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.     

Role of DopR in the molecular mechanism of the dopamine control of juvenile hormone metabolism in female Drosophila

The effect of a decreased availability of the D1-like dopamine receptor (DopR) in Drosophila (caused by DopR antagonist added into food) on the juvenile hormone (JH) synthesis rate in young female D. melanogaster has been studied. The JH degradation rate and the alkaline phosphatase (ALP) and tyrosine decarboxylase (TDC) activities were used as indicators of the JH synthesis rate. Treatment of the flies with butaclamol, a specific DopR antagonist, has been demonstrated to increase the JH degradation rate, and the stress reactivity of the system of JH metabolism and to decrease the ALP activity and stress reactivity, and to increase the TDC activity and stress reactivity. As shown earlier, all this indicates a decrease in the JH synthesis rate in young female drosophila with a decreased DopR availability. It is concluded that the activating effect of dopamine on JH synthesis in Drosophila is mediated by D1-like receptors.     

Mutation of Drosophila dopamine receptor DopR leads to male-male courtship behavior

In Drosophila, dopamine plays important roles in many biological processes as a neuromodulator. Previous studies showed that dopamine level could affect fly courtship behaviors. Disturbed dopamine level leads to abnormal courtship behavior in two different ways. Dopamine up-regulation induces male-male courtship behavior, while down-regulation of dopamine level results in increased sexual attractiveness of males towards other male flies. Until now, the identity of the dopamine receptor involved in this abnormal male-male courtship behavior remains unknown. Here we used genetic approaches to investigate the role of dopamine receptors in fly courtship behavior. We found that a dopamine D1-like receptor, DopR, was involved in fly courtship behavior. DopR mutant male flies display male-male courtship behavior. This behavior is mainly due to the male's increased propensity to court other males. Expression of functional DopR successfully rescued this mutant phenotype. Knock-down of D2-like receptor D2R and another D1-like receptor, DAMB, did not induce male-male courtship behavior, indicating the receptor-type specificity of this phenomenon. Our findings provide insight into a possible link between dopamine level disturbance and the induced male-male courtship behavior.     

Probable role of dopamine in the regulation of ventral nephrocyte functioning in Drosophila melanogaster adults

Filtration of haemolymph in insects to remove waste products is performed by nephrocytes, which comprise accessory cells of the circulatory system that are not connected to Malpighian tubules. There are two types of nephrocytes in Drosophila: ventral cells, situated around the junction between the cardia and the oesophagus, and pericardial cells, situated around the heart. In the present study, the expression of dopamine D1 (DopR) and D2‐like (DD2R) receptors in the ventral nephrocytes of Drosophila melanogaster Meigen (Diptera: Drosophilidae) is investigated. Immunohistochemical staining with polyclonal antibodies against DopR and DD2R demonstrates the presence of these receptors in adult nephrocytes. The functioning of D. melanogaster nephrocytes is investigated by evaluation of mortality rates in flies treated with silver nitrate (AgNO₃) compared with untreated controls. To determine whether a change in the level of dopamine receptors has an effect on the functioning of nephrocytes, the antisense suppressor of DD2R gene together with a nephrocyte‐specific driver is used in the UAS‐GAL4 system. The suppression of DD2R in nephrocytes results in a significant decrease of mortality under toxic conditions. Taken together, the data obtained in the present study indicate that dopamine takes part in the control of ventral nephrocyte functioning in D. melanogaster.     

