Neurons and behavior: ex uno, plures.

Li et al. demonstrate that a single interneuron can regulate analog- and digital-like behaviors guided by two different postsynaptic neurons. Releasing a single neurotransmitter onto downstream neurons that express receptors with distinct biophysical properties enables a small set of neurons to direct a range of functional responses.

Neurofibromin 1 mediates sleep depth in Drosophila.

Neural regulation of sleep and metabolic homeostasis are critical in many aspects of human health. Despite extensive epidemiological evidence linking sleep dysregulation with obesity, diabetes, and metabolic syndrome, little is known about the neural and molecular basis for the integration of sleep and metabolic function. The RAS GTPase-activating gene Neurofibromin (Nf1) has been implicated in the regulation of sleep and metabolic rate, raising the possibility that it serves to integrate these processes, but the effects on sleep consolidation and physiology remain poorly understood. A key hallmark of sleep depth in mammals and flies is a reduction in metabolic rate during sleep. Here, we examine multiple measures of sleep quality to determine the effects of Nf1 on sleep-dependent changes in arousal threshold and metabolic rate. Flies lacking Nf1 fail to suppress metabolic rate during sleep, raising the possibility that loss of Nf1 prevents flies from integrating sleep and metabolic state. Sleep of Nf1 mutant flies is fragmented with a reduced arousal threshold in Nf1 mutants, suggesting Nf1 flies fail to enter deep sleep. The effects of Nf1 on sleep can be localized to a subset of neurons expressing the GABAA receptor Rdl. Sleep loss has been associated with changes in gut homeostasis in flies and mammals. Selective knockdown of Nf1 in Rdl-expressing neurons within the nervous system increases gut permeability and reactive oxygen species (ROS) in the gut, raising the possibility that loss of sleep quality contributes to gut dysregulation. Together, these findings suggest Nf1 acts in GABA-sensitive neurons to modulate sleep depth in Drosophila.

Modeling neurodegenerative and neurodevelopmental disorders in the Drosophila mushroom body.

The common fruit fly Drosophila melanogaster provides a powerful platform to investigate the genetic, molecular, cellular, and neural circuit mechanisms of behavior. Research in this model system has shed light on multiple aspects of brain physiology and behavior, from fundamental neuronal function to complex behaviors. A major anatomical region that modulates complex behaviors is the mushroom body (MB). The MB integrates multimodal sensory information and is involved in behaviors ranging from sensory processing/responses to learning and memory. Many genes that underlie brain disorders are conserved, from flies to humans, and studies in Drosophila have contributed significantly to our understanding of the mechanisms of brain disorders. Genetic mutations that mimic human diseases-such as Fragile X syndrome, neurofibromatosis type 1, Parkinson's disease, and Alzheimer's disease-affect MB structure and function, altering behavior. Studies dissecting the effects of disease-causing mutations in the MB have identified key pathological mechanisms, and the development of a complete connectome promises to add a comprehensive anatomical framework for disease modeling. Here, we review Drosophila models of human neurodevelopmental and neurodegenerative disorders via the effects of their underlying mutations on MB structure, function, and the resulting behavioral alterations.

Dopamine, sleep, and neuronal excitability modulate amyloid-ß-mediated forgetting in Drosophila.

Alzheimer disease (AD) is one of the main causes of age-related dementia and neurodegeneration. However, the onset of the disease and the mechanisms causing cognitive defects are not well understood. Aggregation of amyloidogenic peptides is a pathological hallmark of AD and is assumed to be a central component of the molecular disease pathways. Pan-neuronal expression of Aß42Arctic peptides in Drosophila melanogaster results in learning and memory defects. Surprisingly, targeted expression to the mushroom bodies, a center for olfactory memories in the fly brain, does not interfere with learning but accelerates forgetting. We show here that reducing neuronal excitability either by feeding Levetiracetam or silencing of neurons in the involved circuitry ameliorates the phenotype. Furthermore, inhibition of the Rac-regulated forgetting pathway could rescue the Aß42Arctic-mediated accelerated forgetting phenotype. Similar effects are achieved by increasing sleep, a critical regulator of neuronal homeostasis. Our results provide a functional framework connecting forgetting signaling and sleep, which are critical for regulating neuronal excitability and homeostasis and are therefore a promising mechanism to modulate forgetting caused by toxic Aß peptides.

