The function of the central nervous system as it controls sex-specific behaviors in Drosophila has been studied with renewed intensity, in the context of genetic factors that influence the development of sexually differentiated aspects of this insect. Three categories of genetic variations that cause anomalies in courtship and mating behaviors are discussed: (1) mutants isolated with regard to courtship defects, of which putatively courtship-specific variants such as the fruitless mutant are a subset; (2) general behavioral and neurological variants (including sensory and learning mutants), whose defects include subnormal reproductive performance; and (3) mutations of genes within the sex-determination regulatory hierarchy of Drosophila, the analysis of which has included studies of reproductive behavior. Recent studies of mutations in two of these categories have provided new insights into the control of neuronally based aspects of sex-specific behavior. The doublesex gene, the final factor acting in the sex-determination hierarchy, had been previously thought to regulate all aspects of sexual differentiation. Yet, it has been recently shown that doublesex does not control at least one neuronally-determined feature of sex-specific anatomy--a muscle in the male's abdomen, whose normal development is, however, dependent on the action of fruitless. These considerations prompted us to examine further (and in some cases re-examine) the influences exerted by sex-determination hierarchy genes on behavior. Our results--notably those obtained from assessments of doublesex mutations' effects on general reproductive actions and on a particular component of the courtship sequence (male "singing" behavior)--lead to the suggestion that there is a previously unrecognized branch within the sex-determination hierarchy, which controls the differentiation of the male- and female- specific phenotypes of Drosophila. This new branch separates from the doublesex-related one immediately before the action of that gene (just after transformer and transformer-2) and appears to control as least some aspects of neuronally determined sexual differentiation of males.

Animals avoid predators and find the best food and mates by learning from the consequences of their behavior. However, reinforcers are not always uniquely appetitive or aversive but can have complex properties. Most intoxicating substances fall within this category; provoking aversive sensory and physiological reactions while simultaneously inducing overwhelming appetitive properties. Here we describe the subtle behavioral features associated with continued seeking for alcohol despite aversive consequences. We developed an automated runway apparatus to measure how Drosophila respond to consecutive exposures of a volatilized substance. Behavior within this Behavioral Expression of Ethanol Reinforcement Runway (BEER Run) demonstrated a defined shift from aversive to appetitive responses to volatilized ethanol. Behavioral metrics attained by combining computer vision and machine learning methods, reveal that a subset of 9 classified behaviors and component behavioral features associate with this shift. We propose this combination of 9 be

The neural circuit mechanisms underlying emotion states remain poorly understood. Drosophila offers powerful genetic approaches for dissecting neural circuit function, but whether flies exhibit emotion-like behaviors has not been clear. We recently proposed that model organisms may express internal states displaying “emotion primitives,” which are general characteristics common to different emotions, rather than specific anthropomorphic emotions such as “fear” or “anxiety.” These emotion primitives include scalability, persistence, valence, and generalization to multiple contexts. Here, we have applied this approach to determine whether flies’ defensive responses to moving overhead translational stimuli (“shadows”) are purely reflexive or may express underlying emotion states. We describe a new behavioral assay in which flies confined in an enclosed arena are repeatedly exposed to an overhead translational stimulus. Repetitive stimuli promoted graded (scalable) and persistent increases in locomotor velocity and hopping, and occasional freezing. The stimulus also dispersed feeding flies from a food resource, suggesting both negative valence and context generalization. Strikingly, there was a significant delay before the flies returned to the food following stimulus-induced dispersal, suggestive of a slowly decaying internal defensive state. The length of this delay was increased when more stimuli were delivered for initial dispersal. These responses can be mathematically modeled by assuming an internal state that behaves as a leaky integrator of stimulus exposure. Our results suggest that flies’ responses to repetitive visual threat stimuli express an internal state exhibiting canonical emotion primitives, possibly analogous to fear in mammals. The mechanistic basis of this state can now be investigated in a genetically tractable insect species.

Brains encode behaviors using neurons amenable to systematic classification by gene expression. The contribution of molecular identity to neural coding is not understood because of the challenges involved with measuring neural dynamics and molecular information from the same cells. We developed CaRMA (calcium and RNA multiplexed activity) imaging based on recording in vivo single-neuron calcium dynamics followed by gene expression analysis. We simultaneously monitored activity in hundreds of neurons in mouse paraventricular hypothalamus (PVH). Combinations of cell-type marker genes had predictive power for neuronal responses across 11 behavioral states. The PVH uses combinatorial assemblies of molecularly defined neuron populations for grouped-ensemble coding of survival behaviors. The neuropeptide receptor neuropeptide Y receptor type 1 (Npy1r) amalgamated multiple cell types with similar responses. Our results show that molecularly defined neurons are important processing units for brain function.

The behavioral state of an animal can dynamically modulate visual processing. In flies, the behavioral state is known to alter the temporal tuning of neurons that carry visual motion information into the central brain. However, where this modulation occurs and how it tunes the properties of this neural circuit are not well understood. Here, we show that the behavioral state alters the baseline activity levels and the temporal tuning of the first directionally selective neuron in the ON motion pathway (T4) as well as its primary input neurons (Mi1, Tm3, Mi4, Mi9). These effects are especially prominent in the inhibitory neuron Mi4, and we show that central octopaminergic neurons provide input to Mi4 and increase its excitability. We further show that octopamine neurons are required for sustained behavioral responses to fast-moving, but not slow-moving, visual stimuli in walking flies. These results indicate that behavioral-state modulation acts directly on the inputs to the directionally selective neurons and supports efficient neural coding of motion stimuli.

