Kayla Perry discovered a behavioral intervention, raising BTBR juveniles with B6 juveniles, that improved adult sociability in BTBR. These treatment studies provide proof of principle for the usefulness of mouse models of autism in preclinical discovery of novel therapeutics for autism.
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