Hierarchical Control of Drosophila Sleep, Courtship, and Feeding Behaviors by Male-Specific P1 Neurons
Animals choose among sleep, courtship, and feeding behaviors based on the integration of both external sensory cues and internal states; such choices are essential for survival and reproduction. These competing behaviors are closely related and controlled by distinct neural circuits, but whether they are also regulated by shared neural nodes is unclear. Here, we investigated how a set of male-specific P1 neurons controls sleep, courtship, and feeding behaviors in Drosophila males. We found that mild activation of P1 neurons was sufficient to affect sleep, but not courtship or feeding, while stronger activation of P1 neurons labeled by four out of five independent drivers induced courtship, but only the driver that targeted the largest number of P1 neurons affected feeding. These results reveal a common neural node that affects sleep, courtship, and feeding in a threshold-dependent manner, and provide insights into how competing behaviors can be regulated by a shared neural node.
KeywordsDrosophila Courtship Sleep Feeding P1 neurons Neural circuit
A fundamental question in biology is how animals sense environmental cues and alter their physiology and behavior in ways that are beneficial for their survival and reproduction. The amenability of Drosophila melanogaster as a model system using genetic and physiological approaches makes it ideal for exploring the neural mechanisms underlying a variety of behaviors including sleep, courtship, and feeding. Indeed, substantial progress has been made on how these individual behaviors are controlled by specific neuronal circuits, often referred to as sleep circuits, courtship circuits, and feeding circuits [1, 2, 3, 4, 5, 6].
Sleep, courtship, and feeding behaviors are mutually exclusive in principle. Very little is known about how these behaviors cross-talk at the level of neuronal circuits. Recently, we showed that sleep and sexual behaviors interact in a sex-specific way and identified sexually dimorphic neurons that mediate the interplay between sleep and sex . Among these neurons, P1 neurons are of particular interest because they serve as a higher center that integrates both external sensory cues and social experience [8, 9, 10, 11, 12]. It has also been found that P1 neurons regulate male aggression, as a P1-activated male fly is more aggressive if presented with another male . P1 neurons are only present in males and express the male-specific Fruitless (FruM) and Doublesex (DsxM) proteins [11, 14, 15, 16], which are crucial for male sexual development and behaviors [17, 18, 19, 20]. It is generally accepted that the activity of P1 neurons is positively correlated with male sexual arousal in flies [8, 10, 15]. However, it is still unclear whether P1 neurons represent a general arousal state and modulate many other behaviors, and if so, how multiple behaviors are controlled by the same set of neurons.
Here, we used five independent driver lines that labeled P1 neurons ranging from 9 to 23 cells per hemisphere (20%–50% of all P1 neurons) to determine how P1 activation affects sleep, courtship, and feeding behaviors in Drosophila.
Materials and Methods
Flies were maintained at 22°C under a 12 h:12 h light:dark cycle. Split-GAL4 reagents including R15A01-AD, R17D06-AD, R71G01-DBD, and R22D03-DBD, as well as R17D06-LexA and R71G01-LexA have been described previously [21, 22] and were obtained from Janelia Research Campus (Ashburn, VA). dsxGAL4, UAS>stop>dTrpA1, UAS>stop>myrGFP, and LexAop2-FlpL were used as previously described [15, 23].
Sleep Test and Analysis
Individual 2–4 day-old male flies were placed in locomotor activity monitor tubes (DAM2, TriKinetics Inc., Waltham, MA) with fly food (2% agarose and 5% sugar), and entrained under 22°C and 12 h:12 h light:dark conditions for at least 2 days before sleep tests. One day of sleep data were first recorded at 22°C as baseline, then the flies were shifted to 25.5°C, 27°C, 28.5°C, or 30°C for two days, and then returned to 22°C for at least one day. Sleep was analyzed as previously described . Changes in total sleep were calculated as the percentage of sleep change on the first day of temperature shift relative to baseline sleep at 22°C.
