Abstract: A functional neural circuit consists of a group of connected neurons that collaborate to execute a specific function of the brain. The understanding of the molecular mechanisms that underlie the development, maintenance and experience-dependent modification of functional neural circuits is incomplete due to limitations of existing methods. I propose to generate a transgenic mouse that addresses these limitations by exploiting two recent methodological advances. The first advance is the TetTag mouse, which is a transgenic mouse that can be used to genetically tag a single functional neural circuit. This tag enables the selective molecular analysis of neurons that have a shared function. This method is more sensitive to changes within a single functional neural circuit than other available methods, which have to rely on spatial criteria and thereby include neurons that do not participate in the circuit of interest. The second advance is the Translating Ribosome Affinity Purification (TRAP) method, which enables purification of actively translated messenger RNA from a genetically defined group of neurons. TRAP analysis reflects changes at both the transcriptional and translational level, while other available methods only detect transcriptional changes. I will combine TetTag and TRAP within a single transgenic mouse to generate the first tool that enables the comprehensive analysis of all translational events within a single functional neural circuit. Neurons tagged with the TetTag mouse during fear conditioning provide a stable neural correlate of the fear memory. I will use the TetTag/TRAP mouse to purify actively translated messenger RNA from these tagged neurons in order to detect the protein synthesis events that underlie the storage of a memory. The TetTag/TRAP mouse can be used for the molecular analysis of various functional neural circuits, including those involved in memory, addiction, epilepsy, circadian rhythms, spinal cord regeneration, pain, brain development, and neuronal cell death. Public Health Relevance: The proposed research will lead to a better understanding of how the synthesis of new proteins helps neurons in the brain to work together and execute the functions of the brain. This increased understanding can be used to develop therapies for various brain diseases, including memory impairment, addiction and epilepsy.
