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

Dop1R2 orthologs: Biolitmine

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
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
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
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.
Zhou, M., Chen, N., Tian, J., Zeng, J., Zhang, Y., Zhang, X., Guo, J., Sun, J., Li, Y., Guo, A. and Li, Y. (2019). Suppression of GABAergic neurons through D2-like receptor secures efficient conditioning in Drosophila aversive olfactory learning. Proc Natl Acad Sci U S A 116(11): 5118-5125. PubMed ID: 30796183
In Drosophila, the GABAergic anterior paired lateral (APL) neurons mediate a negative feedback essential for odor discrimination; however, their activity is suppressed by learning via unknown mechanisms. In aversive olfactory learning, a group of dopaminergic (DA) neurons is activated on electric shock (ES) and modulates the Kenyon cells (KCs) in the mushroom body, the center of olfactory learning. This work finds that the same group of DA neurons also form functional synaptic connections with the APL neurons, thereby emitting a suppressive signal to the latter through Drosophila dopamine 2-like receptor (DD2R). Knockdown of either DD2R or its downstream molecules in the APL neurons results in impaired olfactory learning at the behavioral level. Results obtained from in vivo functional imaging experiments indicate that this DD2R-dependent DA-to-APL suppression occurs during odor-ES conditioning and discharges the GABAergic inhibition on the KCs specific to the conditioned odor. Moreover, the decrease in odor response of the APL neurons persists to the postconditioning phase, and this change is also absent in DD2R knockdown flies. Taken together, these findings show that DA-to-GABA suppression is essential for restraining the GABAergic inhibition during conditioning, as well as for inducing synaptic modification in this learning circuit.
Akiba, M., Sugimoto, K., Aoki, R., Murakami, R., Miyashita, T., Hashimoto, R., Hiranuma, A., Yamauchi, J., Ueno, T. and Morimoto, T. (2019). Dopamine modulates the optomotor response to unreliable visual stimuli in Drosophila melanogaster. Eur J Neurosci. PubMed ID: 31834948
The precise neural mechanism underlying dopaminergic modulation of behaviour induced by sensory stimuli remains poorly understood. This study used Drosophila melanogaster to show that dopamine can modulate the optomotor response to moving visual stimuli including noise. The optomotor response is the head-turning response to moving objects, which is observed in most sight-reliant animals including mammals and insects. First, the effects of the dopamine system on the optomotor response were investigated in mutant flies deficient in dopamine receptors D1R1 (DopR) or D1R2, which are involved in the modulation of sleep-arousal in flies. This study examined the optomotor response in D1R1 knockout (D1R1 KO) and D1R2 knockout (D1R2 KO) flies and found that it was not affected in D1R1 KO flies; however, it was significantly reduced in D1R2 KO flies compared with the wild type. Using cell-type-specific expression of an RNA interference construct of D1R2, the fan-shaped body, a part of the central complex, was identified as responsible for dopamine-mediated modulation of the optomotor response. In particular, pontine cells in the fan-shaped body seemed important in the modulation of the optomotor response, and their neural activity was required for the optomotor response. These results suggest a novel role of the central complex in the modulation of a behaviour based on the processing of sensory stimulations.
El Kholy, S., Wang, K., El-Seedi, H. R. and Al Naggar, Y. (2021). Dopamine Modulates Drosophila Gut Physiology, Providing New Insights for Future Gastrointestinal Pharmacotherapy. Biology (Basel) 10(10). PubMed ID: 34681083.
Dopamine has a variety of physiological roles in the gastrointestinal tract (GI) through binding to Drosophila dopamine D1-like receptors (DARs) and/or adrenergic receptors and has been confirmed as one of the enteric neurotransmitters. To gain new insights into what could be a potential future promise for GI pharmacology, Drosophila was used as a model organism to investigate the effects of dopamine on intestinal physiology and gut motility. GAL4/UAS system was utilized to knock down specific dopamine receptors using specialized GAL4 driver lines targeting neurons or enterocytes cells to identify which dopamine receptor controls stomach contractions. DARs (Dop1R1 and Dop1R2) were shown by immunohistochemistry to be strongly expressed in all smooth muscles in both larval and adult flies, which could explain the inhibitory effect of dopamine on GI motility. Adult males' gut peristalsis was significantly inhibited by knocking down dopamine receptors Dop1R1, Dop1R2, and Dop2R, but female flies' gut peristalsis was significantly repressed by knocking down only Dop1R1 and Dop1R2. These findings also showed that dopamine drives PLC-β translocation from the cytoplasm to the plasma membrane in enterocytes for the first time. Overall, these data revealed the role of dopamine in modulating Drosophila gut physiology, offering us new insights for the future gastrointestinal pharmacotherapy of neurodegenerative diseases associated with dopamine deficiency.
Miller, H. A., Huang, S., Dean, E. S., Schaller, M. L., Tuckowski, A. M., Munneke, A. S., Beydoun, S., Pletcher, S. D. and Leiser, S. F. (2022). Serotonin and dopamine modulate aging in response to food odor and availability. Nat Commun 13(1): 3271. PubMed ID: 35672307
An organism's ability to perceive and respond to changes in its environment is crucial for its health and survival. This study reveals how the most well-studied longevity intervention, dietary restriction, acts in-part through a cell non-autonomous signaling pathway that is inhibited by the presence of attractive smells. Using an intestinal reporter for a key gene induced by dietary restriction but suppressed by attractive smells, this study identified three compounds that block food odor effects in C. elegans, thereby increasing longevity as dietary restriction mimetics. These compounds clearly implicate serotonin and dopamine in limiting lifespan in response to food odor. A chemosensory neuron that likely perceives food odor, an enteric neuron that signals through the serotonin receptor 5-HT1A/SER-4, and a dopaminergic neuron that signals through the dopamine receptor DRD2/DOP-3. Aspects of this pathway are conserved in D. melanogaster. Thus, blocking food odor signaling through antagonism of serotonin or dopamine receptors is a plausible approach to mimic the benefits of dietary restriction.
Love, C. R., Gautam, S., Lama, C., Le, N. H. and Dauwalder, B. (2023). The Drosophila dopamine 2-like receptor D2R (Dop2R) is required in the blood brain barrier for male courtship. Genes Brain Behav 22(1): e12836. PubMed ID: 36636829
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. This study shows 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. These findings thus identify a specific glial function for the DR2 receptor and dopamine signaling in the regulation of a complex behavior.


