Functional Dissection of the Neural Substrates for Sexual Behaviors in Drosophila melanogaster

The male-specific Fruitless proteins (FruM) act to establish the potential for male courtship behavior in Drosophila melanogaster and are expressed in small groups of neurons throughout the nervous system. We screened ∼1000 GAL4 lines, using assays for general courtship, male–male interactions, and male fertility to determine the phenotypes resulting from the GAL4-driven inhibition of FruM expression in subsets of these neurons. A battery of secondary assays showed that the phenotypic classes of GAL4 lines could be divided into subgroups on the basis of additional neurobiological and behavioral criteria. For example, in some lines, restoration of FruM expression in cholinergic neurons restores fertility or reduces male–male courtship. Persistent chains of males courting each other in some lines results from males courting both sexes indiscriminately, whereas in other lines this phenotype results from apparent habituation deficits. Inhibition of ectopic FruM expression in females, in populations of neurons where FruM is necessary for male fertility, can rescue female infertility. To identify the neurons responsible for some of the observed behavioral alterations, we determined the overlap between the identified GAL4 lines and endogenous FruM expression in lines with fertility defects. The GAL4 lines causing fertility defects generally had widespread overlap with FruM expression in many regions of the nervous system, suggesting likely redundant FruM-expressing neuronal pathways capable of conferring male fertility. From associations between the screened behaviors, we propose a functional model for courtship initiation

Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons.

The use of genetically encoded 'self-labeling tags' with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.This work was supported by Howard Hughes Medical Institute (https://www.hhmi.org), the Medical Research Council (https://mrc.ukri.org; MRC file reference U105188491) and a European Research Council (https://erc.europa.eu) Consolidator grant (649111) to G.S.X.E.J., and a Royal Society (https://royalsociety.org) Dorothy Hodgkin Fellowship to S.C

Mapping tissue-specific <i>fru^M</i> repression to courtship latency phenotypes.

<p>Courtship latencies of <i>P[GawB] UAS-fru^MIR</i> latencies depicted as stacked bars. A) White bars represent latencies in ambient light, while dark gray bars represent latencies in infrared (n = 10–44 males per genotype). Purple bars indicate the difference between dark-light latencies. Green triangles indicate position of control <i>UAS-fru^MIR</i>/+ line. Purple arrows indicate position of <i>P[GawB]4-57 UAS-fru^MIR</i>. B) Colored heat map representing the percent of <i>fru^P1-LexA</i>(+) neurons in a given hemisphere cluster or peripheral segment that also express Gal4. Rows are <i>P[GawB]</i> lines in the same respective order as panel A. Columns represent the <i>fru^P1-LexA</i>(+) clusters in foreleg tarsal segments (T5-T1), maxillary palp (M), labellum (P), 3^rd and 2^nd antennal segments (3, 2), and brain clusters 1–15, nomenclature from 21. C) Three lines that exhibited no or little peripheral Gal4 expression and limited central overlap with <i>fru^P1-LexA</i>. D) Estimated relative contributions of each <i>fru^M</i>(+) cluster to delays in courtship latency in infrared; values are coefficients of a linear regression derived from partial least squares analysis (see Methods). Error bars represent 95% confidence intervals estimated by resampling.</p

Visualization of <i>fru^M</i>(+) and <i>P[GawB]4-57</i> intersection revealed a sexually dimorphic arborization in the tritocerebrum.

<p>A–F) Anterior-posterior, G–J) sagittal, and K–L) dorsal-ventral confocal projections. Unless otherwise stated, images are from males. A–I) Z-projections showing GFP fluorescence from male <i>P[GawB]4-57</i>/<i>UAS>stop>mCD8-GFP</i>; <i>fru^FLP</i>/+ brains. A–F) Merged images showing anti-NC82 and GFP expressions in male and female brains. In all brains two GFP(+) cell bodies, in the ventral medial SOG (mSG, arrows) project to and make extensive arborizations in the tritocerebrum. C) In 7/14 brains, 3 cell bodies (aSG, arrowheads) fluoresced at depths of 17–20 µm without detectable neurites. D–F versus A–C) Z-projection showing the weaker tritocerebral aborizations from the two mSG neurons in female <i>P[GawB]4-57</i>/<i>UAS>stop>mCD8-GFP</i>; <i>fru^FLP</i>/+ brains. F) pSG marks one posterior GFP(+) neuron. G–I), sagittal reconstructions of mSG projections in a G) <i>P[GawB]4-57</i>/<i>UAS>stop>mCD8-GFP</i>; <i>fru^FLP</i>/+ male, H) female, and I) a <i>P[GawB]4-57</i>/<i>UAS>stop>mCD8-GFP</i>; <i>fru^FLP</i>/<i>fru^LexA</i> male mutant brain. Dashed lines mark the path of the esophagus. In <i>fru</i>⁺/<i>fru</i>^- males the tritocerebral arbors are significantly larger and fluoresce brighter compared to <i>fru</i>⁺/<i>fru</i>^- females or <i>fru</i> mutant males (arrows). J) Expression of the dendritic marker UAS>stop>Dscam17-1-GFP in mSG∩4-57 neurons colocalized with the tritocerebral arbors and anterior to medial tracts. L) Presynaptic marker, UAS>stop>nsyb-GFP was expressed mainly in prothoracic/metathoracic boundary. K) The presence of <i>tsh-Gal80</i> repressed expression from <i>fru^M</i>(+) ventral nerve highlighting the descending projections from mSG and pSG cells. Scale bars = 50 µm. eo = esophageal foramen, TC = tritocerebrm, and SOG = subesophageal ganglion.</p