Courtship and Locomotion Assay
We used unilateral wing extension in isolated males to compare courtship induced by P1 activation. Males were individually placed in 2 cm-diameter round chambers with food (2% agarose and 5% sugar) at 25.5°C, 27°C, 28.5°C, or 30°C and video was captured for 24 h, starting at 09:00. We then manually scored the percentage of males displaying unilateral wing extension. The average walking velocity during the 24-h recording was further quantified using the ZebraLab software system (ViewPoint Life Sciences, Montreal, Quebec, Canada).
Feeding was assayed using food with blue dye. In brief, flies were starved for 24 h on 1% aqueous agarose at 22°C, then moved to 25.5°C, 27°C, 28.5°C, or 30°C for 30 min for dTrpA1 activation. Thereafter, they were transferred to 1% FD&C Blue 1 (Sigma-Aldrich, St. Louis, MO) food (2.5% sucrose, 2.5% yeast extract, and 0.5% agar) for 15 min (between 15:00 and 17:00) at the above temperatures for continuous activation while allowing feeding. To quantify the food intake, the absorbance of the ingested blue dye was measured as previously described .
Tissue Dissection, Staining, and Imaging
We dissected the brains of 4–6 day-old male or female flies in Schneider’s insect medium (Thermo Fisher Scientific, Waltham, MA) and fixed them in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature. After 4 × 15-min washes in PAT (0.5% Triton X-100 and 0.5% bovine serum albumin in PBS), tissues were blocked in 3% normal goat serum (NGS) for 60 min, then incubated in primary antibodies diluted in 3% NGS for ~24 h at 4°C, washed (4 × 15-min) in PAT, and incubated in secondary antibodies diluted in 3% NGS for ~24 h at 4°C. Tissues were then washed (4 × 15-min) in PAT and mounted in Vectashield (Vector Laboratories, Burlingame, CA) for imaging. The primary antibodies used were rabbit anti-GFP (1:1000; A11122, Invitrogen, Waltham, MA) and mouse anti-Bruchpilot (1:30; nc82, Developmental Studies Hybridoma Bank, Iowa City, IA). The secondary antibodies used were goat anti-mouse IgG conjugated to Alexa 555 (1:500, A28180, Invitrogen) and goat anti-rabbit IgG conjugated to Alexa 488 (1:500, A11008, Invitrogen). Samples were imaged at 20× magnification on a Zeiss 700 confocal microscope, and processed with ImageJ.
Statistical analysis was performed using Prism GraphPad as indicated in the figure legends.
Results and Discussion
Sleep, courtship, and feeding are competing behaviors that are mediated by external sensory cues and internal states. Whether these competing behaviors are regulated by common neural nodes is an intriguing question. P1 neurons have been established as a center that mediates sexual arousal, but their role in regulating other internal states and behaviors has been underestimated. Our findings that P1 neurons mediate sleep, courtship, and feeding behaviors not only reveal a neural node (P1) that participates in all these competing behaviors, but also how P1 neurons modulate these behaviors in a hierarchical manner (Fig. 4B).
There are nearly 50 pairs of P1 neurons in the male fly brain , and we studied here only 20%–50% of them. Given that mild activation of ~10 P1 neurons was sufficient to inhibit sleep, and stronger activation of ~23 (nearly half) suppressed feeding, what if the other half or all P1 neurons were activated? Do P1 neurons regulate behaviors other than sleep, courtship, aggression, and feeding? Is P1 a center for internal states that coordinate different behaviors? To answer these questions, better tools are needed to subdivide P1 populations, with driver lines that target small and distinct subsets of P1 neurons and driver lines targeting all or the majority of P1 neurons.
We also note that, although males and females play distinct roles in sexual behavior, their differences in non-sexual behaviors (e.g., different amounts of sleep or feeding) are relatively smaller and underestimated, and the mechanism underlying these differences is unclear. That P1 neurons are male-specific and regulate sleep, courtship, aggression, and feeding suggests that sexual dimorphism in these behaviors may be greater than we thought, and our results provide a simple model of how a small number of sex-specific neurons can contribute to various sexually-dimorphic behaviors.
We thank the Rubin lab at Janelia Research Campus for the fly stocks. This work was supported by the Natural Science Foundation of Jiangsu Province of China (BK20150597 and BK20160025), the National Natural Science Foundation of China (31571093 and 31622028), and the Thousand Young Talents Program in China.
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