Interactivefly: GeneBrief

Dopamine 1-like receptor 2: Biological Overview | References


Gene name - Dopamine 1-like receptor 2

Synonyms - Damb, DopR2, DDR2

Cytological map position - 99B5-99B6

Function - Transmembrane protein

Keywords - G-protein coupled receptor, Dopamine receptor, locomotor activity, Malpighian tubules, presynaptic DD2R autoreceptor, mushroom bodies, appetitive and aversive learning

Symbol - Dop1R2

FlyBase ID: FBgn0266137

Genetic map position - chr3R:29,630,304-29,659,984

Classification - 7 transmembrane receptor (rhodopsin family)

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Zhang, S. X., Rogulja, D. and Crickmore, M. A. (2016). Dopaminergic circuitry underlying mating drive. Neuron [Epub ahead of print]. PubMed ID: 27292538
Summary:
This study developed a new system for studying how innate drives are tuned to reflect current physiological needs and capacities, and how they affect sensory-motor processing. The existence of male mating drive is demonstrated in Drosophila that is transiently and cumulatively reduced as reproductive capacity is depleted by copulations. Dopaminergic activity in the anterior of the superior medial protocerebrum (SMPa) is also transiently and cumulatively reduced in response to matings and serves as a functional neuronal correlate of mating drive. The dopamine signal is transmitted through the D1-like DopR2 receptor to P1 neurons, which also integrate sensory information relevant to the perception of females, and which project to courtship motor centers that initiate and maintain courtship behavior. Mating drive therefore converges with sensory information from the female at the point of transition to motor output, controlling the propensity of a sensory percept to trigger goal-directed behavior.

Regna, K., Kurshan, P. T., Harwood, B. N., Jenkins, A. M., Lai, C. Q., Muskavitch, M. A., Kopin, A. S. and Draper, I. (2016). A critical role for the Drosophila dopamine D1-like receptor Dop1R2 at the onset of metamorphosis. BMC Dev Biol 16: 15. PubMed ID: 27184815
Summary:
Insect metamorphosis relies on temporal and spatial cues that are precisely controlled. Previous studies in Drosophila have shown that untimely activation of genes that are essential to metamorphosis results in growth defects, developmental delay and death. Multiple factors exist that safeguard these genes against dysregulated expression. The list of identified negative regulators that play such a role in Drosophila development continues to expand. By using RNAi transgene-induced gene silencing coupled to spatio/temporal assessment, this study has unraveled an important role for the Drosophila dopamine 1-like receptor, Dop1R2, in development. Dop1R2 knockdown leads to pre-adult lethality. In adults that escape death, abnormal wing expansion and/or melanization defects occur. Furthermore salivary gland expression of this GPCR during the late larval/prepupal stage is essential for the flies to survive through adulthood. In addition to RNAi-induced effects, treatment of larvae with the high affinity D1-like receptor antagonist flupenthixol, also results in developmental arrest, and in morphological defects comparable to those seen in Dop1R2 RNAi flies. To examine the basis for pupal lethality in Dop1R2 RNAi flies, transcriptome analysis was carried out. These studies revealed up-regulation of genes that respond to ecdysone, regulate morphogenesis and/or modulate defense/immunity. Taken together these findings suggest a role for Dop1R2 in the repression of genes that coordinate metamorphosis. Premature release of this inhibition is not tolerated by the developing fly.
Pavlowsky, A., Schor, J., Placais, P. Y. and Preat, T. (2018). A GABAergic feedback shapes dopaminergic input on the Drosophila mushroom body to promote appetitive long-term memory. Curr Biol. Pubmed ID: 29779874
Summary:
Memory consolidation is a crucial step for long-term memory (LTM) storage. However, a clear picture of how memory consolidation is regulated at the neuronal circuit level is still lacking. This study took advantage of the Drosophila olfactory memory center, the mushroom body (MB), to address this question in the context of appetitive LTM. The MB lobes, which are made by the fascicled axons of the MB intrinsic neurons, are organized into discrete anatomical modules, each covered by the terminals of a defined type of dopaminergic neuron (DAN) and the dendrites of a corresponding type of MB output neuron (MBON). An essential role has been revealed of one DAN, the MP1 neuron, in the formation of appetitive LTM. The MP1 neuron is anatomically matched to the GABAergic MBON MVP2, which has been attributed feedforward inhibitory functions recently. This study used behavior experiments and in vivo imaging to challenge the existence of MP1-MVP2 synapses and investigate their role in appetitive LTM consolidation. MP1 and MVP2 neurons form an anatomically and functionally recurrent circuit, which features a feedback inhibition that regulates consolidation of appetitive memory. This circuit involves two opposite type 1 and type 2 dopamine receptors (the type 1 DAMB and the type 2 dD2R) in MVP2 neurons and the metabotropic GABAB-R1 receptor in MP1 neurons. It is proposed that this dual-receptor feedback supports a bidirectional self-regulation of MP1 input to the MB. This mechanism displays striking similarities with the mammalian reward system, in which modulation of the dopaminergic signal is primarily assigned to inhibitory neurons.

BIOLOGICAL OVERVIEW

Dopaminergic neurons in Drosophila play critical roles in diverse brain functions such as motor control, arousal, learning, and memory. Using genetic and behavioral approaches, it has been firmly established that proper dopamine signaling is required for olfactory classical conditioning (e.g., aversive and appetitive learning). Dopamine mediates its functions through interaction with its receptors. There are two different types of dopamine receptors in Drosophila: 1) Dopamine 1-like, including Dopamine 1-like receptor 1 and Dopamine 1-like receptor 2 (DDR2, the subject of this report) and 2) Dopamine 2-like receptor. Currently, no study has attempted to characterize the role of DD2R in Drosophila learning and memory. Using a DD2R-RNAi transgenic line, this study has examined the role of DD2R, expressed in dopamine neurons (i.e., the presynaptic DD2R autoreceptor), in larval olfactory learning. The function of postsynaptic DD2R expressed in mushroom body (MB) was also studied as MB is the center for Drosophila learning, with a function analogous to that of the mammalian hippocampus. These results showed that suppression of presynaptic DD2R autoreceptors impairs both appetitive and aversive learning. Similarly, postsynaptic DD2R in MB neurons appears to be involved in both appetitive and aversive learning. The data confirm, for the first time, that DD2R plays an important role in Drosophila olfactory learning (Qi, 2014).

Dopamine (DA) is an important neurotransmitter mediating a variety of brain functions including locomotion, reward, awareness, learning and memory, and cognition. Genetic and pharmacological studies revealed that the dopaminergic system in the fruit fly Drosophila melanogaster plays multiple roles in motor function and associative learning. Using the sophisticated genetic tools available for the fruit fly, it has been firmly established that release of dopamine is required for associative learning in Drosophila adults and larvae. Dopaminergic neural circuits mediating olfactory learning have been also characterized in the fruit fly brain (Qi, 2014).

DA mediates its physiological functions through interaction with its receptors. Analysis of the primary structure of the DA receptors revealed that those receptors belong to the G-protein coupled receptor (GPCR) family. Generally, DA receptors can be divided into two families in vertebrates. The D1-like receptor family stimulates cAMP production by activation of the receptor-coupled Gs subunit of G proteins. The D2-like receptor family belongs to the pertussis toxin (PTX)-sensitive G protein (i.e., Gi and Go)-coupled receptor (GPCR) superfamily. Therefore, actions of D2-like receptor members have been characterized as inhibitory. In relation to regulation of DA signaling, Aghajanian (1997) found a very interesting feature: DA neurons possess receptors for their own transmitter, dopamine, at their synaptic nerve terminals. These DA autoreceptors (autoR) function as self-inhibitory regulators (Qi, 2014).

