Stem cell-specific ecdysone signaling regulates the development and function of a Drosophila sleep homeostat.

Complex behaviors arise from neural circuits that are assembled from diverse cell types. Sleep is a conserved and essential behavior, yet little is known regarding how the nervous system generates neuron types of the sleep-wake circuit. Here, we focus on the specification of Drosophila sleep-promoting neurons-long-field tangential input neurons that project to the dorsal layers of the fan-shaped body neuropil in the central complex (CX). We use lineage analysis and genetic birth dating to identify two bilateral Type II neural stem cells that generate these dorsal fan-shaped body (dFB) neurons. We show that adult dFB neurons express Ecdysone-induced protein E93, and loss of Ecdysone signaling or E93 in Type II NSCs results in the misspecification of the adult dFB neurons. Finally, we show that E93 knockdown in Type II NSCs affects adult sleep behavior. Our results provide insight into how extrinsic hormonal signaling acts on NSCs to generate neuronal diversity required for adult sleep behavior. These findings suggest that some adult sleep disorders might derive from defects in stem cell-specific temporal neurodevelopmental programs.

Homeostatic control of deep sleep and molecular correlates of sleep pressure in Drosophila.

Homeostatic control of sleep is typically addressed through mechanical stimulation-induced forced wakefulness and the measurement of subsequent increases in sleep. A major confound attends this approach: biological responses to deprivation may reflect a direct response to the mechanical insult rather than to the loss of sleep. Similar confounds accompany all forms of sleep deprivation and represent a major challenge to the field. Here, we describe a new paradigm for sleep deprivation in Drosophila that fully accounts for sleep-independent effects. Our results reveal that deep sleep states are the primary target of homeostatic control and establish the presence of multi-cycle sleep rebound following deprivation. Furthermore, we establish that specific deprivation of deep sleep states results in state-specific homeostatic rebound. Finally, by accounting for the molecular effects of mechanical stimulation during deprivation experiments, we show that serotonin levels track sleep pressure in the fly's central brain. Our results illustrate the critical need to control for sleep-independent effects of deprivation when examining the molecular correlates of sleep pressure and call for a critical reassessment of work that has not accounted for such non-specific effects.

The Divider Assay is a high-throughput pipeline for aggression analysis in Drosophila.

Aggression is a complex social behavior that remains poorly understood. Drosophila has become a powerful model system to study the underlying biology of aggression but lack of high throughput screening and analysis continues to be a barrier for comprehensive mutant and circuit discovery. Here we developed the Divider Assay, a simplified experimental procedure to make aggression analysis in Drosophila fast and accurate. In contrast to existing methods, we can analyze aggression over long time intervals and in complete darkness. While aggression is reduced in the dark, flies are capable of intense fighting without seeing their opponent. Twenty-four-hour behavioral analysis showed a peak in fighting during the middle of the day, a drastic drop at night, followed by re-engagement with a further increase in aggression in anticipation of the next day. Our pipeline is easy to implement and will facilitate high throughput screening for mechanistic dissection of aggression.

Multivariate Analysis of Open Field Exploration Identifies Latent Spatial and Social Behavioral Axes in Domestic Dogs.

Recent methodological advances in studying large scale animal movements have let researchers gather rich datasets from behaving animals. Often collected in small sample sizes due to logistical constraints, these datasets are however, ideal for multivariate explorations into behavioral complexity. In behavioral studies of domestic dogs, although automated data loggers have recently seen increasing use, a comprehensive framework to identify complex behavioral axes is lacking. Dog behavioral studies frequently rely on subjective ratings, despite demonstrable evidence that these are insufficient for identifying behavioral variables. Taking advantage of dogs' innate running abilities and readily available GPS data loggers, we extracted latitude-longitude coordinates from running dogs in a large field setup. By extracting multiple variables from each logged coordinate, we generated a complex dataset from limited numbers of dog runs. Individual variables were successful in classifying aerobic competence, social awareness, and different exploratory patterns of dogs. Multivariate analyses identified latent features in movement patterns of dogs which were primarily comprised of two behavioral axes: spatial acuity and social awareness. Individual dogs were then behaviorally classified into independent clusters through unsupervised learning. Interestingly, even though field dogs clustered primarily with each other in varying degrees of energetic exploration and handler focus, some house pets displayed moderately high exploration abilities as well. We expect our proof of principle quantitative pipeline to provide a robust framework for behavioral classification, generating case-control clusters based solely on complex behavioral axes, and greatly benefiting genetic association studies of dog behavior.