Wasp uses venom cocktail to manipulate the behavior of its cockroach prey

The sting of the parasitoid wasp Ampulex compressa is unusual, as it induces a transient paralysis of the front legs followed by grooming behavior and then by a long-term hypokinesia of its cockroach prey. Because the wasp's goal is to provide a living meal for its newborn larva, the behavioral changes in the prey are brought about by manipulating the host behavior in a way beneficial to the wasp and its offspring. To this end, the wasp injects its venom cocktail with two consecutive stings directly into the host's central nervous system. The first sting in the thorax causes a transient front leg paralysis lasting a few minutes. This paralysis is due to the presence of a venom component that induces a postsynaptic block of central cholinergic synaptic transmission. Following the head sting, dopamine identified in the venom appears to induce 30 min of intense grooming. During the long-term hypokinesia that follows the grooming, specific behaviors of the prey are inhibited while others are unaffected. We propose that the venom represses the activity of head ganglia neurons thereby removing the descending excitatory drive to the thoracic neurons.Abbreviations CNS central nervous system - DA dopamine - GI giant interneuron - PSP postsynaptic potential - SEG sub-esophageal ganglion - TI thoracic interneuron     

The mushroom body D1 dopamine receptor controls innate courtship drive

Mating is critical for species survival and is profoundly regulated by neuromodulators and neurohormones to accommodate internal states and external factors. To identify the underlying neuromodulatory mechanisms, we investigated the roles of dopamine receptors in various aspects of courtship behavior in Drosophila. Here, we report that the D1 dopamine receptor dDA1 regulates courtship drive in naïve males. The wild‐type naïve males actively courted females regardless their appearance or mating status. On the contrary, the dDA1 mutant (dumb) males exhibited substantially reduced courtship toward less appealing females including decapitated, leg‐less and mated females. The dumb male's reduced courtship activity was due to delay in courtship initiation and prolonged intervals between courtship bouts. The dampened courtship drive of dumb males was rescued by reinstated dDA1 expression in the mushroom body α/β and γ neurons but not α/β or γ neurons alone, which is distinct from the previously characterized dDA1 functions in experience‐dependent courtship or other learning and memory processes. We also found that the dopamine receptors dDA1, DAMB and dD2R are dispensable for associative memory formation and short‐term memory of conditioned courtship, thus courtship motivation and associative courtship learning and memory are regulated by distinct neuromodulatory mechanisms. Taken together, our study narrows the gap in the knowledge of the mechanism that dopamine regulates male courtship behavior.     

The Drosophila dopamine 2-like receptor D2R (Dop2R) is required in the blood brain barrier for male courtship

The blood brain barrier (BBB) has the essential function to protect the brain from potentially hazardous molecules while also enabling controlled selective uptake. How these processes and signaling inside BBB cells control neuronal function is an intense area of interest. Signaling in the adult Drosophila BBB is required for normal male courtship behavior and relies on male-specific molecules in the BBB. Here we show that the dopamine receptor D2R is expressed in the BBB and is required in mature males for normal mating behavior. Conditional adult male knockdown of D2R in BBB cells causes courtship defects. The courtship defects observed in genetic D2R mutants can be rescued by expression of normal D2R specifically in the BBB of adult males. Drosophila BBB cells are glial cells. Our findings thus identify a specific glial function for the DR2 receptor and dopamine signaling in the regulation of a complex behavior.     

Characterization of [3H]LS‐3‐134, a novel arylamide phenylpiperazine D3 dopamine receptor selective radioligand

LS‐3‐134 is a substituted N‐phenylpiperazine derivative that has been reported to exhibit: (i) high‐affinity binding (K_i value 0.2 nM) at human D3 dopamine receptors, (ii) > 100‐fold D3 versus D2 dopamine receptor subtype binding selectivity, and (iii) low‐affinity binding (K_i > 5000 nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin‐dependent activation of the adenylyl cyclase inhibition assay, LS‐3‐134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [³H]‐labeled LS‐3‐134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10–15 min at 37°C) and binds with high affinity to both human (K_d = 0.06 ± 0.01 nM) and rat (K_d = 0.2 ± 0.02 nM) D3 receptors expressed in HEK293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [³H]LS‐3‐134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies, we propose that [³H]LS‐3‐134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype.     

