Genetic and neuronal mechanisms governing the sex-specific interaction between sleep and sexual behaviors in Drosophila

Abstract

Animals execute one particular behavior among many others in a context-dependent manner, yet the mechanisms underlying such behavioral choice remain poorly understood. Here we studied how two fundamental behaviors, sex and sleep, interact at genetic and neuronal levels in Drosophila. We show that an increased need for sleep inhibits male sexual behavior by decreasing the activity of the male-specific P1 neurons that coexpress the sex determination genes fru ^M and dsx, but does not affect female sexual behavior. Further, we delineate a sex-specific neuronal circuit wherein the P1 neurons encoding increased courtship drive suppressed male sleep by forming mutually excitatory connections with the fru ^M -positive sleep-controlling DN1 neurons. In addition, we find that FRU^M regulates male courtship and sleep through distinct neural substrates. These studies reveal the genetic and neuronal basis underlying the sex-specific interaction between sleep and sexual behaviors in Drosophila, and provide insights into how competing behaviors are co-regulated.Genes and circuits involved in sleep and sexual arousal have been extensively studied in Drosophila. Here the authors identify the sex determination genes fruitless and doublesex, and a sex-specific P1-DN1 neuronal feedback that governs the interaction between these competing behaviors.

Fig. 1

SD suppresses male courtship. a Courtship indices of males that had been sleep-deprived for 12 h (light gray) or 16 h (dark gray) during indicated periods. n = 24 for each. *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA. b Detailed sleep profile of male flies with 16-h SD (15 s/min shaking). c The 16-h SD results in 468-min sleep loss during SD and induces post-SD sleep rebound. The start point of baseline was chosen at the same ZT the day before SD. n = 32. ***p < 0.001, unpaired t-test. d Detailed sleep profile of male flies with 8-h SD (30 s/min shaking), but the amount of mechanical perturbation is the same as the above 16-h SD. e The 8-h SD results in 269-min sleep loss during SD, and does not induce post-SD sleep rebound. n = 32. N.S., not significant; ***p < 0.001, unpaired t-test. f Males that were sleep-deprived for 8 h court more than males that were sleep-deprived for 16 h, although they received the same amount of mechanical perturbation. n = 24 for each. ***p < 0.001, one-way ANOVA. Error bars indicate SEM. Please see Supplementary Fig. 1 for the effect of SD on female sexual behavior

Fig. 2

Sex-promoting neurons regulate sleep. a Diagram of intersectional strategy to label and manipulate subsets of dsx neurons: P1 neurons in males promoting male courtship, pC1 and pCd neurons in females promoting female receptivity. b Activating P1 neurons suppresses total sleep (n = 60), and inhibiting P1 neurons promotes total sleep in male flies. c Detailed sleep plot of activating P1 neurons (in red line, controls in blue and gray) in male flies. d Activating pC1 but not pCd neurons promotes female sleep, but inhibiting pC1 or pCd neurons does not affect female sleep. e Another way of targeting P1 neurons using the splitGAL4 system. f Activation of P1 neurons inhibits male sleep. g Silencing P1 neurons slightly increases male sleep compared to controls. n = 24~32 for each. ***p < 0.001, unpaired t-test. NS, not significant. Error bars indicate SEM. Scale bars, 50 µm

Fig. 3

Activity-dependent regulation of courtship and sleep by P1. a–d Sleep profiles of males with P1 neurons activated at indicated temperatures, as well as control males. n = 32 for each. e–h Velocity of males with P1 neurons activated at indicated temperatures over 24 h of video tracking. n = 24 for each. i–k Percentage of males with P1 neurons activated at indicated temperatures shows wing extension over 24 h of video tracking. n = 48 for each. l Percentage of sleep changes from 21.5 °C to indicated temperatures. m The mean velocity of males with P1 neurons activated at indicated temperatures over 24 h. n Percentage of males with P1 neurons activated at indicated temperatures show wing extension. ***p < 0.001, unpaired t-test. NS, not significant. Error bars indicate SEM

Fig. 4

ACh release by P1 neurons is required for sleep regulation. a Knocking down Ach in P1 neurons abolishes the effect of P1 activation on male sleep. The blue bar indicates P1 activation alone, while bars in magenta indicate P1 activation together with knocking down specific genes in P1 neurons. n = 32 for each. b Detailed sleep profiles of P1 activation alone (in blue line) and P1 activation together with VAChT knocked down (in magenta line). c, d Intersectional expression of fru ^LexA and cha-GAL4 in a male brain c, where P1 neurons are labeled, and in a female brain d. Error bars indicate SEM. Scale bars, 50 µm