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).

Distinct dopamine receptor pathways underlie the temporal sensitivity of associative learning

Animals rely on the relative timing of events in their environment to form and update predictive associations, but the molecular and circuit mechanisms for this temporal sensitivity remain incompletely understood. This study shows that olfactory associations in Drosophila can be written and reversed on a trial-by-trial basis depending on the temporal relationship between an odor cue and dopaminergic reinforcement. Through the synchronous recording of neural activity and behavior, this study shows that reversals in learned odor attraction correlate with bidirectional neural plasticity in the mushroom body, the associative olfactory center of the fly. Two dopamine receptors, DopR1 and DopR2, contribute to this temporal sensitivity by coupling to distinct second messengers and directing either synaptic depression or potentiation. These results reveal how dopamine-receptor signaling pathways can detect the order of events to instruct opposing forms of synaptic and behavioral plasticity, allowing animals to flexibly update their associations in a dynamic environment (Handler, 2019).

While memories are often thought of as windows into the past, their adaptive value lies in the ability to predict the future. This study, took advantage of the concise circuitry of the Drosophila mushroom body to investigate how the precise timing of dopaminergic reinforcement allows animals to form and maintain predictive associations between cues and outcomes. While studies of associative learning have often focused on sensory cues that anticipate punishment or reward, equally informative are cues associated with their termination. This study demonstrates that shifting the relative timing of an odor and reinforcement by <1 s can switch the valence of an olfactory memory, underscoring the exquisite temporal sensitivity of this circuit. As a consequence, flies can form equivalent appetitive associations with odors that anticipate rewards or follow punishments, or aversive associations with odors that predict punishments or follow rewards. The symmetry of this behavioral modulation permits Drosophila to take advantage of all the temporally correlated features of their environment that can be used to infer causal relationships. Together, this work suggests a model in which the steep temporal sensitivity of associative learning arises from the concerted action of two dopamine receptor-signaling pathways that work in opposition to bidirectionally regulate the strength of KC-MBON signaling (see DopR1 and DopR2 Are Required for Behavioral Flexibility), allowing animals to maintain an accurate model of a complex and changing world (Handler, 2019).

In a dynamic environment, memories must be continually retouched and rewritten to maintain their relevance and predictive value. By monitoring how individual flies adapt their odor preferences over 50 conditioning trials, this study has revealed that Drosophila can form and reverse learned associations on a trial-by-trial basis, pointing to the fundamental flexibility of memory updating mechanisms (Handler, 2019).