Why the young sleep longer.

A transcription factor helps young flies to sleep longer by delaying the maturation of a neural network that controls sleep.

Putative transmembrane transporter modulates higher-level aggression in Drosophila.

By selection of winners of dyadic fights for 35 generations, we have generated a hyperaggressive Bully line of flies that almost always win fights against the parental wild-type Canton-S stock. Maintenance of the Bully phenotype is temperature dependent during development, with the phenotype lost when flies are reared at 19 °C. No similar effect is seen with the parent line. This difference allowed us to carry out RNA-seq experiments and identify a limited number of genes that are differentially expressed by twofold or greater in the Bullies; one of these was a putative transmembrane transporter, CG13646, which showed consistent and reproducible twofold down-regulation in Bullies. We examined the causal effect of this gene on the phenotype with a mutant line for CG13646, and with an RNAi approach. In all cases, reduction in expression of CG13646 by approximately half led to a hyperaggressive phenotype partially resembling that seen in the Bully flies. This gene is a member of a very interesting family of solute carrier proteins (SLCs), some of which have been suggested as being involved in glutamine/glutamate and GABA cycles of metabolism in excitatory and inhibitory nerve terminals in mammalian systems.

Handling alters aggression and "loser" effect formation in Drosophila melanogaster.

In Drosophila, prior fighting experience influences the outcome of later contests: losing a fight increases the probability of losing second contests, thereby revealing "loser" effects that involve learning and memory. In these experiments, to generate and quantify the behavioral changes observed as consequences of losing fights, we developed a new behavioral arena that eliminates handling. We compared two commonly used fly handling procedures with this new chamber and demonstrated that handling influences aggressive behavior and prevents "loser" effect formation. In addition, we induced and observed novel aspects of learning associated with aggression such as the formation of robust winner effects.

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster.

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see (1)). This complex behavior is influenced by genetic, hormonal and environmental factors. As in many organisms, previous fighting experience influences the fighting strategy of flies and the outcome of later contests: losing a fight increases the probability of losing later contests, revealing "loser" effects that likely involve learning and memory (2-4). The learning and memory that accompanies expression of complex social behaviors like aggression, is sensitive to pre-test handling of animals (5,6). Many experimental procedures are used in different laboratories to study aggression (7-9), however, no routinely used protocol that excludes handling of flies is currently available. Here, we report a new behavioral apparatus that eliminates handling of flies, using instead their innate negative geotactic responses to move animals into or out of fighting chambers. In this protocol, small circular fight arenas containing a food cup are divided into two equal halves by a removable plastic slider prior to introduction of flies. Flies enter chambers from their home isolation vials via sliding chamber doors and geotaxis. Upon removal of plastic sliders, flies are free to interact. After specified time periods, flies are separated again by sliders for subsequent experimentation. All of this is done easily without handling of individual flies. This apparatus offers a novel approach to study aggression and the associated learning and memory, including the formation of "loser" effects in fly fights. In addition, this new general-purpose behavioral apparatus can be employed to study other social behaviors of flies and should, in general, be of interest for investigating experience-related changes in fundamental behavioral processes.

Evidence for discrete landmark use by pigeons during homing.

Considerable efforts have been made to investigate how homing pigeons (Columba livia f. domestica) are able to return to their loft from distant, unfamiliar sites while the mechanisms underlying navigation in familiar territory have received less attention. With the recent advent of global positioning system (GPS) data loggers small enough to be carried by pigeons, the role of visual environmental features in guiding navigation over familiar areas is beginning to be understood, yet, surprisingly, we still know very little about whether homing pigeons can rely on discrete, visual landmarks to guide navigation. To assess a possible role of discrete, visual landmarks in navigation, homing pigeons were first trained to home from a site with four wind turbines as salient landmarks as well as from a control site without any distinctive, discrete landmark features. The GPS-recorded flight paths of the pigeons on the last training release were straighter and more similar among birds from the turbine site compared with those from the control site. The pigeons were then released from both sites following a clock-shift manipulation. Vanishing bearings from the turbine site continued to be homeward oriented as 13 of 14 pigeons returned home. By contrast, at the control site the vanishing bearings were deflected in the expected clock-shift direction and only 5 of 13 pigeons returned home. Taken together, our results offer the first strong evidence that discrete, visual landmarks are one source of spatial information homing pigeons can utilize to navigate when flying over a familiar area.