UC Riverside

Proper establishment of connectivity in the nervous system is essential for higher cognitive functions. Progressive and regressive developmental events build and sculpt these circuits to reach functional maturity. A key progressive step during the assembly of neural circuits is the guidance of growing axons. The direction that an axon grows is largely influenced by the degree of adhesion to the extracellular matrix (ECM) substrate, which is locally regulated in response to guidance cues. Adhesion must also be regulated during fasciculation into axon bundles and dissociation for innervation of individual targets. The modulation of adhesion by guidance cues is mediated by intracellular cascades of signaling proteins, of which very little is known. In Aim 1, we provide genetic evidence for the requirement of Cas signaling adaptor proteins in guidance and fasciculation of DRG projections in mice. Our results demonstrate that Cas proteins play important roles in allowing sensory axons to distinguish between adhesion to the substrate and to other axons.A critical regressive event during the refinement of neural circuits is the pruning of exuberant synapses and projections. Activity-dependent pruning ensures that only the most functional connections are maintained. Although clearly important for proper connectivity, this developmental process is understudied. An apparent roadblock in the investigation of these refinement events is the need for tools that are able to reversibly modulate neuronal activity for a protracted period of time. In Aim 2, we offer a solution with the design and characterization of the Expression by Boolean Exclusion (ExBoX) system, a simple AAV-based approach to control expression by exclusion logic (AND NOT). This ExBoX system encodes for a gene of interest which is turned ON by a particular recombinase (Cre or FlpO) and turned OFF by another. We show the ability of the ExBoX system to tightly regulate expression of fluorescent reporters both in vitro and in vivo, and demonstrate the adaptability of the system by achieving expression of a variety of virally-delivered functional manipulations in the mouse brain. This simple strategy will expand the molecular toolkit available for cell- and time-specific gene expression.