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Summary 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., Graphical Abstract, Highlights • Red harvester ant colonies vary in how they adjust foraging effort in dry conditions • Colonies that differ in behavior significantly differ in forager brain transcriptome • Pharmacological increases of dopamine increased foraging in field experiments • Foragers from colonies more sensitive to humidity were more stimulated by dopamine, Entomology; Neuroscience; Molecular Neuroscience

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.

Summary 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.

SummaryAnimals employ multiple and distributed neuronal networks with diverse learning rules and synaptic plasticity dynamics to record distinct 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 & 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.

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.

Dopamine (DA) is a neurotransmitter with conserved behavioral roles between invertebrate and vertebrate animals. In addition to its neural functions, in insects DA is a critical substrate for cuticle pigmentation and hardening. Drosophila tyrosine hydroxylase (DTH) is the rate limiting enzyme for DA biosynthesis. Viable brain DA-deficient flies were previously generated using tissue-selective GAL4-UAS binary expression rescue of a DTH null mutation and these flies show specific behavioral impairments. To circumvent the limitations of rescue via binary expression, here we achieve rescue utilizing genomically integrated mutant DTH. As expected, our DA-deficient flies have no detectable DTH or DA in the brain, and show reduced locomotor activity. This deficit can be rescued by l-DOPA/carbidopa feeding, similar to human Parkinson's disease treatment. Genetic rescue via GAL4/UAS-DTH was also successful, although this required the generation of a new UAS-DTH1 transgene devoid of most untranslated regions, as existing UAS-DTH transgenes express in the brain without a Gal4 driver via endogenous regulatory elements. A surprising finding of our newly constructed UAS-DTH1m is that it expresses DTH at an undetectable level when regulated by dopaminergic GAL4 drivers even when fully rescuing DA, indicating that DTH immunostaining is not necessarily a valid marker for DA expression. This finding necessitated optimizing DA immunohistochemistry, showing details of DA innervation to the mushroom body and the central complex. When DA rescue is limited to specific DA neurons, DA does not diffuse beyond the DTH-expressing terminals, such that DA signaling can be limited to very specific brain regions.

Parkinson9s Disease (PD), is a neurodegenerative disorder affecting both cognitive and motor functions. It is characterized by decreased brain dopamine (DA) and a selective and progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc), whereas dopaminergic neurons in the ventral tegmental area (VTA) show reduced vulnerability. The majority of animal models of PD are genetic lesion or neurotoxin exposure models that lead to death of dopaminergic neurons. Here we use a DAT:TH KO mouse model that by inactivation of the tyrosine hydroxylase ( Th ) gene in dopamine transporter-expressing neurons, causes selective depletion of striatal dopamine without affecting DA neuron survival. We analyzed transcriptome responses to decreased DA in both pre- and post-synaptic dopaminergic brain regions of DAT:TH KO animals. None of the post-synaptic regions analyzed - Dorsal Striatum, Ventral Striatum and Prefrontal Cortex - show an overall change in transcriptome as a function of DA deficiency. This suggests that the broad striatal transcriptional changes in neurodegeneration-based PD models are not direct effects of DA depletion, but are rather a result of DA neuronal death. However, we find a number of dopaminergic genes differentially expressed in SNc, and to a lesser extent in VTA, as a function of DA deficiency, providing evidence for a DA-dependent feedback loop. Of particular interest, expression of Nr4a2 , a crucial transcription factor maintaining DA neuron identity, is significantly decreased in SNc, but not VTA, of DAT:TH KO mice. Unexpectedly, we find that half of DAT:TH KO animals show near-normal levels of Th transcription, and also display an overall transcript expression pattern more similar to wild type animals than to the remaining DAT:TH KO mice. These animals could represent recruitment of a pathway attempting to compensate for dopamine loss.

