Failure to mate enhances investment in behaviors that may promote mating reward and impairs the ability to cope with stressors via a subpopulation of Neuropeptide F receptor neurons.

Living in dynamic environments such as the social domain, where interaction with others determines the reproductive success of individuals, requires the ability to recognize opportunities to obtain natural rewards and cope with challenges that are associated with achieving them. As such, actions that promote survival and reproduction are reinforced by the brain reward system, whereas coping with the challenges associated with obtaining these rewards is mediated by stress-response pathways, the activation of which can impair health and shorten lifespan. While much research has been devoted to understanding mechanisms underlying the way by which natural rewards are processed by the reward system, less attention has been given to the consequences of failure to obtain a desirable reward. As a model system to study the impact of failure to obtain a natural reward, we used the well-established courtship suppression paradigm in Drosophila melanogaster as means to induce repeated failures to obtain sexual reward in male flies. We discovered that beyond the known reduction in courtship actions caused by interaction with non-receptive females, repeated failures to mate induce a stress response characterized by persistent motivation to obtain the sexual reward, reduced male-male social interaction, and enhanced aggression. This frustrative-like state caused by the conflict between high motivation to obtain sexual reward and the inability to fulfill their mating drive impairs the capacity of rejected males to tolerate stressors such as starvation and oxidative stress. We further show that sensitivity to starvation and enhanced social arousal is mediated by the disinhibition of a small population of neurons that express receptors for the fly homologue of neuropeptide Y. Our findings demonstrate for the first time the existence of social stress in flies and offers a framework to study mechanisms underlying the crosstalk between reward, stress, and reproduction in a simple nervous system that is highly amenable to genetic manipulation

CD74 is a regulator of hematopoietic stem cell maintenance.

Hematopoietic stem and progenitor cells (HSPCs) are a small population of undifferentiated cells that have the capacity for self-renewal and differentiate into all blood cell lineages. These cells are the most useful cells for clinical transplantations and for regenerative medicine. So far, it has not been possible to expand adult hematopoietic stem cells (HSCs) without losing their self-renewal properties. CD74 is a cell surface receptor for the cytokine macrophage migration inhibitory factor (MIF), and its mRNA is known to be expressed in HSCs. Here, we demonstrate that mice lacking CD74 exhibit an accumulation of HSCs in the bone marrow (BM) due to their increased potential to repopulate and compete for BM niches. Our results suggest that CD74 regulates the maintenance of the HSCs and CD18 expression. Its absence leads to induced survival of these cells and accumulation of quiescent and proliferating cells. Furthermore, in in vitro experiments, blocking of CD74 elevated the numbers of HSPCs. Thus, we suggest that blocking CD74 could lead to improved clinical insight into BM transplant protocols, enabling improved engraftment

LC-MS metabolite results. Compound averaged peak area/total measurable ions results are presented for mated (n = 16), rejected (n = 17), and naïve-single (n = 17) cohorts.

LC-MS metabolite results. Compound averaged peak area/total measurable ions results are presented for mated (n = 16), rejected (n = 17), and naïve-single (n = 17) cohorts.</p

S7 Fig -

A. Behavioral signatures of male-male social interaction within the FlyBowl system during the optogenetic activation of Dh44 neurons. Data is represented as normalized Z scores of 60 behavioral parameters. Dh44 G4/+;UAS-csChrimson/+ males (green, n = 8) and their genetic controls Dh44 G4/+, UAS-csChrimson/+ (black, n = 7 and grey, n = 8, respectively). **pB. Starvation resistance assayed on Dh44 G4/NPFR RNAi (green, n = 70) flies and their genetic controls Dh44 G4/+ (blue, n = 66) and NPFR RNAi/+ (black, n = 64). No significant difference was observed among experimental flies and the controls, p>0.05. Pairwise log-rank test with FDR correction for multiple comparisons was performed. (EPS)</p

Courtship conditioning did not affect TAG and glucose levels and most head metabolites in males.

