An incentive circuit for memory dynamics in the mushroom body of Drosophila melanogaster.

Insects adapt their response to stimuli, such as odours, according to their pairing with positive or negative reinforcements, such as sugar or shock. Recent electrophysiological and imaging findings in Drosophila melanogaster allow detailed examination of the neural mechanisms supporting the acquisition, forgetting, and assimilation of memories. We propose that this data can be explained by the combination of a dopaminergic plasticity rule that supports a variety of synaptic strength change phenomena, and a circuit structure (derived from neuroanatomy) between dopaminergic and output neurons that creates different roles for specific neurons. Computational modelling shows that this circuit allows for rapid memory acquisition, transfer from short term to long term, and exploration/exploitation trade-off. The model can reproduce the observed changes in the activity of each of the identified neurons in conditioning paradigms and can be used for flexible behavioural control.

A neuronal ensemble encoding adaptive choice during sensory conflict in Drosophila.

Feeding decisions are fundamental to survival, and decision making is often disrupted in disease. Here, we show that neural activity in a small population of neurons projecting to the fan-shaped body higher-order central brain region of Drosophila represents food choice during sensory conflict. We found that food deprived flies made tradeoffs between appetitive and aversive values of food. We identified an upstream neuropeptidergic and dopaminergic network that relays internal state and other decision-relevant information to a specific subset of fan-shaped body neurons. These neurons were strongly inhibited by the taste of the rejected food choice, suggesting that they encode behavioral food choice. Our findings reveal that fan-shaped body taste responses to food choices are determined not only by taste quality, but also by previous experience (including choice outcome) and hunger state, which are integrated in the fan-shaped body to encode the decision before relay to downstream motor circuits for behavioral implementation.

C. elegans discriminates colors to guide foraging.

Color detection is used by animals of diverse phyla to navigate colorful natural environments and is thought to require evolutionarily conserved opsin photoreceptor genes. We report that Caenorhabditis elegans roundworms can discriminate between colors despite the fact that they lack eyes and opsins. Specifically, we found that white light guides C. elegans foraging decisions away from a blue-pigment toxin secreted by harmful bacteria. These foraging decisions are guided by specific blue-to-amber ratios of light. The color specificity of color-dependent foraging varies notably among wild C. elegans strains, which indicates that color discrimination is ecologically important. We identified two evolutionarily conserved cellular stress response genes required for opsin-independent, color-dependent foraging by C. elegans, and we speculate that cellular stress response pathways can mediate spectral discrimination by photosensitive cells and organisms-even by those lacking opsins.

Dopaminergic mechanism underlying reward-encoding of punishment omission during reversal learning in Drosophila.

Animals form and update learned associations between otherwise neutral sensory cues and aversive outcomes (i.e., punishment) to predict and avoid danger in changing environments. When a cue later occurs without punishment, this unexpected omission of aversive outcome is encoded as reward via activation of reward-encoding dopaminergic neurons. How such activation occurs remains unknown. Using real-time in vivo functional imaging, optogenetics, behavioral analysis and synaptic reconstruction from electron microscopy data, we identify the neural circuit mechanism through which Drosophila reward-encoding dopaminergic neurons are activated when an olfactory cue is unexpectedly no longer paired with electric shock punishment. Reduced activation of punishment-encoding dopaminergic neurons relieves depression of olfactory synaptic inputs to cholinergic neurons. Synaptic excitation by these cholinergic neurons of reward-encoding dopaminergic neurons increases their odor response, thus decreasing aversiveness of the odor. These studies reveal how an excitatory cholinergic relay from punishment- to reward-encoding dopaminergic neurons encodes the absence of punishment as reward, revealing a general circuit motif for updating aversive memories that could be present in mammals.

Parvalbumin expression affects synaptic development and physiology at the Drosophila larval NMJ.