Developmental loss of neurofibromin across distributed neuronal circuits drives excessive grooming in Drosophila.

Neurofibromatosis type 1 is a monogenetic disorder that predisposes individuals to tumor formation and cognitive and behavioral symptoms. The neuronal circuitry and developmental events underlying these neurological symptoms are unknown. To better understand how mutations of the underlying gene (NF1) drive behavioral alterations, we have examined grooming in the Drosophila neurofibromatosis 1 model. Mutations of the fly NF1 ortholog drive excessive grooming, and increased grooming was observed in adults when Nf1 was knocked down during development. Furthermore, intact Nf1 Ras GAP-related domain signaling was required to maintain normal grooming. The requirement for Nf1 was distributed across neuronal circuits, which were additive when targeted in parallel, rather than mapping to discrete microcircuits. Overall, these data suggest that broadly-distributed alterations in neuronal function during development, requiring intact Ras signaling, drive key Nf1-mediated behavioral alterations. Thus, global developmental alterations in brain circuits/systems function may contribute to behavioral phenotypes in neurofibromatosis type 1.

Cyclic AMP-dependent plasticity underlies rapid changes in odor coding associated with reward learning.

Learning and memory rely on dopamine and downstream cAMP-dependent plasticity across diverse organisms. Despite the central role of cAMP signaling, it is not known how cAMP-dependent plasticity drives coherent changes in neuronal physiology that encode the memory trace, or engram. In Drosophila, the mushroom body (MB) is critically involved in olfactory classical conditioning, and cAMP signaling molecules are necessary and sufficient for normal memory in intrinsic MB neurons. To evaluate the role of cAMP-dependent plasticity in learning, we examined how cAMP manipulations and olfactory classical conditioning modulate olfactory responses in the MB with in vivo imaging. Elevating cAMP pharmacologically or optogenetically produced plasticity in MB neurons, altering their responses to odorants. Odor-evoked Ca2+ responses showed net facilitation across anatomical regions. At the single-cell level, neurons exhibited heterogeneous responses to cAMP elevation, suggesting that cAMP drives plasticity to discrete subsets of MB neurons. Olfactory appetitive conditioning enhanced MB odor responses, mimicking the cAMP-dependent plasticity in directionality and magnitude. Elevating cAMP to equivalent levels as appetitive conditioning also produced plasticity, suggesting that the cAMP generated during conditioning affects odor-evoked responses in the MB. Finally, we found that this plasticity was dependent on the Rutabaga type I adenylyl cyclase, linking cAMP-dependent plasticity to behavioral modification. Overall, these data demonstrate that learning produces robust cAMP-dependent plasticity in intrinsic MB neurons, which is biased toward naturalistic reward learning. This suggests that cAMP signaling may serve to modulate intrinsic MB responses toward salient stimuli.

Cellular and circuit mechanisms of olfactory associative learning in Drosophila.

Recent years have witnessed significant progress in understanding how memories are encoded, from the molecular to the cellular and the circuit/systems levels. With a good compromise between brain complexity and behavioral sophistication, the fruit fly Drosophila melanogaster is one of the preeminent animal models of learning and memory. Here we review how memories are encoded in Drosophila, with a focus on short-term memory and an eye toward future directions. Forward genetic screens have revealed a large number of genes and transcripts necessary for learning and memory, some acting cell-autonomously. Further, the relative numerical simplicity of the fly brain has enabled the reverse engineering of learning circuits with remarkable precision, in some cases ascribing behavioral phenotypes to single neurons. Functional imaging and physiological studies have localized and parsed the plasticity that occurs during learning at some of the major loci. Connectomics projects are significantly expanding anatomical knowledge of the nervous system, filling out the roadmap for ongoing functional/physiological and behavioral studies, which are being accelerated by simultaneous tool development. These developments have provided unprecedented insight into the fundamental neural principles of learning, and lay the groundwork for deep understanding in the near future.

Elongator complex is required for long-term olfactory memory formation in Drosophila.

