Dopamine Signaling in the Suprachiasmatic Nucleus Enables Weight Gain Associated with Hedonic Feeding.

The widespread availability of energy-dense, rewarding foods is correlated with the increased incidence of obesity across the globe. Overeating during mealtimes and unscheduled snacking disrupts timed metabolic processes, which further contribute to weight gain. The neuronal mechanism by which the consumption of energy-dense food restructures the timing of feeding is poorly understood. Here, we demonstrate that dopaminergic signaling within the suprachiasmatic nucleus (SCN), the central circadian pacemaker, disrupts the timing of feeding, resulting in overconsumption of food. D1 dopamine receptor (Drd1)-null mice are resistant to diet-induced obesity, metabolic disease, and circadian disruption associated with energy-dense diets. Conversely, genetic rescue of Drd1 expression within the SCN restores diet-induced overconsumption, weight gain, and obesogenic symptoms. Access to rewarding food increases SCN dopamine turnover, and elevated Drd1-signaling decreases SCN neuronal activity, which we posit disinhibits downstream orexigenic responses. These findings define a connection between the reward and circadian pathways in the regulation of pathological calorie consumption.

Nitric oxide acts as a cotransmitter in a subset of dopaminergic neurons to diversify memory dynamics.

Animals employ diverse learning rules and synaptic plasticity dynamics to record temporal and statistical information about the world. However, the molecular mechanisms underlying this diversity are poorly understood. The anatomically defined compartments of the insect mushroom body function as parallel units of associative learning, with different learning rates, memory decay dynamics and flexibility (Aso and Rubin, 2016). Here, we show that nitric oxide (NO) acts as a neurotransmitter in a subset of dopaminergic neurons in Drosophila. NO's effects develop more slowly than those of dopamine and depend on soluble guanylate cyclase in postsynaptic Kenyon cells. NO acts antagonistically to dopamine; it shortens memory retention and facilitates the rapid updating of memories. The interplay of NO and dopamine enables memories stored in local domains along Kenyon cell axons to be specialized for predicting the value of odors based only on recent events. Our results provide key mechanistic insights into how diverse memory dynamics are established in parallel memory systems.

An inexpensive air stream temperature controller and its use to facilitate temperature-controlled behavior in Drosophila.

Controlling the environment of an organism has many biologically relevant applications. Temperature-dependent inducible biological reagents have proven invaluable for elucidating signaling cascades and dissection of neural circuits. Here we develop a simple and affordable system for rapidly changing temperature in a chamber housing adult Drosophila melanogaster. Utilizing flies expressing the temperature-inducible channel dTrpA1 in dopaminergic neurons we show rapid and reproducible changes in locomotor behavior. This device should have wide application to temperature-modulated biological reagents.

The Role of Dopamine in the Collective Regulation of Foraging in Harvester Ants.

Colonies of the red harvester ant (Pogonomyrmex barbatus) differ in how they regulate collective foraging activity in response to changes in humidity. We used transcriptomic, physiological, and pharmacological experiments to investigate the molecular basis of this ecologically important variation in collective behavior among colonies. RNA sequencing of forager brain tissue showed an association between colony foraging activity and differential expression of transcripts related to biogenic amine and neurohormonal metabolism and signaling. In field experiments, pharmacological increases in forager brain dopamine titer caused significant increases in foraging activity. Colonies that were naturally most sensitive to humidity were significantly more responsive to the stimulatory effect of exogenous dopamine. In addition, forager brain tissue significantly varied among colonies in biogenic amine content. Neurophysiological variation among colonies associated with individual forager sensitivity to humidity may reflect the heritable molecular variation on which natural selection acts to shape the collective regulation of foraging.

ShinyR-DAM: a program analyzing Drosophila activity, sleep and circadian rhythms.

We developed a web application ShinyR-DAM for analyzing Drosophila locomotor activity, sleep, and circadian rhythms recorded by the Drosophila Activity Monitor (DAM) system (TriKinetics, Waltham, MA). Comparing with the existing programs for DAM data analysis, ShinyR-DAM greatly decreases the complexity and time required to analyze the data, producing informative and customizable plots, summary tables, and data files for statistical analysis. Our program has an intuitive graphical user interface that enables novice users to quickly perform complex analyses.

Dopamine Modulates Serotonin Innervation in the Drosophila Brain.

