Elucidating the pattern of connections between neurons is key to understanding nervous system function and dysfunction. Yet, conventional methods to delineate brain circuitry have been limited by their invasiveness, lack of reproducibility, and lack of cell specificity. Similarly limited are methods for modulating neuronal activity, specifically those methods essential for delineating relationships between particular circuits and perception or behavior. Our goal is to address these technical gaps by harnessing the power afforded by genetics -- we propose engineering transgenic mice bearing conditional alleles encoding transneuronal tracers, optical reporters, and/or neuromodulators that, in conjunction with the hundreds of region/cell-specific Cre and Flpe recombinase mice presently (and soon to be) available, will establish a set of enabling reagents for constructing functional connectivity maps of unprecedented resolution. The proposed genetic tools incorporate a high resolution dual recombinase (Cre and FIp)-mediated transgene activation paradigm pioneered recently by my lab in which selectivity can be improved by orders of magnitude relative to that achieved using conventional transgene (or even single recombinase) expression systems, while also providing great versatility. Using this system, tracer or modulator expression can be conditionally activated in as selective a group of neurons as possible so that circuits may be studied in virtual isolation; moreover, the tools are sufficiently versatile to permit any selected circuit to be studied at any stage. Using our strategy, in Aim 1 we generate three sets of transgenics for transneuronal tracing: one set for conditionally visualizing virtually any chain of neural connections in the direction of circuit flow (anterograde); a second set for delineating connections opposite to circuit flow (retrograde); and a third set (informed by the first two) for visualizing simultaneously both anterograde and retrograde connections.
In Aim 2, we generate a similar set of selective, versatile, and conditional transgenics, but rather than having the capacity to trace connections, these tools permit neuronal silencing by suppressing either synaptic transmission or electrical conductance.
In Aim 3, we validate the efficacy of these tools by applying them along with specific Cre- or Flpe-expressing mice to trace and modulate the well-defined neural pathways of the visual system. Additionally, all of these strains permit the simultaneous visualization of plastic changes in neuron morphology and facilitate integration of neurophysiological studies with the generated maps. Completion of these aims will provide tools that can be used by numerous investigators with different sets of recombinase-expressing mice thereby leveraging the work from this grant into a collective and comprehensive circuitry mapping effort.
