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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.

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.

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.

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 adultDrosophilamelanogaster. 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.Method SummaryWe develop widely applicable and affordable solution to rapidly changing temperature within an enclosed chamber using commercially available components.

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.

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.

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/β 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.

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.

The trace biogenic amine tyramine is present in the nervous systems of animals ranging in complexity from nematodes to mammals. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC), a member of the aromatic amino acid family, but this enzyme has not been identified in Drosophila or in higher animals. To further clarify the roles of tyramine and its metabolite octopamine, we have cloned two TDC genes from Drosophila melanogaster, dTdc1 and dTdc2. Although both gene products have TDC activity in vivo, dTdc1 is expressed nonneurally, whereas dTdc2 is expressed neurally. Flies with a mutation in dTdc2 lack neural tyramine and octopamine and are female sterile due to egg retention. Although other Drosophila mutants that lack octopamine retain eggs completely within the ovaries, dTdc2 mutants release eggs into the oviducts but are unable to deposit them. This specific sterility phenotype can be partially rescued by driving the expression of dTdc2 in a dTdc2-specific pattern, whereas driving the expression of dTdc1 in the same pattern results in a complete rescue. The disparity in rescue efficiencies between the ectopically expressed Tdc genes may reflect the differential activities of these gene products. The egg retention phenotype of the dTdc2 mutant and the phenotypes associated with ectopic dTdc expression contribute to a model in which octopamine and tyramine have distinct and separable neural activities.

The fruit fly Drosophila melanogaster is a model system for studying pathways regulating responses to cocaine. We describe a new method for delivering cocaine to Drosophila. Freebase cocaine dissolved in ethanol is sprayed onto cold-anaesthetized flies using a graphic arts airbrush modified to reproducibly control the drug dosage. Cocaine dose response curves were generated to characterize the behavioral responses of flies using the airbrush method or the established cocaine smoke method of drug delivery. The stereotypic responses observed with the airbrush showed a dose-dependent increase and were qualitatively similar to those elicited by cocaine smoke. The variation in behaviors of flies dosed with the airbrush was smaller than that of the smoke-dosed flies, indicating that the airbrush method gives better reproducibility. Since flies are exposed to alcohol as well as cocaine in the airbrush behavioral paradigm, it was important to control for possible effects of ethanol. Control experiments indicated that none of the stereotypies elicited with cocaine were caused by vehicle alone and very little ethanol remains in the flies following this protocol. The utility of the airbrush method was demonstrated by its use in a pilot genetic screen that identified a cocaine resistant mutant.

Hearing inDrosophiladepends on the transduction of antennal vibration into receptor potentials by ciliated sensory neurons in Johnston's organ, the antennal chordotonal organ. We previously found that aDrosophilaprotein in the vanilloid receptor subfamily (TRPV) channel subunit, Nanchung (NAN), is localized to the chordotonal cilia and required to generate sound-evoked potentials (Kim et al., 2003). Here, we show that the only otherDrosophilaTRPV protein is mutated in the behavioral mutantinactive(iav). The IAV protein forms a hypotonically activated channel when expressed in cultured cells; in flies, it is specifically expressed in the chordotonal neurons, localized to their cilia and required for hearing. IAV and NAN are each undetectable in cilia of mutants lacking the other protein, indicating that they both contribute to a heteromultimeric transduction channelin vivo. A functional green fluorescence protein-IAV fusion protein shows that the channel is restricted to the proximal cilium, constraining models for channel activation.

I did not start out to work in neurogenetics. I am only doing what I do now because of Bill Pak's influence in pushing me in that direction, for which I am forever grateful! Here's my recollection ...

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.

A number of the pattern formation genes expressed in the Drosophila epidermis are also expressed in the developing CNS. It is uncertain how these genes contribute to neuronal differentiation. Here we show that the normal differentiation of the serotonin neurons of the Drosophila nerve cord is dependent on the expression of two pattern formation genes, huckebein (hkb) and engrailed (en). hkb and en gene products coexpress uniquely in the serotonin neurons and in neuroblast 7-3 (NB 7-3), indicating that as in the grasshopper, NB 7-3 is the progenitor for these neurons. Both genes are important for differentiation of this lineage, since loss-of-function alleles for hkb or en eliminate expression of several markers that characterize these cells. hkb appears to act at a step subsequent to NB delamination, since NB 7-3 is detectable in a strain containing a severe hkb allele.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are abundant RNA-binding proteins that are implicated in splicing regulation. Here we investigate the role of a Drosophila hnRNP in splicing regulation in living animals. We find that overexpression of the Drosophila hnRNP HRB98DE leads to skipping of all internal exons in the Drosophila dopa decarboxylase (Ddc) pre-mRNA in vivo. These results indicate that HRB98DE has a splicing activity that promotes use of terminal splice sites. The effect of excess HRB98DE on Ddc splicing is transient, even though high levels of HRB98DE persist for at least 24 hr. This suggests that Drosophila larvae can induce a compensating mechanism to counteract the effects of excess HRB98DE.