In Drosophila, there are four DA receptors (dDA1, DAMB, DopEcR, and DD2R) that have been cloned and characterized. Two DA receptors (dDA1, DAMB) were cloned first and appear to be members of the D1-like receptor family on the basis of their ability to stimulate adenylyl cyclase (AC) in a heterologous expression system. In contrast, only one Drosophila DA receptor DD2R gene has been identified (Hearn, 2002). Functional expression of the DD2R gene in HEK293 cells indicated that DA caused a marked decrease in forskolin-induced cAMP level, indicating that DD2R belongs to the inhibitory D2-like receptor family. Interestingly, two recent studies (Vickrey, 2011; Wiemerslage, 2013) confirmed the existence of DA autoreceptors in Drosophila (Qi, 2014).

Olfactory associative learning in adult flies requires expression of Drosophila D1 receptor dDA1 in the mushroom body, the anatomical center for learning and memory. The dDA1 mutant dumb showed impaired appetitive learning as well as aversive learning. These impaired learning behaviors were fully rescued by expression of the wild-type dDA1 transgene in MB neurons in mutant flies, further confirming the role of Drosophila D1-like receptors in learning. However, no previous study has attempted to characterize the role of D2-like DD2R in Drosophila learning and memory. Interestingly, there was one study showing that a D2 agonist eticlopride did not disrupt visual learning (e.g., T maze assay) in adult flies (Qi, 2014).

Drosophila larvae carrying DD2R-RNAi transgene were used to examine the role of D2-like receptors in associative learning. Two different types of tissue-specific drivers were used to examine both presynaptic D2 autoreceptors and postsynaptic D2 receptors. Dopaminergic-specific driver TH-Gal4 was used to induce DD2R-RNAi expression in DA neurons. Since the target of dopaminergic innervation is the mushroom body (MB), the center for learning and memory in Drosophila, MB-specific drivers (201Y-Gal4, 30Y-Gal4) were used to down-regulate postsynaptic DD2R combined with DD2R-RNAi transgene. The results showed that both presynaptic DD2R autoreceptors and postsynaptic receptors are required for aversive and appetitive olfactory learning in Drosophila larvae (Qi, 2014).

The Drosophila D2 receptor DD2R has been shown to play an important role in locomotion, aggression, and neuroprotection (Wiemerslage, 2013; Draper, 2007; Alekseyenko, 2013). Interestingly, no study has shown whether Drosophila DD2R is involved in learning and memory, although dopaminergic (DA) neural circuits and D1 receptors are known to mediate Drosophila aversive learning. The present study, for the first time, demonstrated that DD2R is involved in olfactory associative learning in Drosophila larvae. Further, we showed that both presynaptic and postsynaptic DD2Rs mediate aversive and appetitive learning in the fly larvae as down-regulation of DD2R in DA and mushroom body (MB) neurons resulted in impaired olfactory learning (Qi, 2014).

Multiple studies have proved that dopamine signaling is necessary in Drosophila aversive learning. However, it is uncertain whether dopamine signaling is involved in appetitive learning. Several laboratories reported that DA signaling is not necessary for appetitive learning, which is mediated by another biogenic amine, octopamine In contrast, Selcho (2009) showed that DA signaling is necessary for appetitive learning; inhibition of DA release resulted in reduced appetitive learning. Furthermore, D1 receptor mutants (e.g., dDA1) showed impaired appetitive learning (Qi, 2014).

This study has demonstrated that dopamine mediates not only aversive learning, but also appetitive learning. Both learning behaviors are impaired when DD2R-RNAi is expressed in DA neurons or in MB neurons. Interestingly, aversive learning was completely impaired, while appetitive learning was only partially impaired when DD2R-RNAi was expressed in DA neurons. A possible explanation is that the effect of DD2R-RNAi is partial as RNAi down-regulates the target gene expression. Another possibility is that DA is not the only modulatory neurotransmitter mediating appetitive learning; another biogenic amine, octopamine, is involved in appetitive learning. Therefore, octopamine can mediate appetitive learning to a certain extent even if DA signaling is impaired. In contrast, no modulatory neurotransmitter other than DA is known to be involved in aversive learning (Qi, 2014).

Draper (2007) reported reduced locomotion due to expression of DD2R-RNAi. The current findings do not support that result since the larvae carrying DD2R-RNAi showed no changes in sensory and motor function, compared to WT and control fly strains. This discrepancy can be explained through the following reasons. first, it may be related to developmental-specific effects. This study used third-instar larvae while adult flies were used by Draper. Second, there are differences in locomotion assays. Draper quantified total activity counts, amount of time active, and number of activity-rest bouts. In the current study, crawling speed was measured. Third, DD2R-RNAi expression patterns are different. Draper used Act5C-Gal or elav-Gal4 to express DD2R-RNAi ubiquitously or pan-neuronally, respectively. In contrast, DD2R-RNAi was only expressed in DA or MB neurons in the current study. Therefore, neural circuits affected by DD2R-RNAi can be different, resulting in different behaviors. It is also possible that expression level of DD2R-RNAi is different due to different drivers (Qi, 2014).

Since the identification of Drosophila D2 receptor DD2R (Han, 1996), two studies have revealed the autoreceptor function of DD2R. Vickrey (2011) reported that D2R agonists reduce DA release in the Drosophila larval central nervous system. It was also shown that DD2R autoreceptors suppress excitability of DA neurons in Drosophila primary neuronal cultures (Wiemerslage, 2013). This study showed that DD2R is involved in mediating both appetitive and aversive olfactory learning. DD2R-RNAi in DA neurons down-regulates DD2R autoreceptor function. Thus excitability of DA neurons is increased, leading to an increase of DA release. Our results indicate that excessive DA release impairs olfactory learning. Indeed, Zhang (2008) showed that olfactory learning is impaired in Drosophila DA transporter mutant fumin, likely due to increased synaptic DA levels. In contrast, a lack of DA release is known to cause impaired learning in Drosophila larvae. Therefore, it appears that homeostatic regulation of DA release by DD2R is important for both appetitive and aversive olfactory learning as either too much or too little synaptic DA causes impaired learning. Taking these facts into consideration, a model is proposed to explain the role of presynaptic DD2R autoreceptors. Presynaptic DD2R autoreceptors suppress release of DA at the presynaptic terminals in the MB. If presynaptic DD2R function is suppressed, then more DA is released into MB neurons. Increased DA tone in the MB impairs both aversive and appetitive learning behaviors (Qi, 2014).

This study also showed that olfactory learning in Drosophila larvae is impaired by down-regulation of postsynaptic DD2R in MB neurons. As the role of DD2R is inhibitory, the effects of DD2R-RNAi in MB neurons are expected to increase neuronal excitability, and thus olfactory learning is impaired by hyperexcitability in MB neurons. This observation might not be consistent with the physiological findings that learning and memory are mediated by enhanced neuronal excitability and synaptic transmission. Such well-known examples are long-term facilitation (LTF) and long-term potentiation (LTP). In this study, DD2R-RNAi is expressed throughout the larval stage. Therefore, hyperexcitability is chronic and thus this increased baseline activity interferes with coding new information in the MB. Indeed, olfactory learning is impaired in Drosophila by the chronic increase of excitatory cholinergic synaptic transmission due to the phosphodiesterase gene dunce mutation, resulting in increased cAMP levels. Taken together, temporal increases in excitability are key physiological changes underlying associative learning and thus DD2R-RNAi interferes with this change by inducing chronic hyperexcitability in MB neurons (Qi, 2014).