Insulin signaling plays a pivotal role in metabolic control and aging, and insulin accordingly is a key factor in several human diseases. Despite this importance, the in vivo activity dynamics of insulin-producing cells (IPCs) are poorly understood. Here, we characterized the effects of locomotion on the activity of IPCs in Drosophila. Using in vivo electrophysiology and calcium imaging, we found that IPCs were strongly inhibited during walking and flight and that their activity rebounded and overshot after cessation of locomotion. Moreover, IPC activity changed rapidly during behavioral transitions, revealing that IPCs are modulated on fast timescales in behaving animals. Optogenetic activation of locomotor networks ex vivo, in the absence of actual locomotion or changes in hemolymph sugar levels, was sufficient to inhibit IPCs. This demonstrates that the behavioral state-dependent inhibition of IPCs is actively controlled by neuronal pathways and is independent of changes in glucose concentration. By contrast, the overshoot in IPC activity after locomotion was absent ex vivo and after starvation, indicating that it was not purely driven by feedforward signals but additionally required feedback derived from changes in hemolymph sugar concentration. We hypothesize that IPC inhibition during locomotion supports mobilization of fuel stores during metabolically demanding behaviors, while the rebound in IPC activity after locomotion contributes to replenishing muscle glycogen stores. In addition, the rapid dynamics of IPC modulation support a potential role of insulin in the state-dependent modulation of sensorimotor processing.

Learning is primarily mediated by activity-dependent modifications of synaptic strength within neuronal circuits. We discovered that place fields in hippocampal area CA1 are produced by a synaptic potentiation notably different from Hebbian plasticity. Place fields could be produced in vivo in a single trial by potentiation of input that arrived seconds before and after complex spiking. The potentiated synaptic input was not initially coincident with action potentials or depolarization. This rule, named behavioral time scale synaptic plasticity, abruptly modifies inputs that were neither causal nor close in time to postsynaptic activation. In slices, five pairings of subthreshold presynaptic activity and calcium (Ca(2+)) plateau potentials produced a large potentiation with an asymmetric seconds-long time course. This plasticity efficiently stores entire behavioral sequences within synaptic weights to produce predictive place cell activity.

Behavioral choices that ignore prior experience promote exploration and unpredictability but are seemingly at odds with the brain's tendency to use experience to optimize behavioral choice. Indeed, when faced with virtual competitors, primates resort to strategic counterprediction rather than to stochastic choice. Here, we show that rats also use history- and model-based strategies when faced with similar competitors but can switch to a "stochastic" mode when challenged with a competitor that they cannot defeat by counterprediction. In this mode, outcomes associated with an animal's actions are ignored, and normal engagement of anterior cingulate cortex (ACC) is suppressed. Using circuit perturbations in transgenic rats, we demonstrate that switching between strategic and stochastic behavioral modes is controlled by locus coeruleus input into ACC. Our findings suggest that, under conditions of uncertainty about environmental rules, changes in noradrenergic input alter ACC output and prevent erroneous beliefs from guiding decisions, thus enabling behavioral variation.

Animals need to flexibly respond to stimuli from their environment without compromising behavioural consistency. For example, female crickets orienting toward a conspecific male's calling song in search of a mating partner need to stay responsive to other signals that provide information about obstacles and predators. Here, we investigate how spontaneously walking crickets and crickets engaging in acoustically guided goal-directed navigation, i.e. phonotaxis, respond to mechanosensory stimuli detected by their long antennae. We monitored walking behaviour of female crickets on a trackball during lateral antennal stimulation, which was achieved by moving a wire mesh transiently into reach of one antenna. During antennal stimulation alone, females reduced their walking speed, oriented toward the object and actively explored it with antennal movements. Additionally, some crickets initially turned away from the approaching object. Females responded in a similar way when the antennal stimulus was presented during ongoing phonotaxis: forward velocity was reduced and phonotactic steering was suppressed while the females turned toward and explored the object. Further, rapid steering bouts to individual chirps, typical for female phonotaxis, no longer occurred.Our data reveals that in this experimental situation antennal stimulation overrides phonotaxis for extended time periods. Phonotaxis in natural environments, which require the integration of multiple sensory cues, may therefore be more variable than phonotaxis measured under ideal laboratory conditions. Combining this new behavioural paradigm with neurophysiological methods will show where the sensory-motor integration of antennal and acoustic stimulation occurs and how this is achieved on a mechanistic level.

The visual allure of microscopy makes it an intuitively powerful research tool. Intuition, however, can easily obscure or distort the reality of the information contained in an image. Common cognitive biases, combined with institutional pressures that reward positive research results, can quickly skew a microscopy project towards upholding, rather than rigorously challenging, a hypothesis. The impact of these biases on a variety of research topics is well known. What might be less appreciated are the many forms in which bias can permeate a microscopy experiment. Even well-intentioned researchers are susceptible to bias, which must therefore be actively recognized to be mitigated. Importantly, although image quantification has increasingly become an expectation, ostensibly to confront subtle biases, it is not a guarantee against bias and cannot alone shield an experiment from cognitive distortions. Here, we provide illustrative examples of the insidiously pervasive nature of bias in microscopy experiments - from initial experimental design to image acquisition, analysis and data interpretation. We then provide suggestions that can serve as guard rails against bias.