A mutation in CLOCK leads to altered dopamine receptor function

Mice with a mutation in the Clock gene (ClockΔ19) have a number of behavioral phenotypes that suggest alterations in dopaminergic transmission. These include hyperactivity, increased exploratory behavior, and increased reward value for drugs of abuse. However, the complex changes in dopaminergic transmission that underlie the behavioral abnormalities in these mice remain unclear. Here we find that a loss of CLOCK function increases dopamine release and turnover in striatum as indicated by increased levels of metabolites HVA and DOPAC, and enhances sensitivity to dopamine receptor antagonists. Interestingly, this enlarged dopaminergic tone results in downstream changes in dopamine receptor (DR) levels with a surprising augmentation of both D1‐ and D2‐type DR protein, but a significant shift in the ratio of D1 : D2 receptors in favor of D2 receptor signaling. These effects have functional consequences for both behavior and intracellular signaling, with alterations in locomotor responses to both D1‐type and D2‐type specific agonists and a blunted response to cAMP activation in the ClockΔ19 mutants. Taken together, these studies further elucidate the abnormalities in dopaminergic transmission that underlie mood, activity, and addictive behaviors.     

Modulation of motor behavior by dopamine and the D1-like dopamine receptor AmDOP2 in the honey bee

Determining the specific molecular pathways through which dopamine affects behavior has been complicated by the presence of multiple dopamine receptor subtypes that couple to different second messenger pathways. The observation of freely moving adult bees in an arena was used to investigate the role of dopamine signaling in regulating the behavior of the honey bee. Dopamine or the dopamine receptor antagonist flupenthixol was injected into the hemolymph of worker honey bees. Significant differences between treated and control bees were seen for all behaviors (walking, stopped, upside down, grooming, flying and fanning), and behavioral shifts were dependent on drug dosage and time after injection. To examine the role of dopamine signaling through a specific dopamine receptor in the brain, RNA interference was used to reduce expression levels of a D1-like receptor, AmDOP2. Injection of Amdop2 dsRNA into the mushroom bodies reduced the levels of Amdop2 mRNA and produced significant changes in the amount of time honey bees spent performing specific behaviors with reductions in time spent walking offset by increases in grooming or time spent stopped. Taken together these results establish that dopamine plays an important role in regulating motor behavior of the honey bee.     

Sequential super-stereotypy of an instinctive fixed action pattern in hyper-dopaminergic mutant mice: a model of obsessive compulsive disorder and Tourette's

Background  

Excessive sequential stereotypy of behavioral patterns (sequential super-stereotypy) in Tourette's syndrome and obsessive compulsive disorder (OCD) is thought to involve dysfunction in nigrostriatal dopamine systems. In sequential super-stereotypy, patients become trapped in overly rigid sequential patterns of action, language, or thought. Some instinctive behavioral patterns of animals, such as the syntactic grooming chain pattern of rodents, have sufficiently complex and stereotyped serial structure to detect potential production of overly-rigid sequential patterns. A syntactic grooming chain is a fixed action pattern that serially links up to 25 grooming movements into 4 predictable phases that follow 1 syntactic rule. New mutant mouse models allow gene-based manipulation of brain function relevant to sequential patterns, but no current animal model of spontaneous OCD-like behaviors has so far been reported to exhibit sequential super-stereotypy in the sense of a whole complex serial pattern that becomes stronger and excessively rigid. Here we used a hyper-dopaminergic mutant mouse to examine whether an OCD-like behavioral sequence in animals shows sequential super-stereotypy. Knockdown mutation of the dopamine transporter gene (DAT) causes extracellular dopamine levels in the neostriatum of these adult mutant mice to rise to 170% of wild-type control levels.     

Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans

Dopamine-modulated behaviors, including information processing and reward, are subject to behavioral plasticity. Disruption of these behaviors is thought to support drug addictions and psychoses. The plasticity of dopamine-mediated behaviors, for example, habituation and sensitization, are not well understood at the molecular level. We show that in the nematode Caenorhabditis elegans, a D1-like dopamine receptor gene (dop-1) modulates the plasticity of mechanosensory behaviors in which dopamine had not been implicated previously. A mutant of dop-1 displayed faster habituation to nonlocalized mechanical stimulation. This phenotype was rescued by the introduction of a wild-type copy of the gene. The dop-1 gene is expressed in mechanosensory neurons, particularly the ALM and PLM neurons. Selective expression of the dop-1 gene in mechanosensory neurons using the mec-7 promoter rescues the mechanosensory deficit in dop-1 mutant animals. The tyrosine hydroxylase-deficient C. elegans mutant (cat-2) also displays these specific behavioral deficits. These observations provide genetic evidence that dopamine signaling modulates behavioral plasticity in C. elegans.     

Drosophila yellow-h encodes dopaminechrome tautomerase: A new enzyme in the eumelanin biosynthetic pathway

Melanin is a widely distributed phenolic pigment that is biosynthesized from tyrosine and its hydroxylated product, dopa, in all animals. However, recent studies reveal a significant deviation from this paradigm, as insects appear to use dopamine rather than dopa as the major precursor of melanin. This observation calls for a reconsideration of the insect melanogenic pathway. While phenoloxidases and laccases can oxidize dopamine for dopaminechrome production, the fate of dopaminechrome remains undetermined. Dopachrome decarboxylase/tautomerase, encoded by yellow-f/f2 of Drosophila melanogaster, can convert dopaminechrome into 5,6-dihydroxyindole, but the same enzyme from other organisms does not act on dopaminechrome, suggesting the existence of a specific dopaminechrome tautomerase (DPT). We now report the identification of this novel enzyme that biosynthesizes 5,6-dihydroxyindole from dopaminechrome in Drosophila. Dopaminechrome tautomerase acted on both dopaminechrome and N-methyl dopaminechrome but not on dopachrome or other aminochromes tested. Our biochemical and molecular studies reveal that this enzyme is encoded by the yellow-h gene, a member of the yellow gene family, and advance our understanding of the physiological functions of this gene family. Identification and characterization of DPT clarifies the precursor for melanin biosynthetic pathways and proves the existence of an independent melanogenic pathway in insects that utilizes dopamine as the primary precursor.     

CIN85 regulates dopamine receptor endocytosis and governs behaviour in mice

Despite extensive investigations of Cbl‐interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85^Δex2) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85^Δex2 animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post‐synaptic compartment of striatal neurons in which it co‐clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85^Δex2 mice.     

Characterization of dopamine releasable and reserve pools in Drosophila larvae using ATP/P2X2‐mediated stimulation

Dopaminergic signaling pathways are conserved between mammals and Drosophila, but the factors important for maintaining the functional pool of synaptic dopamine are not fully understood in Drosophila. In this study, we characterized the releasable and reserve dopamine pools in Drosophila larvae using ATP/P2X₂‐mediated stimulation. Dopamine release was stable with stimulations performed at least every 5 min but decayed with stimulations performed 2 min apart or less, indicating the replenishment of the releasable pool occurred on a time scale between 2 and 5 min. Dopamine synthesis or uptake was pharmacologically inhibited with 3‐iodotyrosine and cocaine, respectively, to evaluate their contributions to maintain the releasable dopamine pool. We found that both synthesis and uptake were needed to maintain the releasable dopamine pool, with synthesis playing a major part in long‐term replenishment and uptake being more important for short‐term replenishment. These effects of synthesis and uptake on different time scales in Drosophila are analogous to mammals. However, unlike in mammals, cocaine did not activate a reserve pool of dopamine in Drosophila when using P2X₂ stimulations. Our study shows that both synthesis and uptake replenish the releasable pool, providing a better understanding of dopamine regulation in Drosophila.