The sympathetic nervous system (SNS) regulates energy homeostasis in part by governing fatty acid liberation from adipose tissue. We first examined whether SNS activity toward discrete adipose depots changes in response to a weight loss diet in mice. We found that SNS activity toward each adipose depot is unique in timing, pattern of activation, and habituation with the most dramatic contrast between visceral and subcutaneous adipose depots. Sympathetic drive toward visceral epididymal adipose is more than doubled early in weight loss and then suppressed later in the diet when weight loss plateaued. Coincident with the decline in SNS activity toward visceral adipose is an increase in activity toward subcutaneous depots indicating a switch in lipolytic sources. In response to calorie restriction, SNS activity toward retroperitoneal and brown adipose depots is unaffected. Finally, pharmacological blockage of sympathetic activity on adipose tissue using the β3-adrenergic receptor antagonist, SR59230a, suppressed loss of visceral adipose mass in response to diet. These findings indicate that SNS activity toward discrete adipose depots is dynamic and potentially hierarchical. This pattern of sympathetic activation is required for energy liberation and loss of adipose tissue in response to calorie-restricted diet.

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.

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.

The involvement of mitochondria and the respiratory chain in aging is well established, and reports have uncovered the role of the nervous system in this process. To address whether particular neurons are important in determining longevity, we targeted the genetic components of complex IV and V in the respiratory chain by RNA inhibition (RNAi) in specific subsets of the Drosophila nervous system. We discovered that simultaneous interference in both serotonergic and dopaminergic neurons or selective interference in neurons expressing the serotonin receptor d5-HT1b leads to shortened Drosophila life span. No differences in life span were observed when RNAi was targeted exclusively to dopaminergic neurons or octopamine/tyramine neurons. Neuronal subtype was a very important factor in our experiments, and this study begins to offer clues as to which neurons might mediate longevity in a cell non-autonomous fashion.

BACKGROUND: Relatively low birth weight is associated with elevated heart rate and increased circulation of catecholamines and overall higher sympathetic activation in children. Increased levels of each of these variables in turn may promote poorer systolic blood pressure regulation in response to an orthostatic challenge. The present study examined the relationship between subsyndromal orthostatic systolic change in children and birth weights varying from 4 to 10 pounds. METHODS: Boys and girls (N=34) ranging in age from 7 – 10 years from a culturally diverse population were recruited. After lying supine for five minutes and again after about one minute of standing, blood pressure and pulse were measured. The greater the difference between standing minus supine systolic blood pressure, the stronger the response to orthostatic challenge. RESULTS: As birth weight increased, the systolic increase in blood pressure to an orthostatic challenge grew stronger. CONCLUSIONS: Increasing birth weight is associated with more efficient subsyndromal orthostatic blood pressure responses. Thus, increased heart rate and greater sympathetic nervous system activation found in low birth weight neonates are associated with poorer orthostatic blood pressure regulation that in turn is prognostic of cardiovascular difficulties and hypertension as well as cognitive and affective difficulties in childhood and in adulthood.

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.

The synaptic machinery for neurotransmitter storage is cell-type specific. Although most elements of biosynthesis and transport have been identified, it remains unclear whether additional factors may be required to maintain this specificity. The Drosophila serotonin transporter (dSERT) is normally expressed exclusively in serotonin (5-HT) neurons in the CNS. Here we examine the effects of ectopic transcriptional expression of dSERT in the Drosophila larval CNS. We find a surprising limitation on 5-HT storage following ectopic expression of dSERT and green fluorescence protein-tagged dSERT (GFP-dSERT). When dSERT transcription is driven ectopically in the CNS, 5-HT is detectable only in 5-HT, dopamine (DA), and a very limited number of additional neurons. Addition of exogenous 5-HT does not dramatically broaden neuronal storage sites, so this limitation is only partly due to restricted intercellular diffusion of 5-HT. Furthermore, this limitation is not due to gross mislocalization of dSERT, because cells lacking or containing 5-HT show similar levels and subcellular distribution of GFP-dSERT protein, nor is it due to lack of the vesicular transporter, dVMAT. These data suggest that a small number of neurons selectively express factor(s) required for 5-HT storage, and potentially for function of dSERT.