The evolutionarily conserved Elongator Complex associates with RNA polymerase II for transcriptional elongation. Elp3 is the catalytic subunit, contains histone acetyltransferase activity, and is associated with neurodegeneration in humans. Elp1 is a scaffolding subunit and when mutated causes familial dysautonomia. Here, we show that elp3 and elp1 are required for aversive long-term olfactory memory in Drosophila RNAi knockdown of elp3 in adult mushroom bodies impairs long-term memory (LTM) without affecting earlier forms of memory. RNAi knockdown with coexpression of elp3 cDNA reverses the impairment. Similarly, RNAi knockdown of elp1 impairs LTM and coexpression of elp1 cDNA reverses this phenotype. The LTM deficit in elp3 and elp1 knockdown flies is accompanied by the abolishment of a LTM trace, which is registered as increased calcium influx in response to the CS+ odor in the α-branch of mushroom body neurons. Coexpression of elp1 or elp3 cDNA rescues the memory trace in parallel with LTM. These data show that the Elongator complex is required in adult mushroom body neurons for long-term behavioral memory and the associated long-term memory trace.

Dopaminergic neurons encode a distributed, asymmetric representation of temperature in Drosophila.

Dopaminergic circuits modulate a wide variety of innate and learned behaviors in animals, including olfactory associative learning, arousal, and temperature-preference behavior. It is not known whether distinct or overlapping sets of dopaminergic neurons modulate these behaviors. Here, I have functionally characterized the dopaminergic circuits innervating the Drosophila mushroom body with in vivo calcium imaging and conditional silencing of genetically defined subsets of neurons. Distinct subsets of PPL1 dopaminergic neurons innervating the vertical lobes of the mushroom body responded to decreases in temperature, but not increases, with rapidly adapting bursts of activity. PAM neurons innervating the horizontal lobes did not respond to temperature shifts. Ablation of the antennae and maxillary palps reduced, but did not eliminate, the responses. Genetic silencing of dopaminergic neurons innervating the vertical mushroom body lobes substantially reduced behavioral cold avoidance, but silencing smaller subsets of these neurons had no effect. These data demonstrate that overlapping dopaminergic circuits encode a broadly distributed, asymmetric representation of temperature that overlays regions implicated previously in learning, memory, and forgetting. Thus, diverse behaviors engage overlapping sets of dopaminergic neurons that encode multimodal stimuli and innervate a single anatomical target, the mushroom body.

Unraveling neuronal and metabolic alterations in neurofibromatosis type 1.

Neurofibromatosis type 1 (OMIM 162200) affects ~ 1 in 3,000 individuals worldwide and is one of the most common monogenetic neurogenetic disorders that impacts brain function. The disorder affects various organ systems, including the central nervous system, resulting in a spectrum of clinical manifestations. Significant progress has been made in understanding the disorder's pathophysiology, yet gaps persist in understanding how the complex signaling and systemic interactions affect the disorder. Two features of the disorder are alterations in neuronal function and metabolism, and emerging evidence suggests a potential relationship between them. This review summarizes neurofibromatosis type 1 features and recent research findings on disease mechanisms, with an emphasis on neuronal and metabolic features.

OTUD6 deubiquitination of RPS7/eS7 on the free 40 S ribosome regulates global protein translation and stress.

Ribosomes are regulated by evolutionarily conserved ubiquitination/deubiquitination events. We uncover the role of the deubiquitinase OTUD6 in regulating global protein translation through deubiquitination of the RPS7/eS7 subunit on the free 40 S ribosome in vivo in Drosophila. Coimmunoprecipitation and enrichment of monoubiquitinated proteins from catalytically inactive OTUD6 flies reveal RPS7 as the ribosomal substrate. The 40 S protein RACK1 and E3 ligases CNOT4 and RNF10 function upstream of OTUD6 to regulate alkylation stress. OTUD6 interacts with RPS7 specifically on the free 40 S, and not on 43 S/48 S initiation complexes or the translating ribosome. Global protein translation levels are bidirectionally regulated by OTUD6 protein abundance. OTUD6 protein abundance is physiologically regulated in aging and in response to translational and alkylation stress. Thus, OTUD6 may promote translation initiation, the rate limiting step in protein translation, by titering the amount of 40 S ribosome that recycles.

