Hundreds of genes are mutated in non-syndromic intellectual disability (ID) and autism spectrum disorder (ASD), with each gene often involved in only a handful of cases. Such heterogeneity can be daunting, but rare recessive loss of function (LOF) mutations can be a good starting point to provide insight into the mechanisms of neurodevelopmental disease. Biallelic LOF mutations in the signaling scaffold CC2D1A cause a rare form of autosomal recessive ID, sometimes associated with ASD and seizures. In parallel, we recently reported that Cc2d1a-deficient mice present with cognitive and social deficits, hyperactivity and anxiety. In Drosophila, loss of the only ortholog of Cc2d1a, lgd, is embryonically lethal, while in vertebrates, Cc2d1a has a homolog Cc2d1b which appears to be compensating, indicating that Cc2d1a and Cc2d1b have a redundant function in humans and mice. Here, we generate an allelic series of Cc2d1a and Cc2d1b LOF to determine the relative role of these genes during behavioral development. We generated Cc2d1b knockout (KO), Cc2d1a/1b double heterozygous and double KO mice, then performed behavioral studies to analyze learning and memory, social interactions, anxiety, and hyperactivity. We found that Cc2d1a and Cc2d1b have partially overlapping roles. Overall, loss of Cc2d1b is less severe than loss of Cc2d1a, only leading to cognitive deficits, while Cc2d1a/1b double heterozygous animals are similar to Cc2d1a-deficient mice. These results will help us better understand the deficits in individuals with CC2D1A mutations, suggesting that recessive CC2D1B mutations and trans-heterozygous CC2D1A and CC2D1B mutations could also contribute to the genetics of ID.

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This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different forms of learning. Specifically, viral-mediated delivery is used to express a genetically modified inhibitory G-protein coupled receptor (the Designer Receptor) into a discrete brain region in the rodent. Three weeks later, when designer receptor expression levels are high, a pharmacological agent (the Designer Drug) is administered systemically 30 min prior to a specific behavioral session. The drug has affinity for the designer receptor and thus results in inhibition of neurons that express the designer receptor, but is otherwise biologically inert. The brain region remains silenced for 2-5 hr (depending on the dose and route of administration). Upon completion of the behavioral paradigm, brain tissue is assessed for correct placement and receptor expression. This approach is particularly useful for determining the contribution of individual brain regions to specific components of behavior and can be used across any number of behavioral paradigms. Video LinkThe video component of this article can be found at

No abstract

Hundreds of genes are mutated in non-syndromic intellectual disability (ID) and autism spectrum disorder (ASD), with each gene often involved in only a handful of cases. Such heterogeneity can be daunting, but rare recessive loss of function (LOF) mutations can be a good starting point to provide insight into the mechanisms of neurodevelopmental disease. Biallelic LOF mutations in the signaling scaffold CC2D1A cause a rare form of autosomal recessive ID, sometimes associated with ASD and seizures. In parallel, we recently reported that Cc2d1a-deficient mice present with cognitive and social deficits, hyperactivity and anxiety. In Drosophila loss of the only ortholog of Cc2d1a, lgd, is embryonic lethal, while in vertebrates Cc2d1a has a homolog Cc2d1b which appears to be compensating, indicating that Cc2d1a and Cc2d1b have redundant function in humans and mice. Here, we generate an allelic series of Cc2d1a and Cc2d1b loss of function to determine the relative role of these genes during behavioral development. We generated Cc2d1b knockout (KO), Cc2d1a/1b double heterozygous and double KO mice, then performed behavioral studies to analyze learning and memory, social interactions, anxiety, and hyperactivity. We found that Cc2d1a and Cc2d1b have partially overlapping roles. Overall, loss of Cc2d1b is less severe than loss of Cc2d1a, only leading to cognitive deficits, while Cc2d1a/1b double heterozygous animals are similar to Cc2d1a-deficient mice. These results will help us better understand the deficits in individuals with CC2D1A mutations, suggesting that recessive CC2D1B mutations and trans-heterozygous CC2D1A and CC2D1B mutations could also contribute to the genetics of ID.