Parkinson's disease (PD) results from a progressive degeneration of the dopaminergic nigrostriatal system leading to a decline in movement control, with resting tremor, rigidity and postural instability. Several aspects of PD can be modeled in the fruit fly, Drosophila melanogaster, including α-synuclein-induced degeneration of dopaminergic neurons, or dopamine (DA) loss by genetic elimination of neural DA synthesis. Defective behaviors in this latter model can be ameliorated by feeding the DA precursor L-DOPA, analogous to the treatment paradigm for PD. Secondary complication from L-DOPA treatment in PD patients are associated with ectopic synthesis of DA in serotonin (5-HT)-releasing neurons, leading to DA/5-HT imbalance. Here we examined the neuro-anatomical adaptations resulting from imbalanced DA/5-HT signaling in Drosophila mutants lacking neural DA. We find that, similar to rodent models of PD, lack of DA leads to increased 5-HT levels and arborizations in specific brain regions. Conversely, increased DA levels by L-DOPA feeding leads to reduced connectivity of 5-HT neurons to their target neurons in the mushroom body (MB). The observed alterations of 5-HT neuron plasticity indicate that loss of DA signaling is not solely responsible for the behavioral disorders observed in Drosophila models of PD, but rather a combination of the latter with alterations of 5-HT circuitry.

Novel Focused Ultrasound Gene Therapy Approach Noninvasively Restores Dopaminergic Neuron Function in a Rat Parkinson's Disease Model.

Therapies capable of decelerating, or perhaps even halting, neurodegeneration in Parkinson's disease (PD) remain elusive. Clinical trials of PD gene therapy testing the delivery of neurotrophic factors, such as the glial cell-line derived neurotrophic factor (GDNF), have been largely ineffective due to poor vector distribution throughout the diseased regions in the brain. In addition, current delivery strategies involve invasive procedures that obviate the inclusion of early stage patients who are most likely to benefit from GDNF-based gene therapy. Here, we introduce a two-pronged treatment strategy, composed of MR image-guided focused ultrasound (FUS) and brain-penetrating nanoparticles (BPN), that provides widespread but targeted GDNF transgene expression in the brain following systemic administration. MR image-guided FUS allows circulating gene vectors to partition into the brain tissue by noninvasive and transient opening of the blood-brain barrier (BBB) within the areas where FUS is applied. Once beyond the BBB, BPN provide widespread and uniform GDNF expression throughout the targeted brain tissue. After only a single treatment, our strategy led to therapeutically relevant levels of GDNF protein content in the FUS-targeted regions in the striatum of the 6-OHDA-induced rat model of PD, which lasted at least up to 10 weeks. Importantly, our strategy restored both dopamine levels and dopaminergic neuron density and reversed behavioral indicators of PD-associated motor dysfunction with no evidence of local or systemic toxicity. Our combinatorial approach overcomes limitations of current delivery strategies, thereby potentially providing a novel means to treat PD.

Caffeine promotes wakefulness via dopamine signaling in Drosophila.

Caffeine is the most widely-consumed psychoactive drug in the world, but our understanding of how caffeine affects our brains is relatively incomplete. Most studies focus on effects of caffeine on adenosine receptors, but there is evidence for other, more complex mechanisms. In the fruit fly Drosophila melanogaster, which shows a robust diurnal pattern of sleep/wake activity, caffeine reduces nighttime sleep behavior independently of the one known adenosine receptor. Here, we show that dopamine is required for the wake-promoting effect of caffeine in the fly, and that caffeine likely acts presynaptically to increase dopamine signaling. We identify a cluster of neurons, the paired anterior medial (PAM) cluster of dopaminergic neurons, as the ones relevant for the caffeine response. PAM neurons show increased activity following caffeine administration, and promote wake when activated. Also, inhibition of these neurons abrogates sleep suppression by caffeine. While previous studies have focused on adenosine-receptor mediated mechanisms for caffeine action, we have identified a role for dopaminergic neurons in the arousal-promoting effect of caffeine.

Exquisite light sensitivity of Drosophila melanogaster cryptochrome.

Drosophila melanogaster shows exquisite light sensitivity for modulation of circadian functions in vivo, yet the activities of the Drosophila circadian photopigment cryptochrome (CRY) have only been observed at high light levels. We studied intensity/duration parameters for light pulse induced circadian phase shifts under dim light conditions in vivo. Flies show far greater light sensitivity than previously appreciated, and show a surprising sensitivity increase with pulse duration, implying a process of photic integration active up to at least 6 hours. The CRY target timeless (TIM) shows dim light dependent degradation in circadian pacemaker neurons that parallels phase shift amplitude, indicating that integration occurs at this step, with the strongest effect in a single identified pacemaker neuron. Our findings indicate that CRY compensates for limited light sensitivity in vivo by photon integration over extraordinarily long times, and point to select circadian pacemaker neurons as having important roles.

Phase-shifting the fruit fly clock without cryptochrome.

