Population parameters of Drosophila larval cooperative foraging.

Cooperative foraging behavior can be advantageous when there is a common exploitable resource. By cooperating, members of the group can take advantage of the potential of increased efficiency of working together as well as equitable distribution of the product. An experimental signature of cooperative foraging is an Allee effect where at a certain number of individuals, there is a peak of fitness. What happens when there are intruders especially ones that do not contribute to any work required for foraging? Drosophila larvae secrete digestive enzymes and exodigest food. Under crowded conditions in liquid food these larvae form synchronized feeding clusters which provides a fitness benefit. A key for this synchronized feeding behavior is the visually guided alignment between adjacent larvae in a feeding cluster. Larvae who do not align their movements are excluded from the groups and subsequently lose the benefit. This may be a way of editing the group to include only known members. To test the model, the fitness benefit from cooperative behavior was further investigated to establish an Allee effect for a number of strains including those who cannot exodigest or cluster. In a standard lab vial, about 40 larvae is the optimal number for fitness. Combinations of these larvae were also examined. The expectation was that larvae who do not contribute to exodigestion are obligate cheaters and would be expelled. Indeed, obligate cheaters gain greatly from the hosts but paradoxically, so do the hosts. Clusters that include cheaters are more stable. Therefore, clustering and the benefits from it are dependent on more than just the contribution to exodigestion. This experimental system should provide a rich future model to understand the metrics of cooperative behavior.

Birth temperature followed by a visual critical period determines cooperative group membership.

Cooperative behavior often arises when a common exploitable resource is generated. Cooperation can provide equitable distribution and protection from raiding of a common resource such as processed food. Under crowded conditions in liquid food, Drosophila larvae adopt synchronized feeding behavior which provides a fitness benefit. A key for this synchronized feeding behavior is the visually guided alignment of a 1-2 s locomotion stride between adjacent larvae in a feeding cluster. The locomotion stride is thought to be set by embryonic incubation temperature. This raises a question as to whether sib larvae will only cluster efficiently if they hatch at the same temperature. To test this, larvae were first collected and incubated in outdoor conditions. Morning hatched lower temperature larvae move slower than their afternoon higher temperature sibs. Both temperature types synchronize but tend to exclude the other type of larvae from their clusters. In addition, fitness, as measured by adult wing size, is highest when larvae cluster with their own temperature type. Thus, the temperature at which an egg is laid sets a type of behavioral stamp or password which locks in membership for later cooperative feeding.

Critical periods shaping the social brain: A perspective from Drosophila.

Many sensory processing regions of the central brain undergo critical periods of experience-dependent plasticity. During this time ethologically relevant information shapes circuit structure and function. The mechanisms that control critical period timing and duration are poorly understood, and this is of special importance for those later periods of development, which often give rise to complex cognitive functions such as social behavior. Here, we review recent findings in Drosophila, an organism that has some unique experimental advantages, and introduce novel views for manipulating plasticity in the post-embryonic brain. Critical periods in larval and young adult flies resemble classic vertebrate models with distinct onset and termination, display clear connections with complex behaviors, and provide opportunities to control the time course of plasticity. These findings may extend our knowledge about mechanisms underlying extension and reopening of critical periods, a concept that has great relevance to many human neurodevelopmental disorders.

Cooperative foraging during larval stage affects fitness in Drosophila.

Cooperative behavior can confer advantages to animals. This is especially true for cooperative foraging which provides fitness benefits through more efficient acquisition and consumption of food. While examples of group foraging have been widely described, the principles governing formation of such aggregations and rules that determine group membership remain poorly understood. Here, we take advantage of an experimental model system featuring cooperative foraging behavior in Drosophila. Under crowded conditions, fly larvae form coordinated digging groups (clusters), where individuals are linked together by sensory cues and group membership requires prior experience. However, fitness benefits of Drosophila larval clustering remain unknown. We demonstrate that animals raised in crowded conditions on food partially processed by other larvae experience a developmental delay presumably due to the decreased nutritional value of the substrate. Intriguingly, same conditions promote the formation of cooperative foraging clusters which further extends larval stage compared to non-clustering animals. Remarkably, this developmental retardation also results in a relative increase in wing size, serving an indicator of adult fitness. Thus, we find that the clustering-induced developmental delay is accompanied by fitness benefits. Therefore, cooperative foraging, while delaying development, may have evolved to give Drosophila larvae benefits when presented with competition for limited food resources.