Sleep and arousal are known to be regulated by both homeostatic and circadian processes, but the underlying molecular mechanisms are not well understood. It has been reported that theDrosophilarest/activity cycle has features in common with the mammalian sleep/wake cycle, and it is expected that use of the fly genetic model will facilitate a molecular understanding of sleep and arousal. Here, we report the phenotypic characterization of aDrosophilarest/activity mutant known asfumin(fmn). We show thatfmnmutants have abnormally high levels of activity and reduced rest (sleep); genetic mapping, molecular analyses, and phenotypic rescue experiments demonstrate that these phenotypes result from mutation of theDrosophila dopamine transportergene. Consistent with the rest phenotype,fmnmutants show enhanced sensitivity to mechanical stimuli and a prolonged arousal once active, indicating a decreased arousal threshold. Strikingly,fmnmutants do not show significant rebound in response to rest deprivation as is typical for wild-type flies, nor do they show decreased life span. These results provide direct evidence that dopaminergic signaling has a critical function in the regulation of insect arousal.

Extracellular concentrations of monoamine neurotransmitters are regulated by a family of high-affinity transporters that are the molecular targets for such psychoactive drugs as cocaine, amphetamines, and therapeutic antidepressants. In Drosophila melanogaster, cocaine-induced behaviors show striking similarities to those induced in vertebrate animal models. Although a cocaine-sensitive serotonin carrier exists in flies, there has been no pharmacological or molecular evidence to support the presence of distinct carrier subtypes for other bioactive monoamines. Here we report the cloning and characterization of a cocaine-sensitive fly dopamine transporter (dDAT). In situ hybridization demonstrates that dDAT mRNA expression is restricted to dopaminergic cells in the fly nervous system. The substrate selectivity of dDAT parallels that of the mammalian DATs in that dopamine and tyramine are the preferred substrates, whereas octopamine is transported less efficiently, and serotonin not at all. In contrast, dDAT inhibitors display a rank order of potency most closely resembling that of mammalian norepinephrine transporters. Cocaine has a moderately high affinity to the cloned dDAT (IC50 = 2.6 microM). Voltage-clamp analysis of dDAT expressed in Xenopus laevis oocytes indicates that dDAT-mediated uptake is electrogenic; however, dDAT seems to lack the constitutive leak conductance that is characteristic of the mammalian catecholamine transporters. The combination of a DAT-like substrate selectivity and norepinephrine transporter-like inhibitor pharmacology within a single carrier, and results from phylogenetic analyses, suggest that dDAT represents an ancestral catecholamine transporter gene. The identification of a cocaine-sensitive target linked to dopaminergic neurotransmission in D. melanogaster will serve as a basis for further dissection of the genetic components of psychostimulant-mediated behavior.

We identified a unique type II cAMP-dependent protein kinase regulatory subunit (PKA-RII) gene in Drosophila melanogaster and a severely hypomorphic if not null mutation, pka-RII(EP(2)2162). Extracts from pka- RII(EP(2)2162) flies selectively lack RII-specific autophosphorylation activity and show significantly reduced cAMP binding activity, attributable to the loss of functional PKA-RII. pka-RII(EP(2)2162) shows 2-fold increased basal PKA activity and approximately 40% of normal cAMP-inducible PKA activity. pka-RII(EP(2)2162) is fully viable but displays abnormalities of ovarian development and multiple behavioral phenotypes including arrhythmic circadian locomotor activity, decreased sensitivity to ethanol and cocaine, and a lack of sensitization to repeated cocaine exposures. These findings implicate type II PKA activity in these processes in Drosophila and imply a common role for PKA signaling in regulating responsiveness to cocaine and alcohol.

