Insect neurobiology: Oviposition crowd control.

A new study examines how Helicoverpa armigera females detect chemicals released by conspecific eggs in order to avoid laying more eggs on the same substrate. This work opens new avenues for basic research inquiries and offers a potential strategy for controlling insect pests.

A functional division of Drosophila sweet taste neurons that is value-based and task-specific.

Sucrose is an attractive feeding substance and a positive reinforcer for Drosophila But Drosophila females have been shown to robustly reject a sucrose-containing option for egg-laying when given a choice between a plain and a sucrose-containing option in specific contexts. How the sweet taste system of Drosophila promotes context-dependent devaluation of an egg-laying option that contains sucrose, an otherwise highly appetitive tastant, is unknown. Here, we report that devaluation of sweetness/sucrose for egg-laying is executed by a sensory pathway recruited specifically by the sweet neurons on the legs of Drosophila First, silencing just the leg sweet neurons caused acceptance of the sucrose option in a sucrose versus plain decision, whereas expressing the channelrhodopsin CsChrimson in them caused rejection of a plain option that was "baited" with light over another that was not. Analogous bidirectional manipulations of other sweet neurons did not produce these effects. Second, circuit tracing revealed that the leg sweet neurons receive different presynaptic neuromodulations compared to some other sweet neurons and were the only ones with postsynaptic partners that projected prominently to the superior lateral protocerebrum (SLP) in the brain. Third, silencing one specific SLP-projecting postsynaptic partner of the leg sweet neurons reduced sucrose rejection, whereas expressing CsChrimson in it promoted rejection of a light-baited option during egg-laying. These results uncover that the Drosophila sweet taste system exhibits a functional division that is value-based and task-specific, challenging the conventional view that the system adheres to a simple labeled-line coding scheme.

A neuropeptidergic circuit gates selective escape behavior of Drosophila larvae.

Animals display selective escape behaviors when faced with environmental threats. Selection of the appropriate response by the underlying neuronal network is key to maximizing chances of survival, yet the underlying network mechanisms are so far not fully understood. Using synapse-level reconstruction of the Drosophila larval network paired with physiological and behavioral readouts, we uncovered a circuit that gates selective escape behavior for noxious light through acute and input-specific neuropeptide action. Sensory neurons required for avoidance of noxious light and escape in response to harsh touch, each converge on discrete domains of neuromodulatory hub neurons. We show that acute release of hub neuron-derived insulin-like peptide 7 (Ilp7) and cognate relaxin family receptor (Lgr4) signaling in downstream neurons are required for noxious light avoidance, but not harsh touch responses. Our work highlights a role for compartmentalized circuit organization and neuropeptide release from regulatory hubs, acting as central circuit elements gating escape responses.

Molecular control limiting sensitivity of sweet taste neurons in Drosophila.

To assess the biological value of environmental stimuli, animals' sensory systems must accurately decode both the identities and the intensities of these stimuli. While much is known about the mechanism by which sensory neurons detect the identities of stimuli, less is known about the mechanism that controls how sensory neurons respond appropriately to different intensities of stimuli. The ionotropic receptor IR76b has been shown to be expressed in different Drosophila chemosensory neurons for sensing a variety of chemicals. Here, we show that IR76b plays an unexpected role in lowering the sensitivity of Drosophila sweet taste neurons. First, IR76b mutants exhibited clear behavioral responses to sucrose and acetic acid (AA) at concentrations that were too low to trigger observable behavioral responses from WT animals. Second, IR76b is expressed in many sweet neurons on the labellum, and these neurons responded to both sucrose and AA. Removing IR76b from the sweet neurons increased their neuronal responses as well as animals' behavioral responses to sucrose and AA. Conversely, overexpressing IR76b in the sweet neurons decreased their neuronal as well as animals' behavioral responses to sucrose and AA. Last, IR76b's response-lowering ability has specificity: IR76b mutants and WT showed comparable responses to capsaicin when the mammalian capsaicin receptor VR1 was ectopically expressed in their sweet neurons. Our findings suggest that sensitivity of Drosophila sweet neurons to their endogenous ligands is actively limited by IR76b and uncover a potential molecular target by which contexts can modulate sensitivity of sweet neurons.