In addition to DD2R, there are Drosophila D1-like receptors (dDA1 and DAMB) that are known to be highly expressed in MB neurons. In fact, dDA1 null mutants showed defects in olfactory learning. Since D1-like receptors increase neuronal excitability via the cAMP-PKA signaling pathway, dDA1 mutant MB neurons are less depolarized when DA is released at the synaptic terminal in the MB, and thus cannot mediate olfactory learning. Proper excitability of MB neurons should be maintained by balancing actions of D1- and D2-like receptors in MB neurons (Qi, 2014).

It has been proposed that the adenylyl cyclase gene rutabaga in MB is a coincidence detector for CS and US in Drosophila olfactory learning and memory. Therefore, on the basis of the current results, a model is proposed to explain postsynaptic mechanisms underlying aversive and appetitive learning. Postsynaptic DD2Rs in MBNs inhibit neuronal excitability while dDA1 stimulates neural circuits associating CS with US in MB. DA receptors dDA1 and DD2R regulate AC in MB neurons in the opposite direction to maintain homeostatic balance of MB neuronal excitability, which is an important physiological element for Drosophila larval olfactory learning (Qi, 2014).

In conclusion, this study examined the role of D2-like receptor DD2R in Drosophila olfactory associative learning. The results showed that suppression of presynaptic DD2R autoreceptors impairs both appetitive and aversive learning. Similarly, postsynaptic DD2R in MB neurons appears to be involved in both appetitive and aversive learning (Qi, 2014).

The data strongly support the hypothesis that presynaptic DD2R autoreceptors suppress release of DA at the presynaptic terminals in the MB. If presynaptic DD2R function is suppressed, then more DA is released. Increasing DA tone to MB neurons impairs both aversive and appetitive learning behaviors. Postsynaptically, DD2R-RNAi impaired olfactory associative learning most likely by inducing chronic hyperexcitability in MB neurons. Therefore, the role of postsynaptic DD2R is to maintain the proper excitability in MB neurons during learning. Taken together, this study, for the first time, demonstrated that DD2R plays an important role in Drosophila olfactory associative learning (Qi, 2014).

Dopamine receptor DAMB signals via Gq to mediate forgetting in Drosophila

Prior studies have shown that aversive olfactory memory is acquired by dopamine acting on a specific receptor, dDA1, expressed by mushroom body neurons. Active forgetting is mediated by dopamine acting on another receptor, Damb, expressed by the same neurons. Surprisingly, prior studies have shown that both receptors stimulate cyclic AMP (cAMP) accumulation, presenting an enigma of how mushroom body neurons distinguish between acquisition and forgetting signals. This study surveyed the spectrum of G protein coupling of dDA1 and Damb, and it was confirmed that both receptors can couple to Gs to stimulate cAMP synthesis. However, the Damb receptor uniquely activates Gq to mobilize Ca(2+) signaling with greater efficiency and dopamine sensitivity. The knockdown of Galphaq with RNAi in the mushroom bodies inhibits forgetting but has no effect on acquisition. These findings identify a Damb/Gq-signaling pathway that stimulates forgetting and resolves the opposing effects of dopamine on acquisition and forgetting (Himmelreich, 2017).

This study provides biochemical and behavioral evidence that the Drosophila DA receptor Damb couples preferentially to Gαq to mediate signaling by Damb for active forgetting. This conclusion offers an interesting contrast to the role of the dDA1 receptor in MBns for acquisition, and it resolves the issue of how MBns distinguish DA-mediated instructions to acquire memory versus those to forget. Prior studies had classified both dDA1 and Damb as cAMP-stimulating receptors, similar to mammalian D1/D5 DA receptors that work through Gαs/olf. The results extend prior studies of dDA1 by examining coupling of this receptor with multiple heterotrimeric G proteins to show that the receptor strongly and preferentially couples to Gs proteins. This affirms the receptor's role in the acquisition of memory, consistent with the tight link between acquisition and cAMP signaling. This study found that the Damb receptor can weakly couple to Gs proteins but preferentially engages Gq to trigger the Ca2+-signaling pathway, a feature not displayed by dDA1. Comparing the two Gαq paralogs of Drosophila (G and D) with a human ortholog shows that Drosophila GαqG and human Gαq share a conserved C terminus, essential for selective coupling to GPCRs, but quite distinct in sequence compared to the GαqD C terminus. Since GαqD is a photoreceptor-selective G protein that couples with rhodopsin, it is proposed that GαqG is the isoform that relays Damb's signals to spur forgetting (Himmelreich, 2017).

It is envisioned that memory acquisition triggered by strong DA release from electric shock pulses used for aversive conditioning drives both cAMP and Ca2+ signaling through dDA1 and Damb receptors in the MBns. Forgetting occurs from weaker DA release after the acquisition through restricted Damb/Gαq/Ca2+ signaling in the MBns. The coupling of Damb to Gs at high DA concentrations also explains why Damb mutants have a slight acquisition defect after training with a large number of shocks. Although the model allows the assignment of acquisition and forgetting to two distinct intracellular signaling pathways, it does not preclude the possibility that other differences in signaling distinguish acquisition from forgetting. These include the possible use of different presynaptic signals, such as a co-neurotransmitter released only during acquisition or forgetting (Himmelreich, 2017).

Drosophila Dopamine2-like receptors function as autoreceptors

Dopaminergic signaling pathways are conserved between mammals and Drosophila and D2 receptors have been identified in Drosophila. However, it has not been demonstrated whether Drosophila D2 receptors function as autoreceptors and regulate the release of dopamine. This study determine whether Drosophila D2 receptors act as autoreceptors by probing the extent to which D2 agonists and antagonists affect evoked dopamine release. Fast-scan cyclic voltammetry was used to measure stimulated dopamine release at a carbon-fiber microelectrode implanted in an intact, larval Drosophila nervous system. Dopamine release was evoked using 5-second blue light stimulations that open Channelrhodopsin-2, a blue light activated cation channel that was specifically expressed in dopaminergic neurons. In mammals, administration of a D2 agonist decreases evoked dopamine release by increasing autoreceptor feedback. Similarly, this study found that the D2 agonists bromocriptine and quinpirole decreased stimulated dopamine release in Drosophila. D2 antagonists were expected to increase dopamine release and the D2 antagonists flupenthixol, butaclamol, and haloperidol did increase stimulated release. Agonists did not significantly modulate dopamine uptake although the modulatory effects of D2 drugs on release were affected by prior administration of the uptake inhibitor nisoxetine. These results demonstrate that the D2 receptor functions as an autoreceptor in Drosophila. The similarities in dopamine regulation validate Drosophila as a model system for studying the basic neurobiology of dopaminergic signaling (Vickrey, 2011).