Background: Sensitization to psychostimulant drugs of abuse is thought to be an important aspect of human addiction, yet how it develops is still unclear. The development of sensitization to cocaine in the fruit fly Drosophila melanogaster is strikingly similar to that observed in vertebrates. By taking advantage of the powerful genetic approaches that are possible in Drosophila , we are able to identify and characterize mutants that fail to develop sensitization. Results: We found that the Drosophila mutant inactive ( iav ) failed to become sensitized to cocaine. Mutant flies had reduced amounts of the trace amine tyramine in the brain because of reduced activity of the enzyme tyrosine decarboxylase (TDC), which converts tyrosine to tyramine. Furthermore, cocaine exposure induced TDC enzyme activity in a time-dependent manner that paralleled the development of behavioral sensitization. The sensitization failure of iav flies could be rescued by feeding the flies with tyramine; other biogenic amines or amine precursors did not have the same effect. Conclusions: These results indicate an essential role for tyramine in cocaine sensitization in Drosophila .

Cocaine abuse is a large social and economic problem that has received much public and scientific attention in recent years. Rodent and primate models have been used to study the behavioral and neurological effects of cocaine. Repeated intermittent doses of cocaine lead to progressive increases in both locomotor activity and stereotyped behaviors known as ‘reverse tolerance’ or behavioral sensitization, which may model the behavioral and neurochemical processes occurring in cocaine-addicted humans [1]. The biological basis of sensitization is poorly understood. We report that free-base cocaine administered in volatile form to the fruit fly Drosophila melanogaster induces multiple reflexive motor responses that resemble cocaine-induced behaviors in rodents. These behaviors are both dose dependent and sexually dimorphic. Furthermore, Drosophila develops a behavioral sensitization to intermittent doses of cocaine. These results suggest that the pathways leading to cocaine-induced responses and sensitization are evolutionarily conserved between Drosophila and higher vertebrates, and that this genetically tractable animal can be used as a new model system to help determine the biological mechanisms underlying these processes.

A preparation of decapitated Drosophila melanogaster has been used for direct application of drugs to the nerve cord. Serotonin, dopamine, and octopamine stimulate locomotion and grooming, showing distinguishable effects that often are potentiated by addition of the vertebrate monoamine oxidase-inhibitor hydrazaline. Many of the hydrazaline-induced effects are sexually dimorphic, with males showing greater responses than females. Behaviors similar to those induced by dopamine can be induced by application of the vertebrate dopamine D2-like receptor agonist quinpirole, whose effects are also sexually dimorphic. In contrast, vertebrate D2-like and D1-like dopamine antagonists result in akinesic states, and D1-like agonists selectively stimulate grooming. These data indicate that Drosophila nerve cord amine receptors are coupled to reflexive behaviors similar to those stimulated by brain dopamine receptors in vertebrates.

We present a high-resolution profile of the temporal and spatial immunoreactivity for dopamine and serotonin in the Drosophila embryonic CNS and the expression pattern of two enzymes important in their biosynthesis, DOPA decarboxylase (DDC) and tyrosine hydroxylase (TH). DDC performs the final catalytic step in the synthesis of both biogenic amines and TH is the rate-limiting enzymatic step in the synthesis of dopamine. We show that the DDC-expressing neurons synthesize either serotonin or dopamine, but not both, and that the two neuronal subtypes follow similar axonal pathways. In addition, we describe two DDC-expressing cell types that do not synthesize detectable levels of serotonin or dopamine. We also describe a novel set of TH-expressing neurons that are detected only during embryogenesis. The initial appearance of both enzymes and their metabolites during embryogenesis shows unexpected diversity. The onset of Ddc expression is heterogeneous, such that certain classes of cells express high levels of DDC several hours before others. High levels of TH immunoreactivity are observed at a time when DDC immunoreactivity is barely detectable. Despite low levels of DDC, both dopamine and serotonin are first detected at the earliest stages of DDC expression. We discuss the implications of these observations in the differentiation of dopamine and serotonin neurons.

This study investigated the feasibility and effectiveness of training pediatric residents to conduct a brief clinic-based behavioral intervention in coordination with community dissemination of a health promotion message developed by the Consortium for Lowering Obesity in Chicago Children. A total of 113 residents completed a short (

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. © 2010 Kong et al.

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.