Learning a Spatial Task by Trial and Error in Drosophila.

The ability to use memory to return to specific locations for foraging is advantageous for survival. Although recent reports have demonstrated that the fruit flies Drosophila melanogaster are capable of visual cue-driven place learning and idiothetic path integration [1-4], the depth and flexibility of Drosophila's ability to solve spatial tasks and the underlying neural substrate and genetic basis have not been extensively explored. Here, we show that Drosophila can remember a reward-baited location through reinforcement learning and do so quickly and without requiring vision. After gaining genetic access to neurons (through 0273-GAL4) with properties reminiscent of the vertebrate medial forebrain bundle (MFB) and developing a high-throughput closed-loop stimulation system, we found that both sighted and blind flies can learn-by trial and error-to repeatedly return to an unmarked location (in a rectangularly shaped arena) where a brief stimulation of the 0273-GAL4 neurons was available for each visit. We found that optogenetic stimulation of these neurons enabled learning by employing both a cholinergic pathway that promoted self-stimulation and a dopaminergic pathway that likely promoted association of relevant cues with reward. Lastly, inhibiting activities of specific neurons time-locked with stimulation of 0273-GAL4 neurons showed that mushroom bodies (MB) and central complex (CX) both play a role in promoting learning of our task. Our work uncovered new depth in flies' ability to learn a spatial task and established an assay with a level of throughput that permits a systematic genetic interrogation of flies' ability to learn spatial tasks.

Sweet neurons inhibit texture discrimination by signaling TMC-expressing mechanosensitive neurons in Drosophila.

Integration of stimuli of different modalities is an important but incompletely understood process during decision making. Here, we show that Drosophila are capable of integrating mechanosensory and chemosensory information of choice options when deciding where to deposit their eggs. Specifically, females switch from preferring the softer option for egg-laying when both options are sugar free to being indifferent between them when both contain sucrose. Such sucrose-induced indifference between options of different hardness requires functional sweet neurons, and, curiously, the Transmembrane Channel-like (TMC)-expressing mechanosensitive neurons that have been previously shown to promote discrimination of substrate hardness during feeding. Further, axons of sweet neurons directly contact axons of TMC-expressing neurons in the brain and stimulation of sweet neurons increases Ca2+ influx into axons of TMC-expressing neurons. These results uncover one mechanism by which Drosophila integrate taste and tactile information when deciding where to deposit their eggs and reveal that TMC-expressing neurons play opposing roles in hardness discrimination in two different decisions.

Serotonergic Modulation Enables Pathway-Specific Plasticity in a Developing Sensory Circuit in Drosophila.

How experiences during development cause long-lasting changes in sensory circuits and affect behavior in mature animals are poorly understood. Here we establish a novel system for mechanistic analysis of the plasticity of developing neural circuits by showing that sensory experience during development alters nociceptive behavior and circuit physiology in Drosophila larvae. Despite the convergence of nociceptive and mechanosensory inputs on common second-order neurons (SONs), developmental noxious input modifies transmission from nociceptors to their SONs, but not from mechanosensors to the same SONs, which suggests striking sensory pathway specificity. These SONs activate serotonergic neurons to inhibit nociceptor-to-SON transmission; stimulation of nociceptors during development sensitizes nociceptor presynapses to this feedback inhibition. Our results demonstrate that, unlike associative learning, which involves inputs from two sensory pathways, sensory pathway-specific plasticity in the Drosophila nociceptive circuit is in part established through feedback modulation. This study elucidates a novel mechanism that enables pathway-specific plasticity in sensory systems. VIDEO ABSTRACT.

Sensory integration and neuromodulatory feedback facilitate Drosophila mechanonociceptive behavior.