The blue light photopigment cryptochrome (CRY) is thought to be the main circadian photoreceptor of Drosophila melanogaster. Nevertheless, entrainment to light-dark cycles is possible without functional CRY. Here, we monitored phase response curves of cry(01) mutants and control flies to 1-hour 1000-lux light pulses. We found that cry(01) mutants phase-shift their activity rhythm in the subjective early morning and late evening, although with reduced magnitude. This phase-shifting capability is sufficient for the slowed entrainment of the mutants, indicating that the eyes contribute to the clock's light sensitivity around dawn and dusk. With longer light pulses (3 hours and 6 hours), wild-type flies show greatly enhanced magnitude of phase shift, but CRY-less flies seem impaired in the ability to integrate duration of the light pulse in a wild-type manner: Only 6-hour light pulses at circadian time 21 significantly increased the magnitude of phase advances in cry(01) mutants. At circadian time 15, the mutants exhibited phase advances instead of the expected delays. These complex results are discussed.

Serotonin-mushroom body circuit modulating the formation of anesthesia-resistant memory in Drosophila.

Pavlovian olfactory learning in Drosophila produces two genetically distinct forms of intermediate-term memories: anesthesia-sensitive memory, which requires the amnesiac gene, and anesthesia-resistant memory (ARM), which requires the radish gene. Here, we report that ARM is specifically enhanced or inhibited in flies with elevated or reduced serotonin (5HT) levels, respectively. The requirement for 5HT was additive with the memory defect of the amnesiac mutation but was occluded by the radish mutation. This result suggests that 5HT and Radish protein act on the same pathway for ARM formation. Three supporting lines of evidence indicate that ARM formation requires 5HT released from only two dorsal paired medial (DPM) neurons onto the mushroom bodies (MBs), the olfactory learning and memory center in Drosophila: (i) DPM neurons were 5HT-antibody immunopositive; (ii) temporal inhibition of 5HT synthesis or release from DPM neurons, but not from other serotonergic neurons, impaired ARM formation; (iii) knocking down the expression of d5HT1A serotonin receptors in α/ß MB neurons, which are innervated by DPM neurons, inhibited ARM formation. Thus, in addition to the Amnesiac peptide required for anesthesia-sensitive memory formation, the two DPM neurons also release 5HT acting on MB neurons for ARM formation.

A Drosophila model for alcohol reward.

The rewarding properties of drugs contribute to the development of abuse and addiction. We developed a new assay for investigating the motivational properties of ethanol in the genetically tractable model Drosophila melanogaster. Flies learned to associate cues with ethanol intoxication and, although transiently aversive, the experience led to a long-lasting attraction for the ethanol-paired cue, implying that intoxication is rewarding. Temporally blocking transmission in dopaminergic neurons revealed that flies require activation of these neurons to express, but not develop, conditioned preference for ethanol-associated cues. Moreover, flies acquired, consolidated and retrieved these rewarding memories using distinct sets of neurons in the mushroom body. Finally, mutations in scabrous, encoding a fibrinogen-related peptide that regulates Notch signaling, disrupted the formation of memories for ethanol reward. Our results thus establish that Drosophila can be useful for understanding the molecular, genetic and neural mechanisms underling the rewarding properties of ethanol.

Behavioral consequences of dopamine deficiency in the Drosophila central nervous system.

The neuromodulatory function of dopamine (DA) is an inherent feature of nervous systems of all animals. To learn more about the function of neural DA in Drosophila, we generated mutant flies that lack tyrosine hydroxylase, and thus DA biosynthesis, selectively in the nervous system. We found that DA is absent or below detection limits in the adult brain of these flies. Despite this, they have a lifespan similar to WT flies. These mutants show reduced activity, extended sleep time, locomotor deficits that increase with age, and they are hypophagic. Whereas odor and electrical shock avoidance are not affected, aversive olfactory learning is abolished. Instead, DA-deficient flies have an apparently "masochistic" tendency to prefer the shock-associated odor 2 h after conditioning. Similarly, sugar preference is absent, whereas sugar stimulation of foreleg taste neurons induces normal proboscis extension. Feeding the DA precursor L-DOPA to adults substantially rescues the learning deficit as well as other impaired behaviors that were tested. DA-deficient flies are also defective in positive phototaxis, without alteration in visual perception and optomotor response. Surprisingly, visual tracking is largely maintained, and these mutants still possess an efficient spatial orientation memory. Our findings show that flies can perform complex brain functions in the absence of neural DA, whereas specific behaviors involving, in particular, arousal and choice require normal levels of this neuromodulator.

A pair of dopamine neurons target the D1-like dopamine receptor DopR in the central complex to promote ethanol-stimulated locomotion in Drosophila.