Presynaptic Ca2+ appears to play multiple roles in synaptic development and physiology. We examined the effect of buffering presynaptic Ca2+ by expressing parvalbumin (PV) in Drosophila neurons, which do not normally express PV. The studies were performed on the identified Ib terminal that innervates muscle fiber 5. The volume-averaged, residual Ca2+ resulting from single action potentials (APs) and AP trains was measured using the fluorescent Ca2+ indicator, OGB-1. PV reduced the amplitude and decay time constant (τ) for single-AP Ca2+ transients. For AP trains, there was a reduction in the rate of rise and decay of [Ca2+]i but the plateau [Ca2+]i was not affected. Electrophysiological recordings from muscle fiber 5 showed a reduction in paired-pulse facilitation, particularly the F1 component; this was likely due to the reduction in residual Ca2+. These synapses also showed reduced synaptic enhancement during AP trains, presumably due to less buildup of synaptic facilitation. The transmitter release for single APs was increased for the PV-expressing terminals and this may have been a homeostatic response to the decrease in facilitation. Confocal microscopy was used to examine the structure of the motor terminals and PV expression resulted in smaller motor terminals with fewer synaptic boutons and active zones. This result supports earlier proposals that increased AP activity promotes motor terminal growth through increases in presynaptic [Ca2+]i.

Daily oscillations in expression and responsiveness of Toll-like receptors in splenic immune cells.

Circadian rhythms refer to biologic processes that oscillate with an approximate 24-h period. These rhythms direct nearly all aspects of animal behavior and physiology. The aim of our study was to determine if Toll-like receptor (TLR) expression and responsiveness exhibit time-of-day dependent differences. Therefore, we isolated an adherent splenocyte population, which consisted primarily of B cells, dendritic cells, and macrophages, over the course of a 24-h light-dark period and measured daily changes in Tlr1-8 mRNA levels and cytokine expression after cells were challenged at Zeitgeber time (ZT) 1 or ZT13 with a TLR ligand. In addition, we assessed TLR3 protein levels in adherent splenocytes over the 24-h light-dark period and challenged mice at ZT1 or ZT13 with poly(I:C), the TLR3 ligand. Our study revealed that in this adherent cell population, all Tlrs exhibited rhythmic expression except Tlr2 and Tlr5, and all TLRs, except TLR8, demonstrated daily variations in responsiveness after challenge with their respective ligand. We also revealed that TLR3 protein levels fluctuate over the daily light-dark cycle in adherent splenocytes and mice exhibit a time-of-day dependent immune response when challenged with poly(I:C). Finally, we demonstrated that mRNA levels of Tlr2 and Tlr6 display rhythmic expression in splenic macrophages. Taken together, these findings could have important implications for TLR-directed therapeutics.

Peptide-Mediated Neurotransmission Takes Center Stage.

Today, we understand peptide transmitters to be signaling molecules that modulate neural activity. However, in 1982 little was known about neuropeptides and their role in neural communication. The influential 1982 paper by Jan and Jan reported definitive evidence that a presynaptically released neuropeptide evokes postsynaptic responses in an identified cholinergic synapse, thereby fueling a new era in neuroscience.

Genetic and neuronal mechanisms governing the sex-specific interaction between sleep and sexual behaviors in Drosophila.

Animals execute one particular behavior among many others in a context-dependent manner, yet the mechanisms underlying such behavioral choice remain poorly understood. Here we studied how two fundamental behaviors, sex and sleep, interact at genetic and neuronal levels in Drosophila. We show that an increased need for sleep inhibits male sexual behavior by decreasing the activity of the male-specific P1 neurons that coexpress the sex determination genes fru M and dsx, but does not affect female sexual behavior. Further, we delineate a sex-specific neuronal circuit wherein the P1 neurons encoding increased courtship drive suppressed male sleep by forming mutually excitatory connections with the fru M -positive sleep-controlling DN1 neurons. In addition, we find that FRUM regulates male courtship and sleep through distinct neural substrates. These studies reveal the genetic and neuronal basis underlying the sex-specific interaction between sleep and sexual behaviors in Drosophila, and provide insights into how competing behaviors are co-regulated.Genes and circuits involved in sleep and sexual arousal have been extensively studied in Drosophila. Here the authors identify the sex determination genes fruitless and doublesex, and a sex-specific P1-DN1 neuronal feedback that governs the interaction between these competing behaviors.