     

Different effects of the biogenic amines dopamine,serotonin and octopamine on the thoracic and abdominal portions of the escape circuit in the cockroach

1. The escape behavior of the cockroach, Periplaneta americana, is known to be modulated under various behavioral conditions (Camhi and Volman 1978; Camhi and Nolen 1981; Camhi 1988). Some of these modulatory effects occur in the last abdominal ganglion (Daley and Delcomyn 1981a, b; Libersat et al. 1989) and others in the thoracic ganglia (Camhi 1988). Neuromodulator substances are known to underlie behavioral modulation in various animals. Therefore, we have sought to determine whether topical application of putative neuromodulators of the escape circuit enhance or depress this circuit, and whether these effects differ in the last abdominal vs. the thoracic ganglia. 2. Topical application of the biogenic amines serotonin and dopamine to the metathoracic ganglion modulates the escape circuitry within this ganglion; serotonin decreases and dopamine enhances the response of leg motoneurons to activation of interneurons in the abdominal nerve cord by electrical or wind stimulation. 3. The neuropil of the thoracic ganglia contains many catecholamine-histofluorescent processes bearing varicosities, providing a possible anatomical substrate for dopamine release sites. 4. Topical application of octopamine to the terminal abdominal ganglion enhances the response of abdominal interneurons to wind stimulation of the cerci. In contrast, serotonin and dopamine have no effect at this site. 5. It is proposed that release of these biogenic amines may contribute to the known modulation of the cockroach escape response.     

Projection pattern of sensory neurons in the central nervous system of a homeotic mutation of the moth Manduca sexta

Octopod (Octo) is a mutation of the moth Manduca sexta, which transforms the first abdominal segment (A1) in the anterior direction. Mutant animals are characterized by the appearance of homeotic thoracic-like legs on A1. We exploited this mutation to determine what rules might be used in specifying the fates of sensory neurons located on the body surface of larval Manduca. Mechanical stimulation of homeotic leg sensilla did not cause reflexive movements of the homeotic legs, but elicited responses similar to those observed following stimulation of ventral A1 body wall hairs. Intracellular recordings demonstrated that several of the motoneurons in the A1 ganglion received inputs from the homeotic sensory hairs. The responses of these motoneurons to stimulation of homeotic sensilla resembled their responses to stimulation of ventral body wall sensilla. Cobalt fills revealed that the mutation transformed the segmental projection pattern of only the sensory neurons located on the ventral surface of A1, resulting in a greater number with intersegmental projection patterns typical of sensory neurons found on the thoracic body wall. Many of the sensory neurons on the homeotic legs had intersegmental projection patterns typical of abdominal sensory neurons: an anteriorly directed projection terminating in the third thoracic ganglion (T3). Once this projection reached T3, however, it mimicked the projections of the thoracic leg sensory neurons. These results demonstrate that the same rules are not used in the establishment of the intersegmental and leg-specific projection patterns. Segmental identity influences the intersegmental projection pattern of the sensory neurons of Manduca, whereas the leg-specific projections are consistent with a role for positional information in determining their pattern. © 1995 John Wiley & Sons, Inc.     

Association of seven polymorphisms of the D2 dopamine receptor gene with brain receptor-binding characteristics

Association of alleles at the Taql A, Taql B, intron 6, Taql D, exon 7, exon 8, and promoter-141C sites of the D2 dopamine receptor gene with D2 dopamine receptor binding characteristics in the caudate nucleus of Caucasian alcoholic and nonalcoholic subjects was determined. For the Taql D, exon 7, exon 8, and promoter-141C sites there were no significant allelic differences in Bmax (number of binding sites) or Kd (binding affinity) of the D2 dopamine receptors. However, subjects having the minor alleles at the Taql A, Taql B, and intron 6 sites had significantly lower Bmax than subjects not having them. None of these three polymorphisms had any significant effect on Kd. Highly significant linkage disequilibria were observed among the Taql A, Taql B, and intron 6 polymorphic sites, but linkage disequilibria between these three sites and each of the Taql D, exon 7, exon 8, and promoter-141C sites were of lesser or of no significance. Taken together, these findings suggest that the Taql A, Taql B, and intron 6 polymorphisms, but not the Taql D, exon 7, exon 8, and promoter-141C polymorphisms, are in linkage disequilibrium with a functional allelic variant that affects D2 dopamine receptor expression.