Serotonin signaling plays a key role in the regulation of development, mood and behavior. Drosophila is well suited for the study of the basic mechanisms of serotonergic signaling, but the small size of its nervous system has previously precluded the direct measurements of neurotransmitters. This study demonstrates the first real-time measurements of changes in extracellular monoamine concentrations in a single larval Drosophila ventral nerve cord. Channelrhodopsin2-mediated, neuronal type-specific stimulation is used to elicit endogenous serotonin release, which is detected using fast-scan cyclic voltammetry at an implanted microelectrode. Release is decreased when serotonin synthesis or packaging are pharmacologically inhibited, confirming that the detected substance is serotonin. Similar to tetanus-evoked serotonin release in mammals, evoked serotonin concentrations are 280 – 640 nM in the fly, depending on the stimulation length. Extracellular serotonin signaling is prolonged after administering cocaine or fluoxetine, showing that transport regulates the clearance of serotonin from the extracellular space. When ChR2 is targeted to dopaminergic neurons, dopamine release is measured demonstrating that this method is broadly applicable to other neurotransmitter systems. This study shows that the dynamics of serotonin release and reuptake in Drosophila are analogous to those in mammals, making this simple organism more useful for the study of the basic physiological mechanisms of serotonergic signaling.

The trace biogenic amines tyramine and octopamine are found in the nervous systems of animals ranging in complexity from nematodes to mammals. In insects such as Drosophila melanogaster, the trace amine octopamine is a well-established neuromodulator that mediates a diverse range of physiological processes, but an independent role for tyramine is less clear. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC). We previously reported the identification of two Tdc genes in Drosophila: the peripherally-expressed Tdc1 and the neurally-expressed Tdc2. To further clarify the neural functions of the trace amines in Drosophila, we examined normal and cocaine-induced locomotor activity in flies that lack both neural tyramine and octopamine because of mutation in Tdc2 (Tdc2RO54). Tdc2RO54 flies have dramatically reduced basal locomotor activity levels and are hypersensitive to an initial dose of cocaine. Tdc2-targeted expression of the constitutively active inward rectifying potassium channel Kir2.1 replicates these phenotypes, and Tdc2-driven expression of Tdc1 rescues the phenotypes. However, flies that contain no measurable neural octopamine and an excess of tyramine due to a null mutation in the tyramine β-hydroxylase gene (TβHnM18) exhibit normal locomotor activity and cocaine responses in spite of showing female sterility due to loss of octopamine. The ability of elevated levels of neural tyramine in TβHnM18 flies to supplant the role of octopamine in adult locomotor and cocaine-induced behaviors, but not in functions related to female fertility, indicates mechanistic differences in the roles of trace amines in these processes. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007

We investigated the circadian function of Drosophila dopamine receptors by using a behaviorally active decapitated preparation that allows for direct application of drugs to the nerve cord. Quinpirole, a D2-like dopamine receptor agonist, induces reflexive locomotion in decapitated flies. We show that the amount of locomotion induced changes as a function of the time of day, with the highest responsiveness to quinpirole during the subjective night. Furthermore, dopamine receptor responsiveness is under circadian control and depends on the normal function of the period gene. The head pacemaker is at least partly dispensable for the circadian modulation of quinpirole-induced locomotion, because changes in agonist responsiveness persist in decapitated flies that are aged for 12 h. This finding suggests a role for the period -dependent molecular oscillators in the body in the modulation of amine receptor responsiveness.

The circadian clock consists of a feedback loop in which clock genes are rhythmically expressed, giving rise to cycling levels of RNA and proteins. Four of the five circadian genes identified to date influence responsiveness to freebase cocaine in the fruit fly, Drosophila melanogaster . Sensitization to repeated cocaine exposures, a phenomenon also seen in humans and animal models and associated with enhanced drug craving, is eliminated in flies mutant for period , clock , cycle , and doubletime , but not in flies lacking the gene timeless . Flies that do not sensitize owing to lack of these genes do not show the induction of tyrosine decarboxylase normally seen after cocaine exposure. These findings indicate unexpected roles for these genes in regulating cocaine sensitization and indicate that they function as regulators of tyrosine decarboxylase.