Nociception is an evolutionarily conserved mechanism to encode and process harmful environmental stimuli. Like most animals, Drosophila melanogaster larvae respond to a variety of nociceptive stimuli, including noxious touch and temperature, with stereotyped escape responses through activation of multimodal nociceptors. How behavioral responses to these different modalities are processed and integrated by the downstream network remains poorly understood. By combining trans-synaptic labeling, ultrastructural analysis, calcium imaging, optogenetics and behavioral analyses, we uncovered a circuit specific for mechanonociception but not thermonociception. Notably, integration of mechanosensory input from innocuous and nociceptive sensory neurons is required for robust mechanonociceptive responses. We further show that neurons integrating mechanosensory input facilitate primary nociceptive output by releasing short neuropeptide F, the Drosophila neuropeptide Y homolog. Our findings unveil how integration of somatosensory input and neuropeptide-mediated modulation can produce robust modality-specific escape behavior.

H2O2-Sensitive Isoforms of Drosophila melanogaster TRPA1 Act in Bitter-Sensing Gustatory Neurons to Promote Avoidance of UV During Egg-Laying.

The evolutionarily conserved TRPA1 channel can sense various stimuli including temperatures and chemical irritants. Recent results have suggested that specific isoforms of Drosophila TRPA1 (dTRPA1) are UV-sensitive and that their UV sensitivity is due to H2O2 sensitivity. However, whether such UV sensitivity served any physiological purposes in animal behavior was unclear. Here, we demonstrate that H2O2-sensitive dTRPA1 isoforms promote avoidance of UV when adult Drosophila females are selecting sites for egg-laying. First, we show that blind/visionless females are still capable of sensing and avoiding UV during egg-laying when intensity of UV is high yet within the range of natural sunlight. Second, we show that such vision-independent UV avoidance is mediated by a group of bitter-sensing neurons on the proboscis that express H2O2-sensitive dTRPA1 isoforms. We show that these bitter-sensing neurons exhibit dTRPA1-dependent UV sensitivity. Importantly, inhibiting activities of these bitter-sensing neurons, reducing their dTRPA1 expression, or reducing their H2O2-sensitivity all significantly reduced blind females' UV avoidance, whereas selectively restoring a H2O2-sensitive isoform of dTRPA1 in these neurons restored UV avoidance. Lastly, we show that specifically expressing the red-shifted channelrhodopsin CsChrimson in these bitter-sensing neurons promotes egg-laying avoidance of red light, an otherwise neutral cue for egg-laying females. Together, these results demonstrate a physiological role of the UV-sensitive dTRPA1 isoforms, reveal that adult Drosophila possess at least two sensory systems for detecting UV, and uncover an unexpected role of bitter-sensing taste neurons in UV sensing.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster.

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the "greater of two goods." However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2.

The transient receptor potential A1 (TRPA1) channel is an evolutionarily conserved detector of temperature and irritant chemicals. Here, we show that two specific isoforms of TRPA1 in Drosophila are H2O2 sensitive and that they can detect strong UV light via sensing light-induced production of H2O2. We found that ectopic expression of these H2O2-sensitive Drosophila TRPA1 (dTRPA1) isoforms conferred UV sensitivity to light-insensitive HEK293 cells and Drosophila neurons, whereas expressing the H2O2-insensitive isoform did not. Curiously, when expressed in one specific group of motor neurons in adult flies, the H2O2-sensitive dTRPA1 isoforms were as competent as the blue light-gated channelrhodopsin-2 in triggering motor output in response to light. We found that the corpus cardiacum (CC) cells, a group of neuroendocrine cells that produce the adipokinetic hormone (AKH) in the larval ring gland endogenously express these H2O2-sensitive dTRPA1 isoforms and that they are UV sensitive. Sensitivity of CC cells required dTRPA1 and H2O2 production but not conventional phototransduction molecules. Our results suggest that specific isoforms of dTRPA1 can sense UV light via photochemical production of H2O2. We speculate that UV sensitivity conferred by these isoforms in CC cells may allow young larvae to activate stress response--a function of CC cells--when they encounter strong UV, an aversive stimulus for young larvae.