Fig. 5

P1 regulates male sleep through DN1 neurons. a Diagram for activating P1 neurons in the sex circuitry, and inhibiting subsets of neurons in the sleep circuitry. b Identification of R18H11-LexA DN1 circadian clock neurons as functionally downstream of P1 neurons. Blue bars indicate LexA inhibition alone, while red bars indicate P1 activation together with LexA inhibition. n = 24~56 for each. **p < 0.01, Kruskal–Wallis non-parametric one-way ANOVA followed by post hoc Dunn’s correction. Error bars indicate SEM. c Expression pattern of P1-splitGAL4 and R18H11-LexA driving UAS-myrGFP and LexAop-myrGFP, respectively. d Detailed sleep profiles of P1 activation alone (in black line) and P1 activation together with DN1 inhibition (in red line). e Brain registration of P1 (magenta) and DN1 (green) neurons. f There is no GRASP signal between P1 and DN1 neurons. g GRASP signals between Gr33a-expressing neurons and fru ^M-expressing neurons as a positive control. Scale bars, 50 µm

Fig. 6

P1 and DN1 neurons form mutually excitatory connections. a Movie still of Calcium imaging of DN1 cell bodies after P1 activation via P2X₂. b Averaged traces of ∆F/F ₀ of DN1 neurons after P1 activation. c Peak fluorescence changes (∆F/F ₀) of DN1 neurons after P1 activation. n = 6 for each, ***p < 0.001. Unpaired t-test. d Movie still of calcium imaging of P1 neurites in the lateral protocerebrum region after DN1 activation via P2X₂. e Averaged traces of ∆F/F ₀ of P1 neurons after DN1 activation. f Peak fluorescence changes (∆F/F ₀) of P1 neurons after DN1 activation. n = 10 for each, **p < 0.01. Unpaired t-test. Error bars indicate SEM

Fig. 7

SD decreases P1 activity. a Optical recording of membrane activity in the lateral junction (circled) of P1 neurons in control (non-deprived) males and sleep-deprived males. b SD of the optical signal was plotted, with mean ± SEM. n = 10 for each. **p < 0.01, unpaired t-test. c Power spectrum was computed using fast Fourier transform with 0.2 Hz bin width. Power of the non-deprived group is significantly greater than the sleep-deprived group (two-way ANOVA with repeated measures). d Activating P1 neurons with dTRPA1 overcomes courtship suppression by SD. n = 24 for each. ***p < 0.001, unpaired t-test. NS, not significant. Error bars indicate SEM

Fig. 8

Sex determination genes regulate sleep. a Sex determination pathway in Drosophila. b, c Sex determination genes tra, fru ^M, and dsx regulate sexually dimorphic sleep quantity. Total sleep amounts of males and females b, and the corresponding sexual difference of sleep c are shown for indicated genotypes. n = 24~56 for each. *p < 0.05 and ***p < 0.001, unpaired t-test. NS, not significant. d–g Total sleep in males (d) and females (e) of dsx alleles, and total sleep in males (f) and females (g) of fru ^M alleles. n = 24~32 for each. *p < 0.05, **p < 0.01, and ***p < 0.001; comparisons are made between the genotype and its parental genotypes, one-way ANOVA. NS, not significant. Error bars indicate SEM

Fig. 9

FRU^M differentially regulates courtship and sleep. a Knocking down fru ^M in MB or DN1 neurons, but not P1 neurons, reduces male sleep, and the function of fru ^M in DN1 neurons on male sleep is dependent on Dh31. b–g Detailed sleep profile of males with fru ^M knocked down in all fru ^M-expressing neurons (b), all dsx-expressing neurons (c), MB neurons (d, e), DN1 neurons (f), and DN1 neurons in Dh31-mutated background (g). h Knocking down fru ^M in dsx-expressing neurons but not MB or DN1 neurons impairs male courtship. n = 24~32 for each. *p < 0.05 and ***p < 0.001, unpaired t-test. NS, not significant

Fig. 10

Sex-specific interaction between courtship and sleep. The male-specific P1 neurons integrate external courtship-related sensory cues and internal sleep need, then inhibit male sleep, and promote male courtship in a threshold-dependent manner. P1 neurons form mutually excitatory connections with sleep-controlling DN1 neurons, which might be important for a persistent behavioral state. Furthermore, FRU^M differentially regulates male courtship and sleep in P1 and DN1 neurons. Scale bars, 50 µm