NeuroPath2Path: Classification and elastic morphing between neuronal arbors using path-wise similarity.

Neuron shape and connectivity affect function. Modern imaging methods have proven successful at extracting morphological information. One potential path to achieve analysis of this morphology is through graph theory. Encoding by graphs enables the use of high throughput informatic methods to extract and infer brain function. However, the application of graph-theoretic methods to neuronal morphology comes with certain challenges in term of complex subgraph matching and the difficulty in computing intermediate shapes in between two imaged temporal samples. Here we report a novel, efficacious graph-theoretic method that rises to the challenges. The morphology of a neuron, which consists of its overall size, global shape, local branch patterns, and cell-specific biophysical properties, can vary significantly with the cell's identity, location, as well as developmental and physiological state. Various algorithms have been developed to customize shape based statistical and graph related features for quantitative analysis of neuromorphology, followed by the classification of neuron cell types using the features. Unlike the classical feature extraction based methods from imaged or 3D reconstructed neurons, we propose a model based on the rooted path decomposition from the soma to the dendrites of a neuron and extract morphological features from each constituent path. We hypothesize that measuring the distance between two neurons can be realized by minimizing the cost of continuously morphing the set of all rooted paths of one neuron to another. To validate this claim, we first establish the correspondence of paths between two neurons using a modified Munkres algorithm. Next, an elastic deformation framework that employs the square root velocity function is established to perform the continuous morphing, which, as an added benefit, provides an effective visualization tool. We experimentally show the efficacy of NeuroPath2Path, NeuroP2P, over the state of the art.

A Plastic Visual Pathway Regulates Cooperative Behavior in Drosophila Larvae.

Cooperative behavior emerges in biological systems through coordinated actions among individuals [1, 2]. Although widely observed across animal species, the cellular and molecular mechanisms underlying the establishment and maintenance of cooperative behaviors remain largely unknown [3]. To characterize the circuit mechanisms serving the needs of independent individuals and social groups, we investigated cooperative digging behavior in Drosophila larvae [4-6]. Although chemical and mechanical sensations are important for larval aggregation at specific sites [7-9], an individual larva's ability to participate in a cooperative burrowing cluster relies on direct visual input as well as visual and social experience during development. In addition, vision modulates cluster dynamics by promoting coordinated movements between pairs of larvae [5]. To determine the specific pathways within the larval visual circuit underlying cooperative social clustering, we examined larval photoreceptors (PRs) and the downstream local interneurons (lOLPs) using anatomical and functional studies [10, 11]. Our results indicate that rhodopsin-6-expressing-PRs (Rh6-PRs) and lOLPs are required for both cooperative clustering and movement detection. Remarkably, visual deprivation and social isolation strongly impact the structural and functional connectivity between Rh6-PRs and lOLPs, while at the same time having no effect on the adjacent rhodopsin-5-expressing PRs (Rh5-PRs). Together, our findings demonstrate that a specific larval visual pathway involved in social interactions undergoes experience-dependent modifications during development, suggesting that plasticity in sensory circuits could act as the cellular substrate for social learning, a possible mechanism allowing an animal to integrate into a malleable social environment and engage in complex social behaviors.

Cooperative Behavior Emerges among Drosophila Larvae.

Spectacular examples of cooperative behavior emerge among a variety of animals and may serve critical roles in fitness [1, 2]. However, the rules governing such behavior have been difficult to elucidate [2]. Drosophila larvae are known to socially aggregate [3, 4] and use vision, mechanosensation, and gustation to recognize each other [5-8]. We describe here a model experimental system of cooperative behavior involving Drosophila larvae. While foraging in liquid food, larvae are observed to align themselves and coordinate their movements in order to drag a common air cavity and dig deeper. Large-scale cooperation is required to maintain contiguous air contact across the posterior breathing spiracles. On the basis of a directed genetic screen we find that vision plays a key role in cluster dynamics. Our experiments show that blind larvae form fewer clusters and dig less efficiently than wild-type and that socially isolated larvae behave as if they were blind. Furthermore, we observed that blind and socially isolated larvae do not integrate effectively into wild-type clusters. Behavioral data indicate that vision and social experience are required to coordinate precise movements between pairs of larvae, therefore increasing the degree of cooperativity within a cluster. Hence, we hypothesize that vision and social experience allow Drosophila larvae to assemble cooperative digging groups leading to more effective feeding and potential evasion of predators. Most importantly, these results indicate that control over membership of such a cooperative group can be regulated.