The monoamine neurotransmitter dopamine plays a major role in many human behaviors such as movement, cognition, reward, addiction, and motivation. Abnormalities in dopaminergic signaling are implicated in diseases such as schizophrenia, Parkinson’s disease, and drug addiction. Dopaminergic signaling is mediated by receptors that are located either postsynaptically, where they regulate downstream signaling, or presynaptically, where they act as autoreceptors regulating release. D2 receptors (D2Rs) are the predominant dopamine autoreceptor, and dysfunction of D2 autoreceptors is involved in disease etiology. Therefore, D2 receptors are important drug target sites. For example, patients with schizophrenia have a higher level of expression of D2 receptors and higher basal levels of dopamine; thus, many antipsychotics target the D2 receptor (Abi-Dargham, 2000). Other studies have shown that mice without the D2R gene have significant neurological impairments and Parkinson-like symptoms (Calabresi, 1997). Consequently, D2Rs are targets for Parkinson treatment. In addition to their implication in specific diseases, D2Rs have also been shown to modulate locomotion. Thus, autoreceptors are critical for regulating dopamine release and maintaining dopaminergic function (Vickrey, 2011 and references therein).

Three mammalian isoforms of D2R, differing by up to 29 amino acids, have been isolated: D2 short (D2S), D2 long (D2L), and D2 extra long. The D2S receptor subtype is located presynaptically and functions as an autoreceptor, while the D2L receptor subtype is located postsynaptically (Lindgren, 2003). Both isoforms are found in many species: human, rat, mouse, bovine, Caenorhabditis elegans, and Xenopus. Eight isoforms of a Drosophila D2-like receptor (DD2R) have been identified. These DD2Rs are G-protein-coupled receptors with a high affinity for dopamine that have amino acid sequences homologous to those of mammalian D2-like receptors (Hearn, 2002). It is unclear whether these receptors are D2L or D2S, and identifying the cellular locations and function of these DD2R receptors is difficult. Immunohistochemistry studies have identified DDR2 localization in larva, and DD2R staining is colocalized with both dopaminergic cell bodies and projections, although the expression presynaptically or postsynaptictically has not been determined (Draper, 2007). DD2Rs were expressed in HEK293 cells, and pharmacological evaluation with mammalian D2R agonists and antagonists showed that the agonist bromocriptine and the antagonists flupenthixol and butaclamol exhibited high-affinity binding. In contrast, the agonist quinpirole and the antagonist haloperidol had little to no affinity for the DD2Rs (Hearn, 2002). However, some drugs with poor affinity cause behavioral effects in Drosophila. For example, the agonist quinpirole increases locomotor activity in adults. Molecular biology and behavioral results suggest that D2 autoreceptor functionality may be conserved in Drosophila. Chemical measurements of dopamine release would provide direct evidence and establish the relative effectiveness of DD2R drugs in an intact Drosophila central nervous system (Vickrey, 2011).

This study chemically investigated the effect of the Drosophila Dopamine-2 receptor (Dopamine 1-like receptor 2, the subject of this report) on regulating dopamine release. The decrease in stimulated dopamine release in the presence of dopamine agonists and the increase in release in the presence of antagonists are consistent with DDR2 acting as an autoreceptor. These studies were modeled after mammalian studies, which probed autoreceptor functionality by electrical stimulation of dopaminergic fibers and detection of dopamine with FSCV. This specific stimulation protocol and the fast nature of the detection led to a probing of presynaptic effects. Similarly, in this study, optical stimulation of ChR2 located specifically in dopaminergic terminals would also allow investigation of primarily presynaptic regulation. Thus, the pharmacological effects are unlikely to be due to downstream effects caused by activation of postsynaptic dopamine receptors. The effects of D2 agonists and antagonists on stimulated dopamine release in Drosophila are analogous to results in mammals; this supports the conclusion that the DD2R is functioning as an autoreceptor, regulating the release of dopamine. While no interaction facilitating uptake was observed for D2 receptors and DAT, disruption of dopamine signaling with an uptake inhibitor did alter the effects of D2 drugs on dopamine release. Because autoreceptors play such an important role in human disease etiology, the conservation of autoreceptors between species makes Drosophila a useful model for studying dopaminergic diseases (Vickrey, 2011).

Single dopaminergic neurons that modulate aggression in Drosophila

Monoamines, including dopamine (DA), have been linked to aggression in various species. However, the precise role or roles served by the amine in aggression have been difficult to define because dopaminergic systems influence many behaviors, and all can be altered by changing the function of dopaminergic neurons. In the fruit fly, with the powerful genetic tools available, small subsets of brain cells can be reliably manipulated, offering enormous advantages for exploration of how and where amine neurons fit into the circuits involved with aggression. By combining the GAL4/upstream activating sequence (UAS) binary system with the flippase (flP) recombination technique, it was possible to restrict the numbers of targeted DA neurons down to a single-cell level. To explore the function of these individual dopaminergic neurons, they were inactivated with the tetanus toxin light chain, a genetically encoded inhibitor of neurotransmitter release, or they were activated with dTrpA1, a temperature-sensitive cation channel. Two sets of dopaminergic neurons were found that modulate aggression, one from the T1 cluster and another from the PPM3 cluster. Both activation and inactivation of these neurons resulted in an increase in aggression. It was demonstrated that the presynaptic terminals of the identified T1 and PPM3 dopaminergic neurons project to different parts of the central complex, overlapping with the receptor fields of DD2R and DopR DA receptor subtypes, respectively. These data suggest that the two types of dopaminergic neurons may influence aggression through interactions in the central complex region of the brain involving two different DA receptor subtypes (Alekseyenko, 2013).

Aggression in Drosophila is an innate behavior whose core circuitry is likely to be wired in the nervous system before eclosion. Appropriate displays of aggression rely on the correct identification of a potential competitor, an evaluation of the environmental signals, and the physiological state of the animal. With fixed numbers of neurons and neuronal circuits available, further flexibility in nervous system utilization is added by neuromodulators that can efficiently and reversibly reconfigure the function of networks without changing their 'hardwiring.' (Alekseyenko, 2013).

Dopamine, among other neuromodulators, is released by interneurons and acts at multiple sites within circuitries to alter the output of systems. Aminergic neurons in the fly nervous system display arbors that branch widely and cover multiple neuropil areas, through which they affect virtually all aspects of fly behavior. An open question remains whether individual neurons have selective actions on specific behavioral pathways or generalized actions on multiple behaviors. This paper used an intersectional genetics approach to alter the function of single neurons. This allowed asking whether individual DA neurons are involved in the regulation of aggression, where that regulation is exerted, and whether this is a selective action on aggression or these neurons modulate other behaviors as well (Alekseyenko, 2013).

A previous attempt to examine the role of DA in aggression in Drosophila by acute shutdown of dopaminergic neurotransmission was inconclusive, because the flies became hyperactive and failed to engage in social interactions. A large and complex literature suggests that DA is important for arousal in Drosophila, just as it is important for arousal in other species. In flies, dopaminergic modulation of arousal has been reported at 'endogenous' levels as in sleep/wake daily cycles, and at 'exogenous' stimulus-evoked levels as in higher-order complex behaviors. In some cases, the effects of altered dopaminergic function appear to be simple and linear; in other cases, the responses are distinctly nonlinear. In a recent study (Lebestky, 2009), elimination of one subtype of DA receptor in flies had opposite effects on sleep/wake cycles and on air puff-evoked startle responses. These traits were separately rescued by receptor replacement in different brain areas, leading the authors to propose that a segregation of brain pathways of arousal was likely involved. The current studies sought 'arousal' effects of dopaminergic neurons on the sleep/wake cycle, movement, courtship, and aggression, but now at a single-neuron level (Alekseyenko, 2013).