Prior work in both Drosophila and mammals has suggested memory retention is regulated by multiple mechanisms at different timescales. If not reinforced, memories may passively fade over time, reflecting the natural turnover of molecular and neural hardware. Alternatively, memories can be actively eroded either by re-exposure to the learned odor in the absence of the anticipated dopaminergic reinforcement or the reinforcement in the absence of the odor, violating the expected contingency between these two events. In contrast, the brief episodes of odor and dopaminergic reinforcement (1-2 s) used in the current study are insufficient to overwrite an olfactory association when presented independently but can immediately reverse a prior association when paired together in time. The convergence of olfactory and DAN input to the mushroom body thus conveys information about their causal relationship, offering a mechanism to rapidly update memories to reflect the changing temporal structure of the environment (Handler, 2019).

While memory updating could rely on plasticity at various sites within this circuit, this study demonstrated that the bidirectional modulation of behavior is highly correlated with bidirectional changes in the strength of the same KC-MBON synapses within the mushroom body. Such bidirectional synaptic plasticity has been proposed to confer reversibility to learning circuits. For example, spike-timing dependent plasticity (STDP) can bidirectionally tune the strength of synaptic connections between neurons depending on the relative timing of spikes in pre- and post-synaptic neurons, mirroring the sensitivity to temporal order observed in associative learning. However, STDP requires nearly coincident firing patterns on a millisecond timescale, far more rapid than the temporal relationships between stimuli typically required for associative learning. In this study, by examining neural and behavioral modulation over the same timescales and even concurrently within the same individuals, the modulation of synaptic signaling within the mushroom body is linked to reversible changes in behavior (Handler, 2019).

Within the mushroom body, each compartment serves as a site of convergence between odor signaling conveyed by KCs and dopaminergic reinforcement, allowing dopamine-receptor pathways within KC axons to detect the temporal order of these inputs. The spatial patterns of dopamine release and dopamine receptor second-messenger cascades are found to adhere to the compartmentalized architecture of the lobes, permitting different synapses along the same KC axon to be independently regulated. These observations suggest that within a compartment, multiple neuromodulatory mechanisms tune neurotransmission depending on the temporal structure of conditioning. As a consequence, the distinct complement of KC-MBON synapses activated by odors that precede or follow a reinforcement are differentially regulated, allowing a single dopaminergic reinforcement to drive the synchronous formation of multiple odor associations, effectively enhancing the coding capacity of a compartment (Handler, 2019).

Dopamine shapes circuit function in diverse ways by engaging distinct classes of receptors that couple to different signaling cascades. In Drosophila, DopR1 and DopR2 have been proposed to play opposing roles in olfactory memory regulation at the behavioral level, with DopR1 essential to memory formation and DopR2 necessary for memory erosion. Yet the contribution of these receptors to synaptic modulation within the mushroom body has remained unclear. The current work reveals that the opposing behavioral roles of DopR1 and DopR2 are mirrored by their antagonistic regulation of KC-MBON signaling, with DopR1 required for the depression ensuing from forward pairing, while DopR2 is essential for the potentiation that follows backward pairing. Although DANs selectively innervating the mushroom body are sufficient to instruct bidirectional behavioral modulation, the broader expression of DopR1 and DopR2 leaves open the possibility that these receptors may also act at other sites within the nervous system to shape the temporal sensitivity of associative learning (Handler, 2019).

Using fluorescent sensors of DopR1 and DopR2 second messengers allowed the gaining insight into the spatial and temporal patterning of these intracellular signaling pathways during conditioning. While a potential limitation of optical reporters is their restricted sensitivity and dynamic range, these sensors nevertheless reveal that the selective recruitment of dopamine-receptor signaling cascades is sufficient to account for the temporal dependence of neural and behavioral modulation. Monitoring cAMP production during conditioning reveals that, while the DopR1 pathway serves as a coincidence detector, in accord with the coordinate regulation of adenylate cyclases by Gαs and calcium, it cannot autonomously encode the temporal order of events. In contrast, DopR2 signaling through Gαq strictly depends on the temporal sequence of KC and DAN activation (Handler, 2019).