Neurofibromin deficiency alters the patterning and prioritization of motor behaviors in a state-dependent manner.

Genetic disorders such as neurofibromatosis type 1 increase vulnerability to cognitive and behavioral disorders, such as autism spectrum disorder and attention-deficit/hyperactivity disorder. Neurofibromatosis type 1 results from loss-of-function mutations in the neurofibromin gene and subsequent reduction in the neurofibromin protein (Nf1). While the mechanisms have yet to be fully elucidated, loss of Nf1 may alter neuronal circuit activity leading to changes in behavior and susceptibility to cognitive and behavioral comorbidities. Here we show that mutations decreasing Nf1 expression alter motor behaviors, impacting the patterning, prioritization, and behavioral state dependence in a Drosophila model of neurofibromatosis type 1. Loss of Nf1 increases spontaneous grooming in a nonlinear spatial and temporal pattern, differentially increasing grooming of certain body parts, including the abdomen, head, and wings. This increase in grooming could be overridden by hunger in food-deprived foraging animals, demonstrating that the Nf1 effect is plastic and internal state-dependent. Stimulus-evoked grooming patterns were altered as well, with nf1 mutants exhibiting reductions in wing grooming when coated with dust, suggesting that hierarchical recruitment of grooming command circuits was altered. Yet loss of Nf1 in sensory neurons and/or grooming command neurons did not alter grooming frequency, suggesting that Nf1 affects grooming via higher-order circuit alterations. Changes in grooming coincided with alterations in walking. Flies lacking Nf1 walked with increased forward velocity on a spherical treadmill, yet there was no detectable change in leg kinematics or gait. Thus, loss of Nf1 alters motor function without affecting overall motor coordination, in contrast to other genetic disorders that impair coordination. Overall, these results demonstrate that loss of Nf1 alters the patterning and prioritization of repetitive behaviors, in a state-dependent manner, without affecting motor coordination.

Ex Vivo Brain Imaging in Drosophila.

Analysis of neuronal circuit function in Drosophila can be facilitated with an ex vivo imaging preparation. In this approach, the brain is isolated but intact, preserving neuronal connectivity and function. The preparation has several advantages, including stability, accessibility for pharmacological manipulations, and the ability to image over several hours. The full range of genetic approaches available in Drosophila can be readily combined with pharmacological manipulations in this preparation, and numerous genetically encoded reporters are available to image cellular events, ranging from Ca2+ signaling to neurotransmitter release.

Imaging Olfactory Learning-Induced Plasticity in Vivo in the Drosophila Brain.

In vivo imaging of brain activity in Drosophila allows the dissection of numerous types of biologically important neuronal events. A common paradigm involves imaging neuronal Ca2+ transients, often in response to sensory stimuli. These Ca2+ transients correlate with neuronal spiking activity, which generates voltage-sensitive Ca2+ influx. In addition, there is a range of genetically encoded reporters of membrane voltage and of other signaling molecules, such as second-messenger signaling cascade enzymes and neurotransmitters, enabling optical access to a range of cellular processes. Moreover, sophisticated gene expression systems enable access to virtually any single neuron or neuronal group in the fly brain. The in vivo imaging approach enables the study of these processes and how they change during salient sensory-driven events such as olfactory associative learning, when an animal (fly) is presented an odor (a conditioned stimulus) paired with an unconditioned stimulus (an aversive or appetitive stimulus) and forms an associative memory of this pairing. Optical access to neuronal events in the brain allows one to image learning-induced plasticity following the formation of associative memory, dissecting the mechanisms of memory formation, maintenance, and recall.

Functional Imaging of Learning-Induced Plasticity in the Central Nervous System with Genetically Encoded Reporters in Drosophila.

Learning and memory allow animals to adjust their behavior based on the predictive value of their past experiences. Memories often exist in complex representations, spread across numerous cells and synapses in the brain. Studying relatively simple forms of memory provides insights into the fundamental processes that underlie multiple forms of memory. Associative learning occurs when an animal learns the relationship between two previously unrelated sensory stimuli, such as when a hungry animal learns that a particular odor is followed by a tasty reward. Drosophila is a particularly powerful model to study how this type of memory works. The fundamental principles are widely shared among animals, and there is a wide range of genetic tools available to study circuit function in flies. In addition, the olfactory structures that mediate associative learning in flies, such as the mushroom body and its associated neurons, are anatomically organized, relatively well-characterized, and readily accessible to imaging. Here, we review the olfactory anatomy and physiology of the olfactory system, describe how plasticity in the olfactory pathway mediates learning and memory, and explain the general principles underlying calcium imaging approaches.