The results with chronic inactivation of isolated dopaminergic neurons showed a clear separation of their effects on tested behaviors. To illustrate, inactivation of the pair of DA neurons from the T1 cluster that project to the protocerebral bridge (flP243) yielded more aggressive flies that were not different from controls in their courtship behavior, sleep/wake activity, and locomotion. Inactivation of a pair of PPM3 neurons that innervate the fan-shaped body and noduli (flP447) also increased aggression but, in addition, had small effects on the courtship vigor index and the average waking activity of flies but did not change their sleep patterns. Another DA neuron from the PPM3 cluster innervated the ellipsoid but not the fan-shaped body, and has been reported to promote ethanol-induced locomotion. These data suggest that even within a single cluster, dopaminergic neurons can differ morphologically and functionally from each other. finally, inactivation of a small number of PPL1 neurons that innervate selective regions of the mushroom bodies yielded flies with no aggression phenotype but with increased sleep, decreased locomotion, and lowered negative geotaxis responses. An overlap between DopR-receptor immunostaining and the arborizations of the PPL1 neurons within the mushroom bodies suggests that the observed effects on sleep and activity might be mediated, at least in part, via this receptor subtype. However, another pair of PPL1 neurons project to the dorsal part of the fan-shaped body, and these have been suggested to promote wakefulness through DopR-receptor subtypes. Another receptor subtype, DopR2, is highly expressed in the mushroom bodies as well, and might also mediate the arousal phenotype of PPL1 neurons (Alekseyenko, 2013).

It is interesting that acute and chronic activation of T1 and PPM3 DA neurons via the dTrpA1 channel yield the same enhanced aggression phenotype as does chronic inactivation of these neurons with TNT. This suggests that a 'U-shaped' relationship governs the action of DA on the circuits in which the amine functions to influence aggression. In mammalian systems, a model has been suggested for the relationship between D1-receptor stimulation and working memory performance, in which both sub- and supraoptimal activation of DA receptors impairs working memory function. In Drosophila, a similar effect has been reported with octopaminergic neurons (octopamine is the invertebrate analog of the catecholamine norepinephrine) and courtship behavior. In that example, both lowering and enhancing the function of octopaminergic neurons resulted in increased male–male courtship (Alekseyenko, 2013).

The results from this study suggest that the modulation of aggression by identified DA neurons may be mediated via at least two subtypes of DA receptors, DopR and DD2R, located within different parts of the central complex of the brain. Drosophila DopR receptors reportedly correspond to the postsynaptic D1-receptor type in mammals and mediate responses to environmental stressors and ethanol-induced hyperactivity. These receptors are abundant on neuronal processes within the fan-shaped body, noduli, and ellipsoid body of the central complex, where they appear to be in close contact with TH-positive neurons. The current results also show close proximity between DopR immunostaining and the sites of presynaptic arbors of targeted PPM3 within the fan-shaped body and the noduli. The D2R (DD2R) receptors correspond to the D2 family in mammals that is found at both pre- and postsynaptic locations. These are expressed in only a few cell bodies in the Drosophila brain, but in many neurons in the ventral nerve cord (Draper, 2007). Neurons bearing these receptors have been implicated in the control of locomotion in previous studies (Draper, 2007). DD2R antibody staining overlaps with the presynaptic arborization of T1 neurons in the protocerebral bridge region of the central complex. Despite dense immunostaining of both presynaptic GFP and DD2R, the two types of neuronal endings appear to intermingle but not colocalize, suggesting that the DD2R receptors are postsynaptic to the dopaminergic nerve terminals of T1 neurons or are on presynaptic terminals of other neurons in the region. Further functional and morphological evidence will be required, however, to determine whether the processes of neurons expressing DopR and DD2R receptors within the central complex represent key synaptic linkages in the pathway of regulation of aggression (Alekseyenko, 2013).

Thus, modulation of higher-level aggression seems to include two morphologically distinguishable dopaminergic neurons whose endings are found within different neuroanatomical segments of the central complex. The proximity of dopaminergic endings originating from different types of neurons within one neuroanatomical region offers possible sites where DA neurons might interact to modulate the ability to escalate aggression. The details of how, where, or whether these particular DA neurons interact to exert their behavioral effects, however, remain to be established. The intersectional genetics approach in combination with the other binary systems available (Alekseyenko, 2013).

A dopamine receptor contributes to paraquat-induced neurotoxicity in Drosophila

Long-term exposure to environmental oxidative stressors, like the herbicide paraquat (PQ), has been linked to the development of Parkinson's disease (PD), the most frequent neurodegenerative movement disorder. Paraquat is thus frequently used in the fruit fly Drosophila melanogaster and other animal models to study PD and the degeneration of dopaminergic neurons (DNs) that characterizes this disease. This study shows that a D1-like dopamine (DA) receptor, DAMB, actively contributes to the fast central nervous system (CNS) failure induced by PQ in the fly. First, it was found that a long-term increase in neuronal DA synthesis reduced DAMB expression and protected against PQ neurotoxicity. Secondly, a striking age-related decrease in PQ resistance in young adult flies correlated with an augmentation of DAMB expression. This aging-associated increase in oxidative stress vulnerability was not observed in a DAMB-deficient mutant. Thirdly, targeted inactivation of this receptor in glutamatergic neurons (GNs) markedly enhanced the survival of Drosophila exposed to either PQ or neurotoxic levels of DA, whereas, conversely, DAMB overexpression in these cells made the flies more vulnerable to both compounds. Fourthly, a mutation in the Drosophila ryanodine receptor (RyR), which inhibits activity-induced increase in cytosolic Ca(2+), also strongly enhanced PQ resistance. Finally, it was found that DAMB overexpression in specific neuronal populations arrested development of the fly and that in vivo stimulation of either DNs or GNs increased PQ susceptibility. This suggests a model for DA receptor-mediated potentiation of PQ-induced neurotoxicity. Further studies of DAMB signaling in Drosophila could have implications for better understanding DA-related neurodegenerative disorders in humans (Cassar, 2015).

Operation of a homeostatic sleep switch

In Drosophila, a crucial component of the machinery for sleep homeostasis is a cluster of neurons innervating the dorsal fan-shaped body (dFB) of the central complex. dFB neurons in sleep-deprived flies tend to be electrically active, with high input resistances and long membrane time constants, while neurons in rested flies tend to be electrically silent. This study demonstrates state switching by dFB neurons, identifies dopamine as a neuromodulator that operates the switch, and delineates the switching mechanism. Arousing dopamine causes transient hyperpolarization of dFB neurons within tens of milliseconds and lasting excitability suppression within minutes. Both effects are transduced by Dop1R2 receptors and mediated by potassium conductances. The switch to electrical silence involves the downregulation of voltage-gated A-type currents carried by Shaker and Shab, and the upregulation of voltage-independent leak currents through a two-pore-domain potassium channel that was termed Sandman. Sandman is encoded by the CG8713 gene and translocates to the plasma membrane in response to dopamine. dFB-restricted interference with the expression of Shaker or Sandman decreases or increases sleep, respectively, by slowing the repetitive discharge of dFB neurons in the ON state or blocking their entry into the OFF state. Biophysical changes in a small population of neurons are thus linked to the control of sleep-wake state (Pimentel, 2016).