Which component of the DopR2-signaling cascade is sensitive to the temporal ordering of odor and reinforcement? IP3 receptors that gate calcium release from the ER lumen represent an intriguing candidate, as their complex regulation by both IP3 and cytosolic calcium renders them inherently sensitive to the sequence of agonist binding: IP3 binding unmasks a calcium regulatory site required for channel opening, while high calcium in the absence of IP3 inhibits channel activity. Indeed, this study observed that ER calcium release is time locked to KC stimulation suggesting that the precise order dependence of this pathway relies on calcium entry subsequent to IP3 production. In the cerebellum, bidirectional plasticity at parallel fiber-Purkinje neuron synapses has been proposed to similarly rely on calcium release from the ER lumen via IP3 receptors. While the analogous circuit organization of the mushroom body and cerebellum has been well described, the current observations suggest they may share conserved molecular mechanisms for temporally precise synaptic modulation (Handler, 2019).

Together, this work points to dopamine receptor signaling pathways in KC axons as a key site of temporal coincidence and order detection during associative learning. While this study focused on the role of dopaminergic signaling within the γ4 compartment, timing-dependent bidirectional plasticity was observed to be shared characteristic of KC-MBON synapses in multiple compartments of the γ lobe. Therefore, the reversible modulation of behavior instructed by both the PAM or PPL DANs likely reflects bidirectional plasticity driven synchronously in the multiple compartments innervated by these DAN drivers. Aversive electric shock and sugar rewards evoke distributed patterns of activity across the DAN population, implying that these naturalistic reinforcers likewise instruct coordinated bidirectional plasticity across different compartments to rapidly shape the net output of the mushroom body. Similar patterns of DAN activity are also elicited by a fly's locomotion, raising the possibility that, in the context of an odor plume, an animal's behavior may serve as a reinforcement stimulus that itself drives bidirectional synaptic plasticity to regulate odor processing (Handler, 2019).

The ability to form or overwrite associations on a trial-by-trial basis allows for adaptive behavior in a noisy and uncertain environment where the temporal relationships between events may quickly change. However, animals must also have the capacity to store relevant memories persistently, even for a lifetime. Therefore, the reversible plasticity observed must co-exist with additional molecular and circuit mechanisms that underlie the formation and retention of longer-term associations. Indeed, recent work has described intrinsic differences between mushroom body compartments in their susceptibility to memory erosion as well as differences in second-messenger signaling in distinct KC subpopulations. Together, these results suggest that the differential expression or coupling of dopamine-receptor signaling pathways in different KC classes may tune synaptic plasticity rules to regulate the persistence of information storage. While this work connects molecular pathways within a sub-population of KCs to the emergence of short-term associations, functional dissection of these signaling cascades across the different lobes of the mushroom body will provide insight into the distinct timescales of memory formation and erosion (Handler, 2019).

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).

A neural circuit arbitrates between persistence and withdrawal in hungry Drosophila

In pursuit of food, hungry animals mobilize significant energy resources and overcome exhaustion and fear. How need and motivation control the decision to continue or change behavior is not understood. Using a single fly treadmill, this study shows that hungry flies persistently track a food odor and increase their effort over repeated trials in the absence of reward suggesting that need dominates negative experience. It was further shown that odor tracking is regulated by two mushroom body output neurons (MBONs) connecting the MB to the lateral horn. These MBONs, together with dopaminergic neurons and Dop1R2 signaling, control behavioral persistence. Conversely, an octopaminergic neuron, VPM4, which directly innervates one of the MBONs, acts as a brake on odor tracking by connecting feeding and olfaction. Together, these data suggest a function for the MB in internal state-dependent expression of behavior that can be suppressed by external inputs conveying a competing behavioral drive (Sayin, 2019).

Flexibility is an important factor in an ever in-flux environment, where scarcity and competition are the norm. Without persistence to achieve its goals, however, an animal's strive to secure food, protect its offspring, or maintain its social status is in jeopardy. Therefore, sensory cues related to food or danger often elicit strong impulses. However, these impulses must be strictly controlled to allow for coherent goal-directed behavior and to permit behavioral transitions when sensible. Inhibition of antagonistic behavioral drives at the cognitive and physiological level has been proposed as a major task of a nervous system. Which sensory cues and ultimately which behaviors are prioritized and win depends on the animal's metabolic state, internal motivation, and current behavioral context. How this is implemented at the level of individual neurons, circuit motifs, and mechanisms remains an important open question (Sayin, 2019).

