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内容記述 |
Higher brain functions such as learning and memory are governed by neurotransmitter receptors at synapses. Establishing causal links between receptor function and behavior requires tools that enable precise manipulation of endogenous receptors in vivo. Genetic loss-of-function approaches enable brain region- and cell-type-specific manipulation of receptor function. However, these approaches lack adequate temporal resolution for the analysis of receptor function and often cause severe phenotypes. In contrast, pharmacological approaches enable reversible and acute inhibition of receptor function but lack spatial specificity. To overcome these limitations, we develop a novel chemogenetic method that enables high spatiotemporal control of native receptors. This approach employs a designer inhibitor and an inhibitorinsensitive mutant receptor pair to enable transient and cell-type-specific inhibition of endogenous receptor signaling in vivo, while preserving intrinsic function of the target receptor.As a proof of concept, we focused on metabotropic glutamate receptor subtype 1 (mGlu1), a G protein–coupled receptor implicated in motor function. Previous mGlu1-knockout studies in mice have shown severe motor defects, precluding detailed behavioral analysis. Thus, a new strategy that enables selective and reversible inhibition of mGlu1 without disrupting its physiological function is required. Based on the X-ray crystal structure of mGlu1 in complex with FITM, an mGlu1-selective inhibitor, we designed FITM derivatives and mGlu1 mutants. Cell-based assays identified a designer inhibitor–mutant receptor pair that selectively inhibits wild-type mGlu1 without affecting the mutant receptor, while preserving its responsiveness to glutamate. To extend this strategy in vivo, we generated genetically modified mice that express the inhibitor-insensitive mutant mGlu1 throughout the brain but retain wild-type mGlu1 in cerebellar Purkinje cells. Administration of the designer inhibitor to these mice enabled selective inhibition of mGlu1 in Purkinje cells. Behavioral analyses using accelerated rotarod tests revealed that cerebellar mGlu1 is essential for motor learning during developmental stages. Taken together, these results demonstrate that this novel chemogenetic approach enables causal dissection of receptor function in defined neural circuits in vivo. |