Recordings were made from dFB neurons (which were marked by R23E10-GAL4 or R23E10-lexA-driven green fluorescent protein (GFP) expression) while head-fixed flies walked or rested on a spherical treadmill. Because inactivity is a necessary correlate but insufficient proof of sleep, the analysis was restricted to awakening, which is defined as a locomotor bout after >5 min of rest, during which the recorded dFB neuron had been persistently spiking. To deliver wake-promoting signals, the optogenetic actuator CsChrimson was expressed under TH-GAL4 control in the majority of dopaminergic neurons, including the PPL1 and PPM3 clusters, whose fan-shaped body (FB)-projecting members have been implicated in sleep control. Illumination at 630 nm, sustained for 1.5 s to release a bolus of dopamine, effectively stimulated locomotion. dFB neurons paused in successful (but not in unsuccessful) trials, and their membrane potentials dipped by 2-13 mV below the baseline during tonic activity. When flies bearing an undriven CsChrimson transgene were photostimulated, neither physiological nor behavioural changes were apparent. The tight correlation between the suppression of dFB neuron spiking and the initiation of movement might, however, merely mirror a causal dopamine effect elsewhere, as TH-GAL4 labels dopaminergic neurons throughout the brain. Because localized dopamine applications to dFB neuron dendrites similarly caused awakening, this possibility is considered remote (Pimentel, 2016).

Flies with enhanced dopaminergic transmission exhibit a short-sleeping phenotype that requires the presence of a D1-like receptor in dFB neurons, suggesting that dopamine acts directly on these cells. dFB-restricted RNA interference (RNAi) confirmed this notion and pinpointed Dop1R2 as the responsible receptor, a conclusion reinforced by analysis of the mutant Dop1R2MI08664 allele. Previous evidence that Dop1R1, a receptor not involved in regulating baseline sleep, confers responsiveness to dopamine when expressed in the dFB indicates that either D1-like receptor can fulfill the role normally played by Dop1R2. Loss of Dop1R2 increased sleep during the day and the late hours of the night, by prolonging sleep bouts without affecting their frequency. This sleep pattern is consistent with reduced sensitivity to a dopaminergic arousal signal (Pimentel, 2016).

To confirm the identity of the effective transmitter, avoid dopamine release outside the dFB, and reduce the transgene load for subsequent experiments, optogenetic manipulations of the dopaminergic system were replaced with pressure ejections of dopamine onto dFB neuron dendrites. Like optogenetically stimulated secretion, focal application of dopamine hyperpolarized the cells and suppressed their spiking. The inhibitory responses could be blocked at several nodes of an intracellular signalling pathway that connects the activation of dopamine receptors to the opening of potassium conductances: by RNAi-mediated knockdown of Dop1R2; by the inclusion in the patch pipette of pertussis toxin (PTX), which inactivates heterotrimeric G proteins of the Gi/o family; and by replacing intracellular potassium with caesium, which obstructs the pores of G-protein-coupled inward-rectifier channels. Elevating the chloride reversal potential above resting potential left the polarity of the responses unchanged, corroborating that potassium conductances mediate the bulk of dopaminergic inhibition (Pimentel, 2016).

Coupling of Dop1R2 to Gi/o, although documented in a heterologous system, represents a sufficiently unusual transduction mechanism for a predicted D1-like receptor to prompt verification of its behavioural relevance. Like the loss of Dop1R2, temperature-inducible expression of PTX in dFB neurons increased overall sleep time by extending sleep bout length (Pimentel, 2016).

While a single pulse of dopamine transiently hyperpolarized dFB neurons and inhibited their spiking, prolonged dopamine applications (50 ms pulses at 10 Hz, or 20 Hz optogenetic stimulation, both sustained for 2-10 min) switched the cells from electrical excitability (ON) to quiescence (OFF). The switching process required dopamine as well as Dop1R2, but once the switch had been actuated the cells remained in the OFF state-and flies, awake-without a steady supply of transmitter. Input resistances and membrane time constants dropped to 53.3 ± 1.8 and 24.0 ± 1.3% of their initial values (means ± s.e.m.), and depolarizing currents no longer elicited action potentials (15 out of 15 cells). The biophysical properties of single dFB neurons, recorded in the same individual before and after operating the dopamine switch, varied as widely as those in sleep-deprived and rested flies (Pimentel, 2016).

Dopamine-induced changes in input resistance and membrane time constant occurred from similar baselines in all genotypes and followed single-exponential kinetics with time constants of 1.07-1.10 min. The speed of conversion points to post-translational modification and/or translocation of ion channels between intracellular pools and the plasma membrane as the underlying mechanism(s). In 7 out of 15 cases, recordings were held long enough to observe the spontaneous recommencement of spiking, which was accompanied by a rise to baseline of input resistance and membrane time constant, after 7-60 min of quiescence (mean ± s.e.m. = 25.86 ± 7.61 min). The temporary suspension of electrical output is thus part of the normal activity cycle of dFB neurons and not a dead end brought on by the experimental conditions (Pimentel, 2016).

dFB neurons in the ON state expressed two types of potassium current: voltage-dependent A-type (rapidly inactivating) and voltage-independent non-A-type currents. The current-voltage (I-V) relation of iA resembled that of Shaker, the prototypical A-type channel: no current flowed below -50 mV, the approximate voltage threshold of Shaker; above -40 mV, peak currents increased steeply with voltage and inactivated with a time constant of 7.5 ± 2.1 ms (mean ± s.e.m.). Non-A-type currents showed weak outward rectification with a reversal potential of -80 mV, consistent with potassium as the permeant ion, and no inactivation (Pimentel, 2016).

Switching the neurons OFF changed both types of potassium current. iA diminished by one-third, whereas inon-A nearly quadrupled when quantified between resting potential and spike threshold. The weak rectification of inon-A in the ON state vanished in the OFF state, giving way to the linear I-V relationship of an ideal leak conductance. dFB neurons thus upregulate iA in the sleep-promoting ON state. When dopamine switches the cells OFF, voltage-dependent currents are attenuated and leak currents augmented. This seesaw form of regulation should be sensitive to perturbations of the neurons' ion channel inventory: depletion of voltage-gated A-type (KV) channels (which predominate in the ON state) should tip the cells towards the OFF state; conversely, loss of leak channels (which predominate in the OFF state) should favour the ON state. To test these predictions, sleep was examined in flies carrying R23E10-GAL4-driven RNAi transgenes for dFB-restricted interference with individual potassium channel transcripts (Pimentel, 2016).

RNAi-mediated knockdown of two of the five KV channel types of Drosophila (Shaker and Shab) reduced sleep relative to parental controls, while knockdown of the remaining three types had no effect. Biasing the potassium channel repertoire of dFB neurons against A-type conductances thus tilts the neurons' excitable state towards quiescence, causing insomnia, but leaves transient and sustained dopamine responses unaffected. The seemingly counterintuitive conclusion that reducing a potassium current would decrease, not increase, action potential discharge is explained by a requirement for A-type channels in generating repetitive activity of the kind displayed by dFB neurons during sleep. Depleting Shaker from dFB neurons shifted the interspike interval distribution towards longer values, as would be expected if KV channels with slow inactivation kinetics replaced rapidly inactivating Shaker as the principal force opposing the generation of the next spike. These findings identify a potential mechanism for the short-sleeping phenotypes caused by mutations in Shaker, its β subunit Hyperkinetic, or its regulator sleepless (Pimentel, 2016).