A Peptidergic Circuit Links the Circadian Clock to Locomotor Activity.

The mechanisms by which clock neurons in the Drosophila brain confer an ∼24-hr rhythm onto locomotor activity are unclear, but involve the neuropeptide diuretic hormone 44 (DH44), an ortholog of corticotropin-releasing factor. Here we identified DH44 receptor 1 as the relevant receptor for rest:activity rhythms and mapped its site of action to hugin-expressing neurons in the subesophageal zone (SEZ). We traced a circuit that extends from Dh44-expressing neurons in the pars intercerebralis (PI) through hugin+ SEZ neurons to the ventral nerve cord. Hugin neuropeptide, a neuromedin U ortholog, also regulates behavioral rhythms. The DH44 PI-Hugin SEZ circuit controls circadian locomotor activity in a daily cycle but has minimal effect on feeding rhythms, suggesting that the circadian drive to feed can be separated from circadian locomotion. These findings define a linear peptidergic circuit that links the clock to motor outputs to modulate circadian control of locomotor activity.

Multisensory integration in C. elegans.

Multisensory integration is a neural process by which signals from two or more distinct sensory channels are simultaneously processed to form a more coherent representation of the environment. Multisensory integration, especially when combined with a survey of internal states, provides selective advantages for animals navigating complex environments. Despite appreciation of the importance of multisensory integration in behavior, the underlying molecular and cellular mechanisms remain poorly understood. Recent work looking at how Caenorhabditis elegans makes multisensory decisions has yielded mechanistic insights into how a relatively simple and well-defined nervous system employs circuit motifs of defined features, synaptic signals and extrasynaptic neurotransmission, as well as neuromodulators in processing and integrating multiple sensory inputs to generate flexible and adaptive behavioral outputs.

Membrane Currents, Gene Expression, and Circadian Clocks.

Neuronal circadian oscillators in the mammalian and Drosophila brain express a circadian clock comprised of interlocking gene transcription feedback loops. The genetic clock regulates the membrane electrical activity by poorly understood signaling pathways to generate a circadian pattern of action potential firing. During the day, Na+ channels contribute an excitatory drive for the spontaneous activity of circadian clock neurons. Multiple types of K+ channels regulate the action potential firing pattern and the nightly reduction in neuronal activity. The membrane electrical activity possibly signaling by changes in intracellular Ca2+ and cyclic adenosine monophosphate (cAMP) regulates the activity of the gene clock. A decline in the signaling pathways that link the gene clock and neural activity during aging and disease may weaken the circadian output and generate significant impacts on human health.

Neural Architecture of Hunger-Dependent Multisensory Decision Making in C. elegans.

Little is known about how animals integrate multiple sensory inputs in natural environments to balance avoidance of danger with approach to things of value. Furthermore, the mechanistic link between internal physiological state and threat-reward decision making remains poorly understood. Here we confronted C. elegans worms with the decision whether to cross a hyperosmotic barrier presenting the threat of desiccation to reach a source of food odor. We identified a specific interneuron that controls this decision via top-down extrasynaptic aminergic potentiation of the primary osmosensory neurons to increase their sensitivity to the barrier. We also establish that food deprivation increases the worm's willingness to cross the dangerous barrier by suppressing this pathway. These studies reveal a potentially general neural circuit architecture for internal state control of threat-reward decision making.

Drosophila DH31 Neuropeptide and PDF Receptor Regulate Night-Onset Temperature Preference.

