An intersectional viral-genetic method for fluorescent tracing of axon collaterals reveals details of noradrenergic locus coeruleus structure

Abstract

Understanding the function of broadly projecting neurons depends on comprehensive knowledge of the distribution and targets of their axon collaterals. While retrograde tracers and, more recently, retrograde viral vectors have been used to identify efferent projections, they have limited ability to reveal the full pattern of axon collaterals from complex, heterogeneous neuronal populations. Here we describe TrAC (Tracing Axon Collaterals), an intersectional recombinase-based viral-genetic strategy that allows simultaneous visualization of axons from a genetically defined neuronal population and a projection-based subpopulation. To test this new method, we have applied TrAC to analysis of locus coeruleus norepinephrine (LC-NE)-containing neurons projecting to medial prefrontal cortex (mPFC) and primary motor cortex (M1) in laboratory mice. TrAC allowed us to label each projection-based LC-NE subpopulation, together with all remaining LC-NE neurons, in isolation from other noradrenergic populations. This analysis revealed mPFC- and M1-projecting LC-NE subpopulations differ from each other and from the LC as a whole in their patterns of axon collateralization. Thus, TrAC complements and extends existing axon tracing methods by permitting analyses that have not previously been possible with complex genetically defined neuronal populations.

SIGNIFICANCE STATEMENT We have developed a new method for mapping axon collaterals of genetically defined neuronal subtypes. TrAC uses an intersectional genetic strategy to define a cell population of interest, and a retrograde viral construct to label a subpopulation based on its axonal projections. This method has three major benefits: 1) only one viral injection is required for labeling, 2) axons from a projection-based subpopulation can be visualized together with a broader genetically defined population, and 3) the cell population of interest can be defined by transient developmental genetic information. Our proof-of-principle analysis of noradrenergic locus coeruleus projections to the forebrain extends previous analyses and reveals new details of this complex system.