Like most animals, energy-deprived flies prioritize food seeking and feeding behavior. To find food, flies can follow olfactory or visual cues over long distances. External gustatory cues provide information about the type and quality of the eventually encountered food. However, only internal nutrient levels will provide reliable feedback about the quality and quantity of a food source and ultimately suppress food-seeking behaviors. Therefore, food odor, the taste of food, and post-ingestive internal feedback signals induce sequential and partly antagonistic behaviors. Interestingly, chemosensory and internal feedback systems typically mediated by distinct neuromodulators appear to converge in the mushroom body (MB). How neurons and neural circuits signal and combine external and internal cues to maintain or suppress competing behavioral drives is not well understood (Sayin, 2019).

In mammals, norepinephrine (NE) released by a brain stem nucleus, the locus coeruleus, has been implicated in controlling the balance between persistence and action selection. The potential functional counterpart of NE in insects could be octopamine (OA). Flies lacking OA indeed show reduced arousal, for instance upon starvation. Additionally, OA neurons (OANs) gate appetitive memory formation of odors and also modulate taste neurons and feeding behavior. OANs are organized in distinct clusters and project axons to diverse higher brain regions in a cell type-specific manner. The precise roles and important types of OA and NE neurons in state-dependent action selection remain to be elucidated (Sayin, 2019).

Similar to NE and OA, dopamine (DA) is being studied in many aspects of behavioral adaptation and flexibility. Different classes of DA neurons (DANs) innervating primarily the MB signal negative or positive context, or even wrong predictions (Sayin, 2019 and references therein).

This study took advantage of the small number and discrete organization of neuromodulatory neurons in the fly brain to analyze the mechanistic relationship between motivation-dependent persistence in one behavior and the decision to disengage and change to another behavior. Using a single fly spherical treadmill assay, this study found that hungry flies increase their effort to track a food odor with every unrewarded trial. MB output through two identified MBONs (MBON-γ1pedc>α/β and MBON-α2sc) is required for persistent odor tracking. MBON-α2sc provides a MB connection to the lateral horn (LH), where it can modify innate food odor attraction. Furthermore, this study pinpoints a specific type of OAN, VPM4 (ventral paired medial), which connects feeding centers directly to MBON-γ1pedc>α/β and disrupts food odor tracking. Finally, the experimental data suggest that persistent tracking depends on DANs, including PPL1-γ1pedc, and signaling through dopamine receptor Dop1R2 in αβ-type KCs. Based on these results, it is proposed that MB output and a direct external input, depending on internal state and motivation, gradually promote or interrupt ongoing behavior (Sayin, 2019).

What drives gradually increasing persistence in behavior? For the fly, a model is proposed by which a circuit module of KCs, MBONs, and DANs drive gradually increasing odor tracking, which can be efficiently suppressed by extrinsic MBON-innervating feeding-related OANs. Behavioral persistence has been previously analyzed in flies in a different context. For instance, courtship of fly males and copulation with a female are maintained by dopaminergic neurons in the ventral nerve cord, where they counteract GABAergic neurons. In that scenario, DANs in the ventral nerve cord maintain an ongoing behavior and prevent male premature disengagement before successful insemination (Sayin, 2019).

The experimental data also implicate DANs, primarily from within the PPL1 (e.g., PPL1-γ1pedc) and PPL2ab clusters, and Dop1R2 signaling. In particular, inactivation of synaptic output of DANs positive for TH-Gal4 as well as loss of Dop1R2 in αβ-type KCs reduced the increase in odor tracking from trial to trial, while not affecting the speed at first odor stimulation. These data suggest that TH+ DANs promote goal-directed movement, i.e., odor tracking, through a Dop1R2-dependent mechanism in KCs (Sayin, 2019).

MBON-γ1pedc>αβ, which receives dopaminergic input by PPL1-γ1pedc, is required for odor tracking. Moreover, this study also observed a trial-to-trial decrease in odor response of this MBON, matching the dopamine-induced synaptic depression previously observed in MBONs upon learning. Notably, PPL1-γ1pedc activates Dop1R2 in MBON-γ1pedc>αβ, a signal recently found to be critical for appetitive long-term memory. Nevertheless, it appears that, in addition to PPL1-γ1pedc, other DANs regulate behavioral persistence by modulating in particular αβ-KCs. It is intriguing to speculate about a common function of Dop1R2 in the formation of long-lasting aversive memory induced by repeatedly pairing odor with an aversive experience and the behavior examined in this study: increased and persistent expression of a behavior induced by the experience of repeated failure to reach a goal (Sayin, 2019).