Body temperature exhibits rhythmic fluctuations over a 24 h period (Refinetti and Menaker, 1992) and decreases during the night, which is associated with sleep initiation (Gilbert et al., 2004; Kräuchi, 2007a,b). However, the underlying mechanism of this temperature decrease is largely unknown. We have previously shown that Drosophila exhibit a daily temperature preference rhythm (TPR), in which their preferred temperatures increase during the daytime and then decrease at the transition from day to night (night-onset) (Kaneko et al., 2012). Because Drosophila are small ectotherms, their body temperature is very close to that of the ambient temperature (Stevenson, 1985), suggesting that their TPR generates their body temperature rhythm. Here, we demonstrate that the neuropeptide diuretic hormone 31 (DH31) and pigment-dispersing factor receptor (PDFR) contribute to regulate the preferred temperature decrease at night-onset. We show that PDFR and tethered-DH31 expression in dorsal neurons 2 (DN2s) restore the preferred temperature decrease at night-onset, suggesting that DH31 acts on PDFR in DN2s. Notably, we previously showed that the molecular clock in DN2s is important for TPR. Although PDF (another ligand of PDFR) is a critical factor for locomotor activity rhythms, Pdf mutants exhibit normal preferred temperature decreases at night-onset. This suggests that DH31-PDFR signaling specifically regulates a preferred temperature decrease at night-onset. Thus, we propose that night-onset TPR and locomotor activity rhythms are differentially controlled not only by clock neurons but also by neuropeptide signaling in the brain. SIGNIFICANCE STATEMENT: Body temperature rhythm (BTR) is fundamental for the maintenance of functions essential for homeostasis, such as generating metabolic energy and sleep. One major unsolved question is how body temperature decreases dramatically during the night. Previously, we demonstrated that a BTR-like mechanism, referred to as temperature preference rhythm (TPR), exists in Drosophila Here, we demonstrate that the diuretic hormone 31 (DH31) neuropeptide and pigment-dispersing factor receptor (PDFR) regulate preferred temperature decreases at night-onset via dorsal neurons 2. This is the first in vivo evidence that DH31 could function as a ligand of PDFR. Although both DH31 and PDF are ligands of PDFR, we show that DH31 regulates night-onset TPR, but PDF does not, suggesting that night-onset TPR and locomotor activity rhythms are controlled by different neuropeptides via different clock cells.

Goggatomy: A Method for Opening Small Cuticular Compartments in Arthropods for Physiological Experiments.

Most sense organs of arthropods are ensconced in small exoskeletal compartments that hinder direct access to plasma membranes. We have developed a method for exposing live sensory and supporting cells in such structures. The technique uses a viscous light cured resin to embed and support the structure, which is then sliced with a sharp blade. We term the procedure a "goggatomy," from the Khoisan word for a bug, gogga. To demonstrate the utility of the method we show that it can be used to expose the auditory chordotonal organs in the second antennal segment and the olfactory receptor neurons in the third antennal segment of Drosophila melanogaster, preserving the transduction machinery. The procedure can also be used on other small arthropods, like mosquitoes and mites to expose a variety of cells.

Presynaptic GABA Receptors Mediate Temporal Contrast Enhancement in Drosophila Olfactory Sensory Neurons and Modulate Odor-Driven Behavioral Kinetics.

Contrast enhancement mediated by lateral inhibition within the nervous system enhances the detection of salient features of visual and auditory stimuli, such as spatial and temporal edges. However, it remains unclear how mechanisms for temporal contrast enhancement in the olfactory system can enhance the detection of odor plume edges during navigation. To address this question, we delivered to Drosophila melanogaster flies pulses of high odor intensity that induce sustained peripheral responses in olfactory sensory neurons (OSNs). We use optical electrophysiology to directly measure electrical responses in presynaptic terminals and demonstrate that sustained peripheral responses are temporally sharpened by the combined activity of two types of inhibitory GABA receptors to generate contrast-enhanced voltage responses in central OSN axon terminals. Furthermore, we show how these GABA receptors modulate the time course of innate behavioral responses after odor pulse termination, demonstrating an important role for temporal contrast enhancement in odor-guided navigation.