Leak conductances are typically formed by two-pore-domain potassium (K2P) channels. dFB-restricted RNAi of one member of the 11-strong family of Drosophila K2P channels, encoded by the CG8713 gene, increased sleep relative to parental controls; interference with the remaining 10 K2P channels had no effect. Recordings from dFB neurons after knockdown of the CG8713 gene product, which this study termed Sandman, revealed undiminished non-A-type currents in the ON state and intact responses to a single pulse of dopamine but a defective OFF switch: during prolonged dopamine applications, inon-A failed to rise, input resistances and membrane time constants remained at their elevated levels, and the neurons continued to fire action potentials (7 out of 7 cells). Blocking vesicle exocytosis in the recorded cell with botulinum neurotoxin C (BoNT/C) similarly disabled the OFF switch. This, combined with the absence of detectable Sandman currents in the ON state, suggests that Sandman is internalized in electrically active cells and recycled to the plasma membrane when dopamine switches the neurons OFF (Pimentel, 2016).

Because dFB neurons lacking Sandman spike persistently even after prolonged dopamine exposure, voltage-gated sodium channels remain functional in the OFF state. The difficulty of driving control cells to action potential threshold in this state must therefore be due to a lengthening of electrotonic distance between sites of current injection and spike generation. This lengthening is an expected consequence of a current leak, which may uncouple the axonal spike generator from somatodendritic synaptic inputs or pacemaker currents when sleep need is low (Pimentel, 2016).

The two kinetically and mechanistically distinct actions of dopamine on dFB neurons-instant, but transient, hyperpolarization and a delayed, but lasting, switch in excitable state-ensure that transitions to vigilance can be both immediate and sustained, providing speedy alarm responses and stable homeostatic control. The key to stability lies in the switching behaviour of dFB neurons, which is driven by dopaminergic input accumulated over time. Unlike bistable neurons, in which two activity regimes coexist for the same set of conductances, dFB neurons switch regimes only when their membrane current densities change. This analysis of how dopamine effects such a change, from activity to silence, has uncovered elements familiar from other modulated systems: simultaneous, antagonistic regulation of multiple conductances; reduction of iA; and modulation of leak currents. Currently little is known about the reverse transition, from silence to activity, except that mutating the Rho-GTPase-activating protein Crossveinless-c locks dFB neurons in the OFF state, resulting in severe insomnia and an inability to correct sleep deficits. Discovering the signals and processes that switch sleep-promoting neurons back ON will hold important clues to the vital function of sleep (Pimentel, 2016).

Selective degeneration of dopaminergic neurons by MPP(+) and its rescue by D2 autoreceptors in Drosophila primary culture

Drosophila melanogaster is widely used to study genetic factors causing Parkinson's disease (PD) largely because of the use of sophisticated genetic approaches and the presence of a high conservation of gene sequence/function between Drosophila and mammals. However, in Drosophila, little has been done to study the environmental factors which cause over 90% of PD cases. This study used Drosophila primary neuronal culture to study degenerative effects of a well-known PD toxin MPP(+) . Dopaminergic (DA) neurons were selectively degenerated by MPP(+) , whereas cholinergic and GABAergic neurons were not affected. This DA neuronal loss was because of post-mitotic degeneration, not by inhibition of DA neuronal differentiation. This study also found that MPP(+) -mediated neurodegeneration was rescued by D2 agonists quinpirole and bromocriptine. This rescue was through activation of Drosophila D2 receptor DD2R, as D2 agonists failed to rescue MPP(+) -toxicity in neuronal cultures prepared from both a DD2R deficiency line and a transgenic line pan-neuronally expressing DD2R RNAi. Furthermore, DD2R autoreceptors in DA neurons played a critical role in the rescue. When DD2R RNAi was expressed only in DA neurons, MPP(+) toxicity was not rescued by D2 agonists. This study study also showed that rescue of DA neurodegeneration by Drosophila DD2R activation was mediated through suppression of action potentials in DA neurons (Wiemerslage, 2013).

Locomotor activity is regulated by D2-like receptors in Drosophila: an anatomic and functional analysis

In mammals, dopamine 2-like receptors are expressed in distinct pathways within the central nervous system, as well as in peripheral tissues. Selected neuronal D2-like receptors play a critical role in modulating locomotor activity and, as such, represent an important therapeutic target (e.g. in Parkinson's disease). Previous studies have established that proteins required for dopamine (DA) neurotransmission are highly conserved between mammals and the fruit fly Drosophila melanogaster. These include a fly dopamine 2-like receptor (DD2R; Hearn, 2002) that has structural and pharmacologic similarity to the human D2-like (D2R). The current study defined the spatial expression pattern of DD2R, and functionally characterize flies with reduced DD2 receptor levels. DD2R was shown to be expressed in the larval and adult nervous systems, in cell groups that include the Ap-let cohort of peptidergic neurons, as well as in peripheral tissues including the gut and Malpighian tubules. To examine DD2R function in vivo, RNA-interference (RNAi) flies were generated with reduced DD2R expression. Behavioral analysis revealed that these flies show significantly decreased locomotor activity, similar to the phenotype observed in mammals with reduced D2R expression. The fly RNAi phenotype can be rescued by administration of the DD2R synthetic agonist bromocriptine, indicating specificity for the RNAi effect. These results suggest Drosophila as a useful system for future studies aimed at identifying modifiers of dopaminergic signaling/locomotor function (Draper, 2007).

Since the cloning and pharmacologic characterization of the fly D2R (Hearn, 2002), multiple invertebrate D2-like receptors have been identified including the C. elegans DOP-2 and DOP-3 as well as the Apis mellifera AmDOP3. Phylogenic analysis has shown that these proteins cluster with the human D2-like receptors, thus indicating that this group of proteins has been well conserved through evolution (Draper, 2007).

In mammals, the spectrum of physiologic functions and behaviors mediated by dopamine 2-like receptors is in part reflected by the tissue and cell specific expression of these proteins. Distinct neuronal D2R circuits have been linked to the control of motor function, hormone release, and aggressive behavior. In addition, mammalian D2-like receptors in renal tubules and the gastrointestinal tract have been implicated in sodium transport, and intestinal motility, respectively (Draper, 2007).

In insects, as in mammals, proteins with different neuronal functions have been mapped to distinct CNS cell types, providing an anatomical framework on which to begin linking physiologic/behavioral functions to cell-specific expression patterns (e.g., locomotor activity to dopaminergic neurons, learning and memory to the mushroom bodies, and circadian rhythms to lateral and dorsal neuronal cell groups). This study has shown that DD2R is expressed in discrete cell populations of the larval and adult CNS (i.e., within and outside the LIM- homeobox apterous cell population), as well as in peripheral organs (i.e., in the intestinal tract and Malpighian tubules of larvae). This pattern potentially reflects the functions of the receptor in different tissues and/or developmental stages (Draper, 2007).