Associative learning drives longitudinally graded presynaptic plasticity of neurotransmitter release along axonal compartments.

Anatomical and physiological compartmentalization of neurons is a mechanism to increase the computational capacity of a circuit, and a major question is what role axonal compartmentalization plays. Axonal compartmentalization may enable localized, presynaptic plasticity to alter neuronal output in a flexible, experience-dependent manner. Here, we show that olfactory learning generates compartmentalized, bidirectional plasticity of acetylcholine release that varies across the longitudinal compartments of Drosophila mushroom body (MB) axons. The directionality of the learning-induced plasticity depends on the valence of the learning event (aversive vs. appetitive), varies linearly across proximal to distal compartments following appetitive conditioning, and correlates with learning-induced changes in downstream mushroom body output neurons (MBONs) that modulate behavioral action selection. Potentiation of acetylcholine release was dependent on the CaV2.1 calcium channel subunit cacophony. In addition, contrast between the positive conditioned stimulus and other odors required the inositol triphosphate receptor, which maintained responsivity to odors upon repeated presentations, preventing adaptation. Downstream from the MB, a set of MBONs that receive their input from the γ3 MB compartment were required for normal appetitive learning, suggesting that they represent a key node through which reward learning influences decision-making. These data demonstrate that learning drives valence-correlated, compartmentalized, bidirectional potentiation, and depression of synaptic neurotransmitter release, which rely on distinct mechanisms and are distributed across axonal compartments in a learning circuit.

Neurofibromin regulates metabolic rate via neuronal mechanisms in Drosophila.

Neurofibromatosis type 1 is a chronic multisystemic genetic disorder that results from loss of function in the neurofibromin protein. Neurofibromin may regulate metabolism, though the underlying mechanisms remain largely unknown. Here we show that neurofibromin regulates metabolic homeostasis in Drosophila via a discrete neuronal circuit. Loss of neurofibromin increases metabolic rate via a Ras GAP-related domain-dependent mechanism, increases feeding homeostatically, and alters lipid stores and turnover kinetics. The increase in metabolic rate is independent of locomotor activity, and maps to a sparse subset of neurons. Stimulating these neurons increases metabolic rate, linking their dynamic activity state to metabolism over short time scales. Our results indicate that neurofibromin regulates metabolic rate via neuronal mechanisms, suggest that cellular and systemic metabolic alterations may represent a pathophysiological mechanism in neurofibromatosis type 1, and provide a platform for investigating the cellular role of neurofibromin in metabolic homeostasis.

The Excitatory, the Inhibitory, and the Modulatory: Mapping Chemical Neurotransmission in the Brain.

In this issue of Neuron, Deng et al. (2019) report the generation of a new set of tools to manipulate the entire set of neurotransmitters, neuromodulators, neuropeptides, and their receptors-the "chemoconnectome"-in Drosophila.

Independent Contributions of Discrete Dopaminergic Circuits to Cellular Plasticity, Memory Strength, and Valence in Drosophila.

Dopaminergic neurons play a key role in encoding associative memories, but little is known about how these circuits modulate memory strength. Here we report that different sets of dopaminergic neurons projecting to the Drosophila mushroom body (MB) differentially regulate valence and memory strength. PPL2 neurons increase odor-evoked calcium responses to a paired odor in the MB and enhance behavioral memory strength when activated during olfactory classical conditioning. When paired with odor alone, they increase MB responses to the paired odor but do not drive behavioral approach or avoidance, suggesting that they increase the salience of the odor without encoding strong valence. This contrasts with the role of dopaminergic PPL1 neurons, which drive behavioral reinforcement but do not alter odor-evoked calcium responses in the MB when stimulated. These data suggest that different sets of dopaminergic neurons modulate olfactory valence and memory strength via independent actions on a memory-encoding brain region.