Control of Sleep by Dopaminergic Inputs to the Drosophila Mushroom Body.

The Drosophila mushroom body (MB) is an associative learning network that is important for the control of sleep. We have recently identified particular intrinsic MB Kenyon cell (KC) classes that regulate sleep through synaptic activation of particular MB output neurons (MBONs) whose axons convey sleep control signals out of the MB to downstream target regions. Specifically, we found that sleep-promoting KCs increase sleep by preferentially activating cholinergic sleep-promoting MBONs, while wake-promoting KCs decrease sleep by preferentially activating glutamatergic wake-promoting MBONs. Here we use a combination of genetic and physiological approaches to identify wake-promoting dopaminergic neurons (DANs) that innervate the MB, and show that they activate wake-promoting MBONs. These studies reveal a dopaminergic sleep control mechanism that likely operates by modulation of KC-MBON microcircuits.

Propagation of Homeostatic Sleep Signals by Segregated Synaptic Microcircuits of the Drosophila Mushroom Body.

The Drosophila mushroom body (MB) is a key associative memory center that has also been implicated in the control of sleep. However, the identity of MB neurons underlying homeostatic sleep regulation, as well as the types of sleep signals generated by specific classes of MB neurons, has remained poorly understood. We recently identified two MB output neuron (MBON) classes whose axons convey sleep control signals from the MB to converge in the same downstream target region: a cholinergic sleep-promoting MBON class and a glutamatergic wake-promoting MBON class. Here, we deploy a combination of neurogenetic, behavioral, and physiological approaches to identify and mechanistically dissect sleep-controlling circuits of the MB. Our studies reveal the existence of two segregated excitatory synaptic microcircuits that propagate homeostatic sleep information from different populations of intrinsic MB "Kenyon cells" (KCs) to specific sleep-regulating MBONs: sleep-promoting KCs increase sleep by preferentially activating the cholinergic MBONs, while wake-promoting KCs decrease sleep by preferentially activating the glutamatergic MBONs. Importantly, activity of the sleep-promoting MB microcircuit is increased by sleep deprivation and is necessary for homeostatic rebound sleep (i.e., the increased sleep that occurs after, and in compensation for, sleep lost during deprivation). These studies reveal for the first time specific functional connections between subsets of KCs and particular MBONs and establish the identity of synaptic microcircuits underlying transmission of homeostatic sleep signals in the MB.

Exploring the Biology of G Protein-Coupled Receptors from In Vitro to In Vivo.

In August 2014, an international group of researchers gathered for 5 days at the Lorentz Center in Leiden, The Netherlands, to explore the technical and conceptual issues associated with the analysis of G protein-coupled receptor functions utilizing information from crystal structure models to the use of model organisms. This collection of review articles evolved from the 5-day meeting, with brief presentations and structured discussion periods that were designed to identify key questions remaining in understanding G protein-coupled receptor function and to propose novel strategies by integrating scientific disciplines to guide future research.

Sensory determinants of behavioral dynamics in Drosophila thermotaxis.

Complex animal behaviors are built from dynamical relationships between sensory inputs, neuronal activity, and motor outputs in patterns with strategic value. Connecting these patterns illuminates how nervous systems compute behavior. Here, we study Drosophila larva navigation up temperature gradients toward preferred temperatures (positive thermotaxis). By tracking the movements of animals responding to fixed spatial temperature gradients or random temperature fluctuations, we calculate the sensitivity and dynamics of the conversion of thermosensory inputs into motor responses. We discover three thermosensory neurons in each dorsal organ ganglion (DOG) that are required for positive thermotaxis. Random optogenetic stimulation of the DOG thermosensory neurons evokes behavioral patterns that mimic the response to temperature variations. In vivo calcium and voltage imaging reveals that the DOG thermosensory neurons exhibit activity patterns with sensitivity and dynamics matched to the behavioral response. Temporal processing of temperature variations carried out by the DOG thermosensory neurons emerges in distinct motor responses during thermotaxis.