in vivo imaging of circuit remodeling in mouse visual cortex

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Many brain disorders manifest impaired synaptic integrity, stability, and experience-dependent selection, resulting in wiring deficits and perturbed function. Unfortunately, our ability to monitor synaptic or circuit failures as they occur has been hindered by the difficulty of visualizing synapses in vivo. Here we propose in vivo monitoring of the ‘order of operations’ in synapse formation and elimination, and identifying the steps and molecules controlling experience-dependent synapse selection. We focus on the visual system, where there is a well-characterized toolkit for manipulating experience. We hypothesize that the dynamics of a synapse's assembly and disassembly, and its propensity to remodel, are intimately linked to its connection identity and proteomic content. To test this, we propose the following aims: Aim1: To track the structural remodeling of inhibitory synapses and how it relates to their afferent input specificity and proteomic content. We will label Somatostatin and Parvalbumin inputs onto the full dendritic arbor of single L2/3 pyramidal neurons in mouse visual cortex, track their daily dynamics and their response to monocular deprivation, and analyze their proteomic content in relation to dynamic history and afferent identity. To this purpose, we will implement triple color two- photon microscopy to simultaneously track, in vivo, both pre- and postsynaptic elements of inhibitory synapses, followed by Magnified Analysis of Proteome (MAP), a combination of tissue clearing and expansion microscopy, for super resolution analysis of synaptic protein content across the entire neuron. Aim 2: To track the structural remodeling of excitatory synapses and how it relates to their afferent input specificity and proteomic content. Using a similar strategy as in Aim 1, we will discriminate general thalamic, LGN, and LP inputs to excitatory synapses across the arbor of L2/3 pyramidal neurons, track their daily dynamics and response to dark adaptation, and analyze their proteomic content in relation to dynamic history and afferent identity. Aim 3: To dissect, at a molecular level, experience-dependent selection and stabilization of excitatory synapses. CPG15/neuritin is an activity-regulated gene product critical for synapse stabilization and maturation. In vivo imaging in WT and CPG15 knockout mice revealed that while spine formation occurs normally in the absence of visual experience or CPG15, in both cases PSD95 recruitment to nascent spines is deficient. CPG15 expression in the absence of activity is sufficient to restore normal PSD95 recruitment and spine stabilization, suggesting it acts as an activity-dependent synapse selector. We ask how CPG15 loss impacts molecular events in synapse formation and maturation. Aim 4: To develop and implement spectrally resolved two-photon microscopy for simultaneous tracking of four distinct genetically encoded fluorophores marking different cellular proteins. We will develop new labeling and two-photon microscope configurations for in vivo monitoring of up to four synaptic components at once, with options for addressing a variety of experimental questions.