Authors:

Zachary Conroy

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About Paper:

Structural neuroplasticity is the physical remodeling of synapses in the nervous system:. Key elements of structural plasticity are dendritic spines — small, compartmentalized membrane continuum of dendrites. Structural plasticity involves a dynamic change in the number, density, and morphology of these dendritic spines, which directly affect synaptic strength'. Neuroplasticity is central in neurodevelopment and learning, while aberrations are associated with a variety of neurological disorders*. Despite its importance, approaches for quantifying structural neuroplasticity are limited and face difficulties either in scalability or in capturing the nuances of plasticity networks®. To address this, the Kozorovitskiy lab has developed a genetically encoded, nanoluciferase-based neuroplasticity biosensor which reports activity-dependent structural remodeling in an accurate, direct, scalable, and highly efficient manner. However, the current sensor lacks the neuron subtype specificity required to analyze differences in neuroplasticity across neuronal subclasses. This project focuses on creating and validating a next-gen cre-dependent version of the neuroplasticity biosensor, which would enable neuronal-subclass specific expression. We hypothesized that cre-dependent design of the biosensor would preserve the sensor's efficacy and range while enabling neuronal-subclass specific expression. After successfully inserting loxp sites around nanoluciferase and a V5 tag using Gibson assembly cloning, the new next-gen sensor was packaged in-house in an AAV (adeno-associated virus) and was validated using a variety of experiments in HT22 cells (mouse hippocampal cell line). The presence of the sensor was confirmed through immunocytochemistry and luciferase activity. Luminescence signals between the cre-dependent biosensor and the original biosensor were measured in response to known plasticity modulator, Forskolin, and DMSO as negative control and the sensor types were found to have comparable sensitivity and effectiveness. The next-gen sensor presents a unique opportunity to dissect plasticity across micro-circuit components in different neuromodulators and complex behaviors.

Source:

Northwestern University

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Co-authors:

Zachary Conroy