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 cry01 mutants and control flies to 1-hour 1000-lux light pulses. We found that cry01 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 cry01 mutants. At circadian time 15, the mutants exhibited phase advances instead of the expected delays. These complex results are discussed.

We report that two isoforms of Drosophila tyrosine hydroxylase protein are encoded via alternatively spliced exons. The major isoform (Type II) contains a novel acidic extension of 71 amino acids in the amino-terminal regulatory domain, which is likely to alter the regulatory properties of the tyrosine hydroxylase protein. The minor isoform (Type I) corresponds to the cDNA sequence reported previously. We also report the structure of the Drosophila tyrosine hydroxylase (DTH) gene and the diversity and tissue localization of its transcripts. At least three types of DTH mRNA are generated from a single primary transcript through alternative splicing and polyadenylation. Type II mRNA is the most abundant tyrosine hydroxylase transcript in Drosophila and is found predominantly in the hypoderm throughout all stages of development. Type I mRNA is present only in the CNS, where it is the primary form. The DTH transcripts detected in the CNS contain a longer 3'-untranslated region than the transcript expressed in the hypoderm, due to differential polyadenylation. In contrast, the same start site is used for DTH gene transcription in both tissues. These results show unexpected diversity in the DTH transcripts and point out possible mechanisms for differential regulation of tyrosine hydroxylase activity in the CNS and in the hypoderm.

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.

We have studied a 40-bp upstream regulatory region of the DOPA decarboxylase gene (Ddc) which is important for cell-specific expression in the Drosophila central nervous system (CNS). This region contains two redundant elements which when simultaneously mutated result in lowered DDC expression in serotonin neurons. We uncovered a protein binding site within one of these elements and have cloned a factor which binds to the site. This factor is the product of the zfh-2 gene, a complex homeodomain/zinc finger protein previously identified by binding to an opsin regulatory element. The in vivo profile of ZFH-2 in the larval CNS shows intriguing overlap with DDC in specific serotonin and dopamine neurons. We show that ZFH-2 is related to a human transcription factor ATBF1. The multiple homeodomain and zinc finger motifs in these two proteins show a similar linear arrangement which implies coordinate action among the motifs. In addition, the homology defines a new homeodomain subtype.

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.

Several genes have been identified that affect the specification of unique neuronal identities during development of the Drosophila central nervous system. At least two of these genes, fushi tarazu (ftz) and even-skipped (eve) share a common structural motif, the homoeobox, which encodes a sequence-specific DNA-binding domain (homoeodomain). A family of related proteins has been recently characterized in mammals and nematodes that contain a diverged homoedomain as part of a structure referred to as a POU domain. Mammalian genes encoding POU domains have region-specific patterns of expression in the central nervous system, indicating a potential role for them in the regulation of neuronal development. The nematode POU-domain gene, unc-86, is involved in the determination of neuroblast lineages leading to serotonergic and dopaminergic neurons. We have now identified a Drosophila gene, Cfla, encoding a sequence-specific DNA-binding protein containing a highly conserved POU domain. The Cfla gene product binds to a DNA element required for expression of the dopa decarboxylase gene (Ddc) in selected dopaminergic neurons, implying that it functions as a neuron-specific transcription factor.

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

Biogenic amines are critically important neuromodulators in both vertebrates and invertebrates. Quantification of these amines can be difficult, particularly in neural extracts of Drosophila melanogaster that contain interfering electroactive compounds. We have developed a method for the reliable separation and quantification of the biogenic amines dopamine, serotonin, tyramine, and octopamine in Drosophila brain extracts using high performance liquid chromatography with electrochemical detection. Our method obviates the need for complex preparatory procedures or instrumentation, and can reproducibly detect picogram quantities of these amines. By optimizing the composition of the mobile phase and the electrode potential, and by examining common complications in the analysis of biological samples, we have developed a reliable technique for monitoring levels of biogenic amines in the Drosophila brain.