Hosted by the Wellcome Neural Dynamics PhD Programme
Synaptic plasticity is a key process for brain function and memory formation. This depends on the ability of neurons to remodel their synaptic proteomes with high temporal and spatial resolution, promptly delivering and degrading the right proteins at the correct locations. Decades of work have shown that local protein synthesis at synapses is one of the mechanisms facilitating remodeling of the local proteome. In vitro and ex vivo studies have shown that neurons locally translate thousands of mRNAs, and that this process is required for several forms of long-term synaptic plasticity. In order to understand the role of local translation in physiological brain function and learning, one must to study this phenomenon in vivo, in an intact neuronal circuit. Here, we present our efforts to visualize directly the protein synthesis dynamics from mRNAs in awake mice. We focus on cortical layer 1, a structure critical for learning that is devoid of neuronal cell bodies but enriched in their dendrites and axons. We show that layer 1 is metabolically active and contains more than 2600 neuronal transcripts. In order to visualize local translation in layer 1 dynamically, we created translation reporters by fusing the mRNA encoding a fast-folding fluorescent protein to the 3’untranslated regions of two synaptic transcripts, Beta actin and Psd95. We sparsely express these reporters in layer 5 excitatory neurons and image their dendrites in layer 1, approximately 400 m away from their cell bodies. Using 2-photon imaging and fluorescence recovery after photobleaching (FRAP), we detect a rapid (protein synthesis dependent) emergence of reporter fluorescence in layer 1 dendrites. With this novel toolset, for the first time, we visualize protein synthesis in intact brains in awake mice and are in a position to study its functional relevance in future studies.
If joining online: https://bristol-ac-uk.zoom.us/j/94138286231?pwd=MlRURE1SWjR6OTZCR1Fnak9QbGxhUT09, Meeting ID: 941 3828 6231, Passcode: 277162