Visual attraction in Drosophila larvae develops during a critical period and is modulated by crowding conditions.

The development of social behavior is poorly understood. Many animals adjust their behavior to environmental conditions based on a social context. Despite having relatively simple visual systems, Drosophila larvae are capable of identifying and are attracted to the movements of other larvae. Here, we show that Drosophila larval visual recognition is encoded by the movements of nearby larvae, experienced during a specific developmental critical period. Exposure to moving larvae, only during a specific period, is sufficient for later visual recognition of movement. Larvae exposed to wild-type body movements, during the critical period, are not attracted to the movements of tubby mutants, which have altered morphology. However, exposure to tubby, during the critical period, results in tubby recognition at the expense of wild-type recognition indicating that this is true learning. Visual recognition is not learned in excessively crowded conditions, and this is emulated by exposure, during the critical period, to food previously used by crowded larvae. We propose that Drosophila larvae have a distinct critical period, during which they assess both social and resource conditions, and that this irreversibly determines later visually guided social behavior. This model provides a platform towards understanding the regulation and development of social behavior.

Tubularity flow field--a technique for automatic neuron segmentation.

A segmentation framework is proposed to trace neurons from confocal microscopy images. With an increasing demand for high throughput neuronal image analysis, we propose an automated scheme to perform segmentation in a variational framework. Our segmentation technique, called tubularity flow field (TuFF) performs directional regional growing guided by the direction of tubularity of the neurites. We further address the problem of sporadic signal variation in confocal microscopy by designing a local attraction force field, which is able to bridge the gaps between local neurite fragments, even in the case of complete signal loss. Segmentation is performed in an integrated fashion by incorporating the directional region growing and the attraction force-based motion in a single framework using level sets. This segmentation is accomplished without manual seed point selection; it is automated. The performance of TuFF is demonstrated over a set of 2D and 3D confocal microscopy images where we report an improvement of >75% in terms of mean absolute error over three extensively used neuron segmentation algorithms. Two novel features of the variational solution, the evolution force and the attraction force, hold promise as contributions that can be employed in a number of image analysis applications.

Segmentation and tracing of single neurons from 3D confocal microscope images.

In order to understand the brain, we need to first understand the morphology of neurons. In the neurobiology community, there have been recent pushes to analyze both neuron connectivity and the influence of structure on function. Currently, a technical road block that stands in the way of these studies is the inability to automatically trace neuronal structure from microscopy. On the image processing side, proposed tracing algorithms face difficulties in low contrast, indistinct boundaries, clutter, and complex branching structure. To tackle these difficulties, we develop Tree2Tree, a robust automatic neuron segmentation and morphology generation algorithm. Tree2Tree uses a local medial tree generation strategy in combination with a global tree linking to build a maximum likelihood global tree. Recasting the neuron tracing problem in a graph-theoretic context enables Tree2Tree to estimate bifurcations naturally, which is currently a challenge for current neuron tracing algorithms. Tests on cluttered confocal microscopy images of Drosophila neurons give results that correspond to ground truth within a margin of ±2.75% normalized mean absolute error.

The simple fly larval visual system can process complex images.

Animals that have simple eyes are thought to only detect crude visual detail such as light level. However, predatory insect larvae using a small number of visual inputs seem to distinguish complex image targets. Here we show that Drosophila melanogaster larvae, which have 12 photoreceptor cells per hemisphere, are attracted to distinct motions of other, tethered larvae and that this recognition requires the visual system but not the olfactory system. In addition, attraction to tethered larvae still occurs across a clear plastic barrier, does not occur significantly in the dark and attraction occurs to a computer screen movie of larval motion. By altering the artificial attractant movie, we conclude that visual recognition involves both spatial and temporal components. Our results demonstrate that a simple but experimentally tractable visual system can distinguish complex images and that processing in the relatively large central brain may compensate for the simple input.

Serotonin: a regulator of neuronal morphology and circuitry.

Serotonin is an important neuromodulator associated with a wide range of physiological effects in the central nervous system. The exact mechanisms whereby serotonin influences brain development are not well understood, although studies in invertebrate and vertebrate model organisms are beginning to unravel a regulatory role for serotonin in neuronal morphology and circuit formation. Recent data suggest a developmental window during which altered serotonin levels permanently influence neuronal circuitry, however, the temporal constraints and molecular mechanisms responsible are still under investigation. Growing evidence suggests that alterations in early serotonin signaling contribute to a number of neurodevelopmental and neuropsychiatric disorders. Thus, understanding how altered serotonin signaling affects neuronal morphology and plasticity, and ultimately animal physiology and pathophysiology, will be of great significance.