The experimental data further implicated MBON-α2sc, which is connected to MBON-γ1pedc>αβ. Calcium imaging data are consistent with an inhibitory interaction between the two MBONs. However, some of the behavioral data and prior imaging data do not support an inhibitory connection. Furthermore, MBON-γ1pedc>αβ projects to other brain regions and downstream targets, and similarly MBON-α2sc receives additional inputs—all of which could be equally or more important for persistent behavior than a direct connection between these two MBONs. Finally, some DANs respond to movement, including PPL1-γ2α'1/MV1. Although no essential role of this particular neuron was found in odor tracking persistence, movement might contribute to the activity of MBONs responding the odorant (Sayin, 2019).

Remarkably, MBON-α2sc connects the MB to neurons within the LH. Thus, it is speculated that the LH might assign an odor to its corresponding behavioral category, such as 'food-related' for vinegar, while the MB acts as a top-down control to gauge the expression of an innate behavior (i.e., tracking an appetitive odor) according to state and experience (Sayin, 2019).

The behavioral data led to the proposal of a circuit model. Using computational modeling, this study tested whether the MB network including DANs and MBONs could, in theory, produce the observed behavior. Indeed, it was found that a simplified recurrent circuit of KCs, DANs, and MBONs can account for the observed behavioral persistence and also the measured MBON-γ1pedc>αβ odor responses. While this model cannot replace experimental evidence, it forms a useful theoretical framework for future studies on the role of the MB in behavioral persistence (Sayin, 2019).

Based on the present data and computational predictions, a model is proposed by which the recurrent circuit architecture of the MB, in addition to storing information for future behavior, is ideally suited to maintain and gradually change ongoing behavior, for instance by modulating output of the LH, according to the animal's internal state and needs (Sayin, 2019).

The use of an olfactory treadmill has allowed dissection of the different aspects of a food search. In particular, how does food and feeding suppress food search if the sensory cue, the odor, is still present? OA-VPM4 connects feeding centers (i.e., SEZ) directly with odor tracking-promoting MBON-γ1pedc>αβ and inhibits its activity suggesting an inhibitory connection between VPM4 and the MBON. Nevertheless, it cannot be excluded that OA-VPM4 signals through multiple mechanisms including OA and possibly other neurotransmitters. In addition, a recent study showed that activation of VPM4 promotes proboscis extension to sugar. Although a direct role in taste detection through pharynx or labellum appears unlikely, it is possible that feeding behavior itself (e.g., lymphatic sugar, food texture, activity of feeding muscles) are detected and/or promoted by these neurons and then brought to the MB. It is proposef that VPM4 is a direct mediator between olfactory-guided food search and the rewarding experience of feeding and related behavior (Sayin, 2019).

The data provide a neural circuit mechanism empowering flies to express and prioritize behavior in a need- and state-dependent manner. It is exciting to speculate that fundamentally similar circuit motifs might exist in NE and DA neuron-containing circuits in the mammalian brain, governing the organization of behavior in a flexible and context-dependent manner by integrating internal and external context. For instance, noradrenergic neurons of the brainstem nucleus of the solitary tract (NST) receive taste information, and input from the gastrointestinal tracts, lungs, and heart. Neurons in the NST project to multiple brain regions including the amygdala, hypothalamus, and insular cortex, all of which receive internal state as well as other sensory information (Sayin, 2019).

The data in the fly provide an experimental and theoretical framework for a better understanding of the fundamental circuit mechanisms underpinning neuromodulation of context-dependent behavioral persistence and withdrawal (Sayin, 2019).

Dopamine receptor Dop1R2 stabilizes appetitive olfactory memory through the Raf/MAPK pathway in Drosophila

In Drosophila, dopamine signaling to the mushroom body intrinsic neurons, Kenyon cells (KCs), is critical to stabilize olfactory memory. Little is known about the downstream intracellular molecular signaling underlying memory stabilization. This study addresses this question in the context of sugar-rewarded olfactory long-term memory (LTM). Associative conditioning increases the phosphorylation of MAPK in KCs, via Dop1R2 signaling. Consistently, the attenuation of Dop1R2, Raf or MAPK expression in KCs selectively impairs LTM but not short-term memory. Moreover, this study shows that the LTM deficit caused by the knockdown of Dop1R2 can be rescued by expressing active Raf in KCs. Thus, the Dop1R2/Raf/MAPK pathway is a pivotal downstream effector of dopamine signaling for stabilizing appetitive olfactory memory (Sun, 2020)