Analyzing animal behavior via classifying each video frame using convolutional neural networks.

High-throughput analysis of animal behavior requires software to analyze videos. Such software analyzes each frame individually, detecting animals' body parts. But the image analysis rarely attempts to recognize "behavioral states"-e.g., actions or facial expressions-directly from the image instead of using the detected body parts. Here, we show that convolutional neural networks (CNNs)-a machine learning approach that recently became the leading technique for object recognition, human pose estimation, and human action recognition-were able to recognize directly from images whether Drosophila were "on" (standing or walking) or "off" (not in physical contact with) egg-laying substrates for each frame of our videos. We used multiple nets and image transformations to optimize accuracy for our classification task, achieving a surprisingly low error rate of just 0.072%. Classifying one of our 8 h videos took less than 3 h using a fast GPU. The approach enabled uncovering a novel egg-laying-induced behavior modification in Drosophila. Furthermore, it should be readily applicable to other behavior analysis tasks.

Long-duration animal tracking in difficult lighting conditions.

High-throughput analysis of animal behavior requires software to analyze videos. Such software typically depends on the experiments' being performed in good lighting conditions, but this ideal is difficult or impossible to achieve for certain classes of experiments. Here, we describe techniques that allow long-duration positional tracking in difficult lighting conditions with strong shadows or recurring "on"/"off" changes in lighting. The latter condition will likely become increasingly common, e.g., for Drosophila due to the advent of red-shifted channel rhodopsins. The techniques enabled tracking with good accuracy in three types of experiments with difficult lighting conditions in our lab. Our technique handling shadows relies on single-animal tracking and on shadows' and flies' being accurately distinguishable by distance to the center of the arena (or a similar geometric rule); the other techniques should be broadly applicable. We implemented the techniques as extensions of the widely-used tracking software Ctrax; however, they are relatively simple, not specific to Drosophila, and could be added to other trackers as well.

Behavioral and circuit basis of sucrose rejection by Drosophila females in a simple decision-making task.

Drosophila melanogaster egg-laying site selection offers a genetic model to study a simple form of value-based decision. We have previously shown that Drosophila females consistently reject a sucrose-containing substrate and choose a plain (sucrose-free) substrate for egg laying in our sucrose versus plain decision assay. However, either substrate is accepted when it is the sole option. Here we describe the neural mechanism that underlies females' sucrose rejection in our sucrose versus plain assay. First, we demonstrate that females explored the sucrose substrate frequently before most egg-laying events, suggesting that they actively suppress laying eggs on the sucrose substrate as opposed to avoiding visits to it. Second, we show that activating a specific subset of DA neurons triggered a preference for laying eggs on the sucrose substrate over the plain one, suggesting that activating these DA neurons can increase the value of the sucrose substrate for egg laying. Third, we demonstrate that neither ablating nor inhibiting the mushroom body (MB), a known Drosophila learning and decision center, affected females' egg-laying preferences in our sucrose versus plain assay, suggesting that MB does not mediate this specific decision-making task. We propose that the value of a sucrose substrate- as an egg-laying option-can be adjusted by the activities of a specific DA circuit. Once the sucrose substrate is determined to be the lesser valued option, females execute their decision to reject this inferior substrate not by stopping their visits to it, but by actively suppressing their egg-laying motor program during their visits.

Egg-laying demand induces aversion of UV light in Drosophila females.