Tissue-specific knockdown of <i>fru^M</i> expression or silencing synaptic transmission in <i>P[GawB]4-57</i>∩<i>fru^M</i> neurons lengthened courtship latency, reduced courtship performance, and prevented progression to copulation.

<p>A–E) Horizontal bars indicate average time to the first unilateral wing extension by males in 10 minutes. F–J) Blue bars represent courtship index, measured as proportion of time spent performing courtship behaviors for 2 minutes after initiation of courtship. K–N) Vertical bars represent the fraction of males that attempt any copulation behaviors in 15 minutes. Darker bars represent courtship in infrared light versus ambient light. B) Under infrared light, <i>P[GawB]4-57</i>-driven <i>UAS-fru^M</i>IR expression significantly lengthened courtship latency, unless <i>Cha-Gal80</i> (green arrow) was present; G) reduced courtship index; and L) led to most males failing to progress to copulation behaviors. D) Silencing <i>fru^FLP</i>∩4-57 neurons via tetanus toxin (TNT) resulted in lengthened infrared courtship latency, H–I) reduced courtship index regardless of light conditions, and M) prevented males from exhibiting copulatory behaviors under infrared. E, J, N) Activation of <i>fru^FLP</i>∩4-57 neurons, using UAS>stop>TRPA1 did not result in abnormal courtship. * =  p<.05, ** = p<.01, and *** = p<.001. Samples ranged from 23–44 males for latency, 11–28 for courtship index, and 17–44 for copulation.</p

Projections <i>fru^M</i> SOG∩4-57 neurons likely receive gustatory and descending protocerebral inputs.

<p>A) Sagittal views showing mSG∩4-57 neurons projections in the brain.. Gustatory inputs from the labellum, mouthparts innervate the anterior-medial SOG and the tritocerebrum. Inputs from the superior medial protocerebrum also innervate the tritocerebrum. Descending tracts from the mSG∩4-57 innervate prothoracic/metathoracic and mesothoracic/abdominal ganglia boundaries B). The mSG∩4-57, and possibly aSG∩4-57 neurons, function to regulate initiation of wing extension and copulatory behaviors. SMPR = superior medial protocerebrum, and CC = cervical connective. TC marks the tritocerebrum. The SOG is marked by a dashed blue line.</p

<i>Cha</i>-driven Gal80 inhibits <i>P[GawB]4-57</i>-driven Gal4 activity mainly in <i>fru^M</i> SOG neurons.

<p>A–E) Partial Z projections showing GFP expression in <i>P[GawB]</i>4-57/<i>UAS-mCD8-GFP</i> expression compared to F–J) GFP expression in the presence of <i>ChaGal80/+</i>. Panels show confocal images of anti-GFP, anti-Fru^M (panels marked by ′), and merged fluorescence (marked by ′′). A) Two GFP(+), Fru^M(+) were detected in the mcAl/DT6 cluster (arrows). Two to four smaller somas are found at 5–30 µm in depth, designated aSG (arrowheads). B) Two larger somas are found from 15–60 µm in depth, designated mSG. C) Two-three large somas are found at 60–100 µm in depth, designated pSG. D) Two GFP(+), Fru^M(+) somas are found near the prothoracic/metathoracic boundary of the ventral nerve cord. E) Three to four GFP(+), Fru^M(+) somas are located in the abdominal ganglion. F) The two mcAl/DT6 somas showed reduced, but detectable GFP expression. Insets show 2 µm Z-slices that highlight the mcAl/DT6 GFP(+), <i>ChaGal80</i>(+), Fru^M(+) cells. F–G) <i>Cha-Gal80</i> repressed GFP fluorescence in all mSG neurons and a subset of aSG cells (hollow arroweads). Hollow arrowheads point to GFP(-), Fru^M(+) cells, indicative of Cha-Gal80 repression, while filled arrowheads point to GFP(+), Fru^M(+) cells. H–J) Fru^M(+) pSG and ventral nerve cord cells still expressed GFP. K) Locations of imaged regions are depicted on the diagram of the nervous system. L) Quantification of cells that express <i>P[GawB]4-57</i>-driven GFP in the presence and absence of Cha-Gal80, (n = 8, asterisk denotes p<.05). Scale bars = 50 µm.</p