Serotonergic dystrophy induced by excess serotonin.

Administration of certain serotonin-releasing amphetamine derivatives (fenfluramine and/or 3,4-methylenedioxymethamphetamine, MDMA, 'ecstasy') results in dystrophic serotonergic morphology in the mammalian brain. In addition to drug administration, dystrophic serotonergic neurites are also associated with neurodegenerative disorders. We demonstrate here that endogenously elevated serotonin in the Drosophila CNS induces aberrant enlarged varicosities, or spheroids, that are morphologically similar to dystrophic mammalian serotonergic fibers. In Drosophila these spheroids are specific to serotonergic neurons, distinct from typical varicosities, and form only after prolonged increases in cytoplasmic serotonin. Our results also suggest that serotonin levels during early development determine later sensitivity of spheroid formation to manipulations of the serotonin transporter (SERT). Elevated serotonin also interacts with canonical protein aggregation and autophagic pathways to form spheroids. The data presented here support a model in which excess cytoplasmic neurotransmitter triggers a cell-specific pathway inducing aberrant morphology in fly serotonergic neurons that may be shared in certain mammalian pathologies.

Both synthesis and reuptake are critical for replenishing the releasable serotonin pool in Drosophila.

The two main sources of serotonin available for release are expected to be newly synthesized serotonin and serotonin recycled after reuptake by the serotonin transporter. However, their relative importance for maintaining release and the time course of regulation are unknown. We studied serotonin signaling in the ventral nerve cord of the larval Drosophila CNS. Fast-scan cyclic voltammetry at implanted microelectrodes was used to detect serotonin elicited by channelrhodopsin2-mediated depolarization. The effects of reuptake were probed by incubating in cocaine, which is selective for the serotonin transporter in Drosophila. p-chlorophenylalanine, an inhibitor of tryptophan hydroxylase2, was used to investigate the effects of synthesis. Stimulations were repeated at various intervals to assess the time course of recovery of the releasable pool. Reuptake is important for the rapid replenishment of the releasable pool, on the 1 min time scale. Synthesis is critical to the longer-term replenishment (10 min) of the releasable pool, especially when reuptake is also inhibited. Concurrent synthesis and reuptake inhibition decreased both serotonin tissue content measured by immunohistochemistry (by 50%) and the initial amount of evoked serotonin (by 65%). Decreases in evoked serotonin are rescued by inhibiting action potential propagation with tetrodotoxin, implicating endogenous activity in the depletion. These results show synthesis is necessary to replenish part of the releasable serotonin pool that is depleted after reuptake inhibition, suggesting that regulation of synthesis may modulate the effects of serotonin reuptake inhibitors.

Detection of endogenous dopamine changes in Drosophila melanogaster using fast-scan cyclic voltammetry.

Drosophila melanogaster, the fruit fly, is a commonly used model organism because of its homology to mammals and facile genetic manipulations. However, the size of the nervous system is very small. We report a method to evoke and detect rapid changes in extracellular dopamine in a single nerve cord isolated from a Drosophila larva. Flies were genetically modified to express Channelrhodopsin-2, a blue-light activated cation channel, in only dopaminergic neurons. Extracellular dopamine changes were measured with fast-scan cyclic voltammetry at an implanted carbon-fiber microelectrode. Stimulations of 7 s with blue light result in an average peak dopamine concentration of 810 +/- 60 nM, similar to electrically-stimulated release in mammals. Stimulations repeated at 15 min intervals are stable for 65 min, allowing pharmacological experiments in the same sample. Peak duration is extended after cocaine or nisoxetine, inhibitors of the dopamine transporter (DAT). Release was reduced upon exposure to reserpine, which inhibits vesicular packaging. Chronic administration of NSD-1015, a dopamine synthesis inhibitor, decreased dopamine release and inhibited pupation, showing a link between neurotransmission and physiology. This is the first method to measure endogenous dopamine in an intact larval Drosophila nervous system and will allow studies of genetic and pharmacological manipulations of dopamine release and uptake.

Drosophila serotonergic varicosities are not distributed in a regular manner.