Drosophila melanogaster females are highly selective about the chemosensory quality of their egg-laying sites, an important trait that promotes the survival and fitness of their offspring. How egg-laying females respond to UV light is not known, however. UV is a well-documented phototactic cue for adult Drosophila, but it is an aversive cue for larvae. Here, we show that female flies exhibit UV aversion in response to their egg-laying demand. First, females exhibit egg-laying aversion of UV: they prefer to lay eggs on dark sites when choosing between UV-illuminated and dark sites. Second, they also exhibit movement aversion of UV: positional tracking of single females suggests that egg-laying demand increases their tendency to turn away from UV. Genetic manipulations of the retina suggest that egg-laying and movement aversion of UV are both mediated by the inner (R7) and not the outer (R1-R6) photoreceptors. Finally, we show that the Dm8 amacrine neurons, a synaptic target of R7 photoreceptors and a mediator of UV spectral preference, are dispensable for egg-laying aversion but essential for movement aversion of UV. This study suggests that egg-laying demand can temporarily convert UV into an aversive cue for female Drosophila and that R7 photoreceptors recruit different downstream targets to control different egg-laying-induced behavioral modifications.

Mechanosensitive neurons on the internal reproductive tract contribute to egg-laying-induced acetic acid attraction in Drosophila.

Selecting a suitable site to deposit their eggs is an important reproductive need of Drosophila females. Although their choosiness toward egg-laying sites is well documented, the specific neural mechanism that activates females' search for attractive egg-laying sites is not known. Here, we show that distention and contraction of females' internal reproductive tract triggered by egg delivery through the tract plays a critical role in activating such search. We found that females start to exhibit acetic acid (AA) attraction prior to depositing each egg but no attraction when they are not laying eggs. Artificially distending the reproductive tract triggers AA attraction in non-egg-laying females, whereas silencing the mechanosensitive neurons we identified that can sense the contractile status of the tract eliminates such attraction. Our work uncovers the circuit basis of an important reproductive need of Drosophila females and provides a simple model for dissecting the neural mechanism that underlies a reproductive need-induced behavioral modification.

Unveiling the secrets to her heart.

Before consenting to copulate, a female fruit fly gauges both her mating status and her suitor's quality. Three recent studies, Bussell et al. (2014) in Current Biology and Feng et al. (2014) and Zhou et al. (2014) in this issue of Neuron, dissected the receptivity circuit of female Drosophila, providing insights into how she integrates courtship cues, assesses her internal state, and directs the motor programs that facilitate copulation.

Female contact modulates male aggression via a sexually dimorphic GABAergic circuit in Drosophila.

Intraspecific male-male aggression, which is important for sexual selection, is regulated by environment, experience and internal states through largely undefined molecular and cellular mechanisms. To understand the basic neural pathway underlying the modulation of this innate behavior, we established a behavioral assay in Drosophila melanogaster and investigated the relationship between sexual experience and aggression. In the presence of mating partners, adult male flies exhibited elevated levels of aggression, which was largely suppressed by prior exposure to females via a sexually dimorphic neural mechanism. The suppression involved the ability of male flies to detect females by contact chemosensation through the pheromone-sensing ion channel ppk29 and was mediated by male-specific GABAergic neurons acting on the GABAA receptor RDL in target cells. Silencing or activating this circuit led to dis-inhibition or elimination of sex-related aggression, respectively. We propose that the GABAergic inhibition represents a critical cellular mechanism that enables prior experience to modulate aggression.

The Drosophila Cadherin Fat regulates tissue size and planar cell polarity through different domains.

The Drosophila Cadherin Fat (Ft) has been identified as a crucial regulator of tissue size and Planar Cell Polarity (PCP). However, the precise mechanism by which Ft regulates these processes remains unclear. In order to advance our understanding of the action of Ft, we have sought to identify the crucial Ft effector domains. Here we report that a small region of the Ft cytoplasmic domain (H2 region) is both necessary and sufficient, when membrane localized, to support viability and prevent tissue overgrowth. Interestingly, the H2 region is dispensable for regulating PCP signaling, whereas the mutant Ft lacking the H2 region is fully capable of directing PCP. This result suggests that Ft's roles in PCP signaling and tissue size control are separable, and each can be carried out independently. Surprisingly, the crucial regions of Ft identified in our structure-function study do not overlap with the previously reported interaction regions with Atrophin, Dco, or Lowfat.