A-D. Metabolic indices of rejected males (red) compared to naïve-single (green) or mated (blue) males. No differences were observed for measurements of (A) triglycerides (TAG, n = 11 for all groups, 5 males/ sample). (B) weight (n = 10 naïve-single, 9 mated, 9 rejected, 5 males/sample). (C) hemolymph, or (D) body glucose (n = 3 for all groups, 5 males/body sample, and 40 males/hemolymph sample). ANOVA or Kruskal-Wallis with post-hoc Tukey’s or Friedman test were performed. NS p>0.05. E. % difference from average (peak area/ total measurable ions) of metabolites detected using LC-MS in rejected, mated, and naïve-single males’ heads. 5-aminolevulinic acid, acetyl-glutamine, glycine, tryptophan, and stearic acid levels were higher in rejected males’ heads (red triangles, n = 17) compared to naïve-single (green circles, n = 17) and mated (blue squares, n = 16, 5 heads/sample). Metabolites of the kynurenine pathways are highlighted in orange; serotonin is highlighted in yellow. NS p>0.05, *ppp (EPS)</p

A movie of the confocal stacks of the shared NPFR^TK neurons (Fig 5C).

Shared NPFRTK neurons were visualized using genetic interaction between NPFR and TK drivers: NPFRG4;+, TK-LexA;+, +;LexAop-FlpL, +;UAScs-Chrimson-mVenus flies. Green marks NPFRTK neurons, magenta marks nc-82. (MP4)</p

Sexual rejection promotes social avoidance.

A. Illustration of network parameters. Density of networks represents how saturated they are compared to the maximum possible. Strength is proportional to vertex size (high in red individual). B-E. Social network analysis of groups composed of mated (blue) naïve-single (green) and rejected (red). Network density, and strength calculated by network weights according to duration (B-C) or number of interactions (D-E). Kruskal–Wallis test followed by Wilcoxon signed-rank test and FDR correction for multiple tests *p F. Rejected male flies maintain large distances between flies along as measured by anglesub which is the Maximum total angle of animal’s field of view (fov) occluded by another animal, statistical significance was tested on averages across time. one-way ANOVA followed by Tukey’s and FDR correction for multiple tests *p G. Average number of flies close to a focal fly. n = 15, 8, 15 for mated, naïve and rejected respectively. One-way ANOVA followed by Tukey’s and FDR correction for multiple tests *p < 0.05, **p < 0.01, ***p < 0.001. Error bars signify SEM.</p

Repeated sexual rejection events increase sensitivity to stressors via NPFR neurons.

A. Schematic representation of courtship conditioning: control males were introduced to either virgin, sexually receptive or sexually non-receptive females. As a result, males were either mated or rejected. The third cohort consisted of control single housed males that did not experience any social or sexual event (naïve-single). Encounters with females were repeated 3x a day for two days. B. Starvation resistance assay: rejected males (red, n = 91) compared to mated (blue, n = 102) and single housed (green, n = 110) males, ***ppC. Resistance to oxidative stress (20mM Paraquat): rejected males (red, n = 50) compared to mated (blue, n = 53) ppD. Starvation resistance assayed on NPFR>RNAi mated (blue, n = 68), rejected (red, n = 68) and naïve-single (green, n = 63) male flies. *ppE. Activation of NPFR neurons promotes sensitivity to starvation. NPFR>csChrimson flies underwent three times a day (5 minutes each) optogenetic activation for two days (pink), NPFR>csChrimson w/o activation (grey) serve as controls. Starvation resistance of experimental (n = 55) and control flies (n = 72) was assayed. Log-rank test was performed, ***pF. Male flies expressing UAS-csChrimson and UAS-Shibirets in NPFR neurons were subjected to three 5 min long optogenetic activations for three days, and their synaptic signaling was blocked at 28–29°C (light+ heat, orange, n = 52). Positive control males (light+cold, pink, n = 52), synaptic release block control (dark+heat, light gray, n = 52), negative control (dark+cold, dark gray, n = 50). Experimental and positive control flies showed no significant difference in resistance to starvation (p>0.05). Both experimental and positive control flies were significantly more sensitive to starvation than ‘dark+heat’, and ‘dark+cold’ flies (**p<0.01). Pairwise log-rank test with FDR correction for multiple comparisons was performed.</p