Neurons of the brain form complex tree-like structures that are critical for function. Here we examine the spatial pattern of serotonergic varicosities, the synaptic sites of serotonin release in the central nervous system (CNS). These varicosities are thought to form largely nonjunctional-type connections that partition in a grid-like manner in order to distribute evenly the neuromodulatory neurotransmitter serotonin. We describe the neuropil distribution of serotonergic varicosities in the brain and ventral nerve cord (VNC) of the larval Drosophila CNS. In the brain, we find evidence for avoidance between varicosities at distances lower than 1.75 microm. However, in the VNC, we find a clustered distribution. A similar clustered pattern is found in the Xenopus brain. This pattern produces many varicosities that are clustered together but also includes some varicosities that are very isolated. These isolated varicosities are not found along particular topological sections of the neurite tree or in particular locations in the CNS. In addition, the pattern breaks down when serotonergic branches of adjacent segments invade each other's territory. The pattern is similar to those described by a power law.

Quantitative evaluation of serotonin release and clearance in Drosophila.

Serotonin signaling plays a key role in the regulation of development, mood and behavior. Drosophila is well suited for the study of the basic mechanisms of serotonergic signaling, but the small size of its nervous system has previously precluded the direct measurements of neurotransmitters. This study demonstrates the first real-time measurements of changes in extracellular monoamine concentrations in a single larval Drosophila ventral nerve cord. Channelrhodopsin-2-mediated, neuronal type-specific stimulation is used to elicit endogenous serotonin release, which is detected using fast-scan cyclic voltammetry at an implanted microelectrode. Release is decreased when serotonin synthesis or packaging are pharmacologically inhibited, confirming that the detected substance is serotonin. Similar to tetanus-evoked serotonin release in mammals, evoked serotonin concentrations are 280-640nM in the fly, depending on the stimulation length. Extracellular serotonin signaling is prolonged after administering cocaine or fluoxetine, showing that transport regulates the clearance of serotonin from the extracellular space. When ChR2 is targeted to dopaminergic neurons, dopamine release is measured demonstrating that this method is broadly applicable to other neurotransmitter systems. This study shows that the dynamics of serotonin release and reuptake in Drosophila are analogous to those in mammals, making this simple organism more useful for the study of the basic physiological mechanisms of serotonergic signaling.

Branch architecture of the fly larval abdominal serotonergic neurons.

In the metazoan central nervous system (CNS), serotonergic neurons send projections throughout the synaptic neuropil. Little is known about the rules that govern these widespread neuromodulatory branching patterns. In this study, we utilize the Drosophila as a model to examine serotonergic branching. Using single cell GFP labeling we show that within each segment of the Drosophila ventral nerve cord (VNC), each of two serotonergic neurons tiles distinct innervation patterns in the contralateral neuropil. In addition, branches extend only a short distance from the target segment. Through ablation-mediated isolation of serotonergic cells, we demonstrate that the distinct areas of innervation are not maintained through competition between neighboring like-serotonergic neurites. Furthermore, the basic branching pattern of serotonergic neurons within the neuropil remains unchanged despite alterations of initial axonal trajectories.

A freeware java tool for spatial point analysis of neuronal structures.

Spatial point analysis is an analytical approach towards understanding patterns in the distribution of single points, such as synapses. To aid in this type of analysis of neuronal structures, a freeware tool, called PAJ, has been developed. This Java-based tool takes 3D Cartesian coordinates as input and performs a range of analyses to test for underlying patterns. In addition, Monte Carlo analysis is performed to compare experimental input with randomized input. This tool should be especially useful in determining whether neuronal structures are spatially patterned such that individual units interact with each other.

Developmental effects of SSRIs: lessons learned from animal studies.

Selective serotonin reuptake inhibitors (SSRIs) are utilized in the treatment of depression in pregnant and lactating women. SSRIs may be passed to the fetus through the placenta and the neonate through breastfeeding, potentially exposing them to SSRIs during peri- and postnatal development. However, the long-term effects of this SSRI exposure are still largely unknown. The simplicity and genetic amenability of model organisms provides a critical experimental advantage compared to studies with humans. This review will assess the current research done in animals that sheds light on the role of serotonin during development and the possible effects of SSRIs. Experimental studies in rodents show that administration of SSRIs during a key developmental window creates changes in brain circuitry and maladaptive behaviors that persist into adulthood. Similar changes result from the inhibition of the serotonin transporter or monoamine oxidase, implicating these two regulators of serotonin signaling in developmental changes. Understanding the role of serotonin in brain development is critical to identifying the possible effects of SSRI exposure.