Dopamine is a mediator of the stimulant properties of drugs of abuse, including ethanol, in mammals and in the fruit fly Drosophila. The neural substrates for the stimulant actions of ethanol in flies are not known. We show that a subset of dopamine neurons and their targets, through the action of the D1-like dopamine receptor DopR, promote locomotor activation in response to acute ethanol exposure. A bilateral pair of dopaminergic neurons in the fly brain mediates the enhanced locomotor activity induced by ethanol exposure, and promotes locomotion when directly activated. These neurons project to the central complex ellipsoid body, a structure implicated in regulating motor behaviors. Ellipsoid body neurons are required for ethanol-induced locomotor activity and they express DopR. Elimination of DopR blunts the locomotor activating effects of ethanol, and this behavior can be restored by selective expression of DopR in the ellipsoid body. These data tie the activity of defined dopamine neurons to D1-like DopR-expressing neurons to form a neural circuit that governs acute responding to ethanol.

Corazonin neurons function in sexually dimorphic circuitry that shape behavioral responses to stress in Drosophila.

All organisms are confronted with dynamic environmental changes that challenge homeostasis, which is the operational definition of stress. Stress produces adaptive behavioral and physiological responses, which, in the Metazoa, are mediated through the actions of various hormones. Based on its associated phenotypes and its expression profiles, a candidate stress hormone in Drosophila is the corazonin neuropeptide. We evaluated the potential roles of corazonin in mediating stress-related changes in target behaviors and physiologies through genetic alteration of corazonin neuronal excitability. Ablation of corazonin neurons confers resistance to metabolic, osmotic, and oxidative stress, as measured by survival. Silencing and activation of corazonin neurons lead to differential lifespan under stress, and these effects showed a strong dependence on sex. Additionally, altered corazonin neuron physiology leads to fundamental differences in locomotor activity, and these effects were also sex-dependent. The dynamics of altered locomotor behavior accompanying stress was likewise altered in flies with altered corazonin neuronal function. We report that corazonin transcript expression is altered under starvation and osmotic stress, and that triglyceride and dopamine levels are equally impacted in corazonin neuronal alterations and these phenotypes similarly show significant sexual dimorphisms. Notably, these sexual dimorphisms map to corazonin neurons. These results underscore the importance of central peptidergic processing within the context of stress and place corazonin signaling as a critical feature of neuroendocrine events that shape stress responses and may underlie the inherent sexual dimorphic differences in stress responses.

Roles of dopamine in circadian rhythmicity and extreme light sensitivity of circadian entrainment.

Light has profound behavioral effects on almost all animals, and nocturnal animals show sensitivity to extremely low light levels [1-4]. Crepuscular, i.e., dawn/dusk-active animals such as Drosophila melanogaster are thought to show far less sensitivity to light [5-8]. Here we report that Drosophila respond to extremely low levels of monochromatic blue light. Light levels three to four orders of magnitude lower than previously believed impact circadian entrainment and the light-induced stimulation of locomotion known as positive behavioral masking. We use GAL4;UAS-mediated rescue of tyrosine hydroxylase (DTH) mutant (ple) flies to study the roles of dopamine in these processes. We present evidence for two roles of dopamine in circadian behaviors. First, rescue with either a wild-type DTH or a DTH mutant lacking neural expression leads to weak circadian rhythmicity, indicating a role for strictly regulated DTH and dopamine in robust circadian rhythmicity. Second, the DTH rescue strain deficient in neural dopamine selectively shows a defect in circadian entrainment to low light, whereas another response to light, positive masking, has normal light sensitivity. These findings imply separable pathways from light input to the behavioral outputs of masking versus circadian entrainment, with only the latter dependent on dopamine.

Writing memories with light-addressable reinforcement circuitry.

Dopaminergic neurons are thought to drive learning by signaling changes in the expectations of salient events, such as rewards or punishments. Olfactory conditioning in Drosophila requires direct dopamine action on intrinsic mushroom body neurons, the likely storage sites of olfactory memories. Neither the cellular sources of the conditioning dopamine nor its precise postsynaptic targets are known. By optically controlling genetically circumscribed subsets of dopaminergic neurons in the behaving fly, we have mapped the origin of aversive reinforcement signals to the PPL1 cluster of 12 dopaminergic cells. PPL1 projections target restricted domains in the vertical lobes and heel of the mushroom body. Artificially evoked activity in a small number of identifiable cells thus suffices for programming behaviorally meaningful memories. The delineation of core reinforcement circuitry is an essential first step in dissecting the neural mechanisms that compute and represent valuations, store associations, and guide actions.