This study has demonstrated that a subpopulation of fly D2-like receptors colocalizes with the transcription factor apterous (ap), in the dorsal chain of interneurons and the thoracic (T) ventral clusters, but not within the FMRF-producing Tv neuroendocrine cells and SP2 interneurons. This profile corresponds to that described for the 'Ap-let' cohort. It is hypothesized that dopamine may fine-tune neurosecretory functions in these cells through the activation of the D2-like receptor, DD2R. In addition to DD2R, it has been postulated that the D1-like receptor DopR (also known as dDA1, or DmDop1), which is present in the 'Ap- let' cohort of interneurons expressing apterous, has a role in modulating the synthesis and/or release of neuropeptides (Draper, 2007).

The presence of D1 and D2 receptors on a single cell type has been observed both in mammals and in C. elegans. Previous work has shown that DD2R can signal in vitro through Gi/o, leading to a decrease of cAMP levels, as well as through Gq, resulting in increased IP3 formation (Hearn, 2002). Others have demonstrated that stimulation of fly D1-like receptors triggers a Gs mediated increase in cAMP, as well as alters intracellular Ca2 levels. Coexpression of dopamine receptors in a single cell type provides the potential for diversity in downstream signal transduction pathways. This, in turn, may offer a means to fine tune the physiology or behavior that is linked to a specific G- protein coupled receptor (Draper, 2007).

It is of interest that in addition to CNS expression, DD2R-IR was observed in the larval midintestine. In mammals, D2 receptors have recently been identified in the gastrointestinal tract, where they have been shown to modulate motility. Consistent with these findings, D2R subtype-selective small molecule ligands (i.e., domperidone, metoclopramide) are found among the arsenal of gastrokinetic and antiemetic compounds. In addition to expression in the intestine, this study also detected DD2R-IR on Malpighian (renal) tubules. The involvement of biogenic amine receptors in the control of fly renal function has previously been suggested. Studies have demonstrated that tyramine, octopamine, and dopamine can modulate chloride conductance in isolated Malpighian tubules. In mammals, it is well known that D2-like receptors are expressed in the kidney where they modulate dopamine induced sodium excretion. Taken together, these observations suggest that the fly may be utilized to investigate mechanisms underlying a range of D2 receptor-mediated physiological processes (Draper, 2007).

Targeted gene disruption in mammals has provided an effective means to define the in vivo function of dopamine receptors. D2 receptor knockout mice display neurologic impairments including postural abnormalities, slow movement, decreased locomotor activity, absent rearing, and catalepsy. Heterozygous animals with reduced receptor levels display intermediate motor abnormalities. With the identification of DD2R, it became possible to explore whether the functions mediated by D2-like receptors are conserved in fruit flies. Analyses of DD2R RNAi flies show significant deficits in locomotor activity, and demonstrate that neuronal expression of this receptor is important for the modulation of motor function. Thus, the D2 receptor has a role in controlling activity in both flies and mammals. Depending on the Gal4 driver utilized to express UAS-ds-DD2R (Actin5C vs. Elav), the motor deficit of the RNAi flies is significant in either young or old adults (vs. controls). These two drivers produce high and low levels of Gal4 expression, respectively, as reflected by the level of GFP fluorescence used as a marker for Gal4 expression. It is hypothesized that the natural age-dependent decline in dopamine exacerbates the locomotor phenotype of the DD2R interference flies, accounting for the age-dependent effects of Elav-Gal4-driven expression. In mammals decreases in dopamine and dopamine receptors during aging have been documented. Of interest, D2R knockdown mice become hypoactive only after 41 weeks of age, while the homozygous mutants display significant motor deficits at 10 weeks of age (Draper, 2007).

In Drosophila, a deficit for the dopamine transporter dDAT (in the fumin mutant), leads to hyperactivity and decreased rest presumably through abnormal accumulation of synaptic dopamine. Similar results have been obtained with exposure of wild-type flies to the dDAT blockers cocaine or methamphetamine. Conversely, a narcolepsy-like state has been reported after treatment of flies with inhibitors of dopamine biosynthesis. In addition, dopaminergic drugs delivered to the nerve cord (VNS) of decapitated Drosophila can elicit motor responses, consistent with the presence of dopamine receptors in VNS cells. Indeed, flies are amenable to pharmacologic manipulations which may complement results obtained from genetic alterations. On the basis of these precedents, this study administered bromocriptine (an anti-parkinsonian agent that activates both the human and fly dopamine 2-like receptors with high potency) to the DD2R RNAi flies. Treated flies exhibited normal levels of locomotor activity, illustrating that the D2 receptor deficit can be pharmacologically rescued (Draper, 2007).

The current results, together with published studies in mammals, demonstrate conservation of D2 receptor- mediated control of motor behavior. Drosophila emerges as a useful system for future studies aimed at identifying modifiers of dopaminergic signaling (Draper, 2007).

A Drosophila dopamine 2-like receptor: Molecular characterization and identification of multiple alternatively spliced variants

Dopamine is an important neurotransmitter in the central nervous system of both Drosophila and mammals. Despite the evolutionary distance, functional parallels exist between the fly and mammalian dopaminergic systems, with both playing roles in modulating locomotor activity, sexual function, and the response to drugs of abuse. In mammals, dopamine exerts its effects through either dopamine 1-like (D1-like) or D2-like G protein-coupled receptors. Although pharmacologic data suggest the presence of both receptor subtypes in insects, only cDNAs encoding D1-like proteins have been isolated previously. This study reports the cloning and characterization of a newly discovered Drosophila dopamine receptor. Sequence analysis reveals that this putative protein shares highest homology with known mammalian dopamine 2-like receptors. Eight isoforms of the Drosophila D2-like receptor (DD2R) transcript have been identified, each the result of alternative splicing. The encoded heptahelical receptors range in size from 461 to 606 aa, with variability in the length and sequence of the third intracellular loop. Pharmacologic assessment of three DD2R isoforms, DD2R-606, DD2R-506, and DD2R-461, revealed that among the endogenous biogenic amines, dopamine is most potent at each receptor. As established for mammalian D2-like receptors, stimulation of the Drosophila homologs with dopamine triggers pertussis toxin-sensitive Gi/o-mediated signaling. The D2-like receptor agonist, bromocriptine, has nanomolar potency at DD2R-606, -506, and -461, whereas multiple D2-like receptor antagonists (as established with mammalian receptors) have markedly reduced if any affinity when assessed at the fly receptor isoforms. The isolation of cDNAs encoding Drosophila D2-like receptors extends the range of apparent parallels between the dopaminergic system in flies and mammals. Pharmacologic and genetic manipulation of the DD2Rs will provide the opportunity to better define the physiologic role of these proteins in vivo and further explore the utility of invertebrates as a model system for understanding dopaminergic function in higher organisms (Hearn, 2002).

DAMB, a novel dopamine receptor expressed specifically in Drosophila mushroom bodies

The modulatory neurotransmitters that trigger biochemical cascades underlying olfactory learning in Drosophila mushroom bodies have remained unknown. To identify molecules that may perform this role, putative biogenic amine receptors were cloned using the polymerase chain reaction (PCR) and single-strand conformation polymorphism analysis. One new receptor, DAMB, was identified as a dopamine D1 receptor by sequence analysis and pharmacological characterization. In situ hybridization and immunohistochemical analyses revealed highly enriched expression of DAMB in mushroom bodies, in a pattern coincident with the rutabaga-encoded adenylyl cyclase. The spatial coexpression of DAMB and the cyclase, along with DAMB's capacity to mediate dopamine-induced increases in cAMP make this receptor an attractive candidate for initiating biochemical cascades underlying learning (Han, 1996).


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Biological Overview

date revised: 4 December 2015

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