Spinal cord interneurons play critical roles shaping motor output, but their precise identity and connectivity remain unclear. Focusing on the V1 interneuron cardinal class we defined four major V1 subsets in the mouse according to neurogenesis, genetic lineage-tracing, synaptic output to motoneurons, and synaptic inputs from muscle afferents. Sequential neurogenesis delineates different V1 subsets: two early born (Renshaw and Pou6f2) and two late born (Foxp2 and Sp8). Early born Renshaw cells and late born Foxp2-V1 interneurons are tightly coupled to motoneurons, while early born Pou6f2-V1 and late born Sp8-V1 interneurons are not, indicating that timing of neurogenesis does not correlate with motoneuron targeting. V1 clades also differ in cell numbers and diversity. Lineage labeling shows that the Foxp2-V1 clade contains over half of all V1 interneurons, provides the largest inhibitory input to motoneuron cell bodies, and includes subgroups that differ in birthdate, location, and proprioceptive input. Notably, one Foxp2-V1 subgroup, defined by postnatal Otp expression, is positioned near the LMC and receives substantial input from proprioceptors, consistent with an involvement in reciprocal inhibitory pathways. Combined tracing of ankle flexor sensory afferents and interneurons monosynaptically connected to ankle extensors confirmed placement of Foxp2-V1 interneurons in reciprocal inhibitory pathways. Our results validate previously proposed V1 clades as unique functional subtypes that differ in circuit placement, with Foxp2-V1 cells forming the most heterogeneous subgroup. We discuss how V1 organizational diversity enables understanding of their roles in motor control, with implications for their diverse ontogenetic and phylogenetic origins.

Monitoring neuronal activity at single-cell resolution in freely moving Drosophila engaged in social behaviors is challenging because of their small size and lack of transparency. Extant methods, such as Flyception, are highly invasive. Whole-brain calcium imaging in head-fixed, walking flies is feasible but the animals cannot perform the consummatory phases of social behaviors like aggression or mating under these conditions. This has left open the fundamental question of whether neurons identified as functionally important for such behaviors using loss- or gain-of-function screens are actually active during the natural performance of such behaviors, and if so during which phase(s). Here, we perform brain-wide mapping of active cells expressing the Immediate Early Gene hr38 using a high-sensitivity/low background fluorescence in situ hybridization (FISH) amplification method called HCR-3.0. Using double-labeling for hr38 mRNA and for GFP, we describe the activity of several classes of aggression-promoting neurons during courtship and aggression, including P1^a cells, an intensively studied population of male-specific interneurons. Using HI-FISH in combination with optogenetic activation of aggression-promoting neurons (opto-HI-FISH), we identify candidate downstream functional targets of these cells in a brain-wide, unbiased manner. Finally, we compare the activity of P1^a neurons during sequential performance of courtship and aggression, using intronic vs. exonic hr38 probes to differentiate newly synthesized nuclear transcripts from cytoplasmic transcripts synthesized at an earlier time. These data provide evidence suggesting that different subsets of P1^a neurons may be active during courtship vs. aggression. HI-FISH and associated methods may help to fill an important lacuna in the armamentarium of tools for neural circuit analysis in Drosophila.