A Small Subset of Fruitless Subesophageal Neurons Modulate Early Courtship in Drosophila

Baker BS (2014) A Small Subset of Fruitless Subesophageal Neurons Modulate Early Courtship in Drosophila. PLoS ONE 9(4): e95472. doi:10.1371/journal.pone.0095472 A Small Subset of Fruitless Subesophageal Neurons Modulate Early Courtship in Drosophila Frederic Marion-Poll, AgroParisTech, France 0 1 Department of Biological Sciences, San Jose State University. San Jose, California, United States of America, 2 Janelia Farm Research Campus, HHMI , Ashburn, Virginia , United States of America We show that a small subset of two to six subesophageal neurons, expressing the male products of the male courtship master regulator gene products fruitlessMale (fruM), are required in the early stages of the Drosophila melanogaster male courtship behavioral program. Loss of fruM expression or inhibition of synaptic transmission in these fruM(+) neurons results in delayed courtship initiation and a failure to progress to copulation primarily under visually-deficient conditions. We identify a fruM-dependent sexually dimorphic arborization in the tritocerebrum made by two of these neurons. Furthermore, these SOG neurons extend descending projections to the thorax and abdominal ganglia. These anatomical and functional characteristics place these neurons in the position to integrate gustatory and higher-order signals in order to properly initiate and progress through early courtship. - Drosophila melanogaster courtship is a multi-step, progressive series of behaviors established by sex-specific genetic and neurobiological components [14]. Work by our lab and others demonstrated that the expression of male products of the gene fruitless (fruM) is both necessary and sufficient to specify the potential for male courtship behavior. Approximately 2000 neurons in the central nervous system (roughly 2% of the CNS neuronal population) express FruM in clusters scattered throughout the central (CNS) and peripheral nervous systems [57]. In the periphery, expression was detected in subsets of primary sensory neurons of the sensory modalities implicated in courtship. Strikingly, fruM(+) neurons are dedicated to courtship as inactivating them disrupts courtship behaviors, but has no discernible effect on non-sexual behaviors. Both fruitlessMale and another component of the sex determination pathway, doublesexMale are involved in establishment of sexually dimorphic neural circuitry [8]. Activity of FruM is required for the survival of several male-specific neurons or sexually dimorphic projection patterns [9]. Multiple female sensory cues combine to regulate the activation and performance of male courtship behaviors. A feature of these multi-modal sensory inputs is the partial redundancy of some modalities: loss of visual, olfactory, or gustatory perception does not block courtship from occurring Dros. melanogaster [1012]. Instead loss of any one of these three modalities delays the initiation and decreases the quantity of courtship. These functional redundancies suggest a compensatory integration of these multiple pathways in the courtship circuitry. Several areas of the CNS have been identified as regions of higher-order processing and integration in the courtship circuitry [1319]. fruM(+) projections densely innervate several regions: the lateral protocerebral complex, the mushroom bodies, the mesothoracic triangle in the ventral nerve cord, and the tritocerebral loop. Neurons projecting to the lateral protocerebral complex and mesothoracic triangle induce wing song behavior; subsets of these neurons require FruM and DsxM for survival in males [9]. The mushroom bodies are well-characterized regions controlling memory and learning. One area of interest is the tritocerebral loopwhich lies just ventral to the subesophageal ganglion (SOG) an area of dense innervations targeted by gustatory, protocerebral/neurosecretory, and stomatogastric inputs [20]. Peripheral gustatory axons, from the mouthparts, subsets of the labellum, and stomatogastric nerves, target the tritocerebrum. The termini of descending tracts from the medial superior protocerebrumnotably the pars interecerebralis, a neurosecretory centerinnervate the dorsal tritocerebrum. The higher-order interneurons that process and regulate gustatory inputs have not been fully characterized; the tritocerebral loop innervations likely integrate chemosensory and protocerebral inputs. Here we targeted subpopulations of fruM(+) neurons that regulate chemosensory-dependent courtship initiation. We screened 72 P[GawB] insertions, driving an RNAi construct targeting fruM, UAS-fruMIR [21], for courtship defects that appear only under conditions where melanogaster is visually deficient [10]. The P[GawB]4-57 line exhibited very limited overlap with fruM(+) neurons. P[GawB]4-57 mainly overlapped with two to six fruM(+) neurons in the subesophageal ganglion (SOG), two clusters in the ventral nerve cord (VNC), and inconsistently an area just medial to the antennal lobe (mAL). Knockdown of fruM or inhibition of synaptic fusion limited to the SOG neurons resulted in infrared-specific courtship delays, and a failure to progress to copulatory behaviors. Strikingly, the tritocerebral projections of these neurons were significantly more extensive in males than in females; this male-specific projection pattern required FruM expression. These fruM(+) SOG neurons likely integrate chemosensory inputs in the tritocerebrum to modulate the initiation and progression of courtship. We identified subpopulations of fruM(+) neurons involved in chemosensory-specific pathways via a behavioral screen for proper courtship initiation in visually-deficient conditions. To do this we built on the findings of Meissner et al. [21] who screened a collection of approximately 1000 Gal4 P-element, P[GawB], insertions driving expression of two copies of a fruM RNAi construct, UAS-fruMIR (one insertion on the 2nd and one insertion on the third chromosomes). We screened 65 P[GawB] and 7 candidate Gal4 lines for significant courtship delays to the first unilateral wing extension (courtship latency) in ambient and infrared light (Figure 1A). Due to lower visual resolution in infrared, we could not reliably identify the first instance of orientation/pursuit, the traditional method of measuring courtship latency. Henceforth courtship latency will refer to the average time to first unilateral wing extension. We targeted lines with infrared-dependent courtship delays and CNS-specific, limited Gal4:fruLexA overlap (Figure 1B) in order to identify candidate fruM(+) interneurons that regulate the activation of courtship. fruLexA is a knock-in insertion of the LexA transcriptional activator that drives expression of LexaOp-GFPnls in fruM(+) cells [21]. Three lines matched those criteria (Figure 1C). One line, P[GawB]4-57, drove Gal4 expression that overlapped fruLexA(+) neurons in only four fruM(+) cell clusters, clusters 1, 7, 8, and 13 in Figure C (Cluster nomenclature from [5]) with extensive ov (...truncated)