Publisher Summary This chapter reviews recent findings to imply that the linkage to dopamine receptors to specific types of behaviors is also conserved between higher vertebrates and insects. These findings have been obtained utilizing decapitated adult fruit flies, Drosophila melanogaster. Serotonin, dopamine, and octopamine show distinguishable effects when added to the decapitated preparations at millimolar concentrations. All three amines stimulate locomotion and hindleg grooming. Whereas the decapitated flies show a basal level of grooming before addition of amines, locomotion is never observed. Behaviors similar to those induced by dopamine can be induced by application of the vertebrate dopamine D2-like receptor agonist quinpirole. Other vertebrate dopamine agonists and antagonists show effects distinguishable from quinpirole: vertebrate D2-like and D1 -like dopamine antagonists result in akinesia, and D1-like agonists result in a selective stimulation of hindleg grooming without locomotion. The responses of decapitated Drosophila show many resemblances to the effects of vertebrate dopamine-receptor agonists and antagonists after injection into rodents: both D1-like and D2-like receptor agonists stimulate locomotion and sterotype behaviors, with differences in the types of stereotypies induced, and both D1-like and D2-like antagonists lead to an akinesic state. These phenotypic similarities are striking, indicating that if these compounds are interacting with receptors analogous to the vertebrate dopamine receptors, the link to locomotor and stereotyped grooming behaviors occurred very early in evolution, before the split between Drosophila and vertebrates. The vertebrate behavioral responses to dopamine depend on striatal dopamine receptors. It, thus, seems possible that the insect nerve cord contains functions that have been taken over by the forebrain in higher vertebrates. Further resolution of these issues should await identification and detailed study of the Drosophila receptors involved in these responses.

Publisher Summary This chapter reviews recent results concerning the transcriptional and post-transcriptional regulation of Drosophila Ddc and discusses the structure and regulation of the vertebrate amino acid decarboxylase (AADC) gene relative to Drosophila Ddc. Biogenic amines are decarboxylated derivatives of tyrosine and tryptophan that are found in animals from simple invertebrates to mammals. These compounds are found in neural tissue where they function as neurotransmitters and in non-neural tissues, where they have a variety of functions. The enzymes involved in biogenic amine synthesis and many receptors for these compounds have been isolated from both invertebrate and vertebrate sources. A key enzyme in biogenic amine biosynthesis is DOPA decarboxylase (DDC), also known as amino acid decarboxylase (AADC) in vertebrates. Functional cis-regulatory elements can be conserved between homologous genes of Drosophila and humans. In the hypoderm, Ddc expression leads to synthesis of dopamine that is further metabolized into quinones that have a vital function in the cross-linking, hardening, and pigmentation of the fly cuticle.

ception, condoms, HIV, STDs, friends, or suicide. Awareness that they could obtain free condoms in school and where to access confidential clinic services in the community was greater at the SBHC school. SBHC school students were much more likely to report a willingness to use a SBHC for a routine physical or for psychological concerns. Conclusions: Overall, SBHC school students were more likely to report better access to higher quality care. Our research design is applicable to the evaluation of existing school-based interventions where true baseline data are unavailable. Sources of Support: Columbia University Heilbrunn Dept of Population and Family Health.

The protein Elf-1 interacts with a cis-acting element that is required specifically for the neuronal expression of the Drosophila dopa decarboxylase gene Ddc. Using protein purified from Drosophila embryos, we raised Elf-1-specific monoclonal antibodies. The expression of Elf-1 during embryogenesis is restricted to nuclei of tissues derived from ectoderm, predominantly the central nervous system (CNS) and the epidermis. Within the CNS, Elf-1 is present in only a small fraction of nuclei, and the pattern of expressing nuclei changes dramatically during development. Elf-1 and Ddc are coexpressed in primary cultures of neural cells. However, we do not detect Elf-1 in Ddc-expressing neurons in vivo, leading to the suggestion that Elf-1 activity is required in vivo for initiation of Ddc expression but not for its maintenance. The antibodies also were used to isolate cDNA clones encoding Elf-1. Alternate forms of Elf-1 mRNA result in at least three protein isoforms.