This study supports the idea that Dop1R2 signaling through the Raf/MAPK pathway in KCs is critical in stabilizing appetitive memory. This could be achieved through acquisition or consolidation of appetitive LTM. How is post-training Dop1R2 signaling triggered in this context? Accumulating evidence implies that Dop1R2 detects the basal dopamine release after learning. In aversive olfactory learning, the post-training enhancement of the oscillatory activity of MB-projecting DANs (MB-MP1 and MB-MV1) underlies LTM consolidation, and Dop1R2 in KCs is responsible for detecting the enhanced dopamine signals. This signaling is also reported to mediate forgetting early labile memory, suggesting distinct neural mechanisms to regulate memories with different temporal dynamics. In appetitive learning, Dop1R2 is suggested to be the mediator of the oscillating DANs, which represent the energy value of the reward and consolidate LTM. Collectively, after conditioning Dop1R2 signaling upon specific reinforcement input is a conserved mechanism to stabilize LTM. As MB-projecting DANs are also engaged in conveying reward information during memory acquisition, the Dop1R2/Raf/MAPK pathway might additionally be involved during the acquisition of LTM (Sun, 2020)

In contrast to the well characterized receptor tyrosine kinase signaling, it is rather unexpected to find the Raf/MAPK pathway as a downstream target of Dop1R2, a G-protein-coupled receptor. Dop1R2 was recently shown to have a preferential affinity to the Gαq subunit to elicit a robust intracellular Ca2+ increase upon ligand stimulation in KCs. There are multiple lines of biochemical evidence suggesting that Gαq-dependent Ca2+ signals could trigger several pathways, such as small GTPase Rap1, protein kinase C, or Ras, to activate Raf. Furthermore, some reports suggested that calcium influx through N-methyl-d-aspartate receptor induces transient MAPK phosphorylation. Hence, intracellular Ca2+ might be the key second-messenger system to link Dop1R2 and Raf/MAPK in appetitive LTM (Sun, 2020)

This study found that MAPK has a pivotal role to stabilize appetitive memory in KCs. MAPK signaling is known to regulate different cellular processes ranging from cytoskeletal dynamics to transcriptional modulation. In Drosophila, a recent work unveiled that MAPK stabilizes presynaptic structural changes in KCs upon associative training with electric shocks, reportedly by changing the activity of an actin cytoskeleton regulator (Zhang, 2018). Such MAPK-induced cytoskeletal change might also occur in appetitive learning. Alternatively, a recent study showed that LTM consolidation involves MAPK translocation to the nuclei in KCs (Li, 2016). Consistently, it is reported that MAPK activates transcription factors like c-Fos and cAMP response element-binding protein (CREB) in KCs to form aversive LTM. Appetitive LTM is also dependent on CREB in KCs. Collectively, it is proposed that MAPK stabilizes appetitive memory by regulating these transcription factors. Future investigation on the downstream of the MAPK pathway should reveal the newly transcribed genes for memory stabilization (Sun, 2020).

Compartment specific regulation of sleep by mushroom body requires GABA and dopaminergic signaling
Sleep is a fundamental behavioral state important for survival and is universal in animals with sufficiently complex nervous systems. As a highly conserved neurobehavioral state, sleep has been described in species ranging from jellyfish to humans. Biogenic amines like dopamine, serotonin and norepinephrine have been shown to be critical for sleep regulation across species but the precise circuit mechanisms underlying how amines control persistence of sleep, arousal and wakefulness remain unclear. The fruit fly, Drosophila melanogaster, provides a powerful model system for the study of sleep and circuit mechanisms underlying state transitions and persistence of states to meet the organisms motivational and cognitive needs. In Drosophila, two neuropils in the central brain, the mushroom body (MB) and the central complex (CX) have been shown to influence sleep homeostasis and receive aminergic neuromodulator input critical to sleep-wake switch. Dopamine neurons (DANs) are prevalent neuromodulator inputs to the MB but the mechanisms by which they interact with and regulate sleep- and wake-promoting neurons within MB are unknown. This study investigated the role of subsets of PAM-DANs that signal wakefulness and project to wake-promoting compartments of the MB. PAM-DANs were found to be GABA responsive and required GABA(A)-Rdl receptor in regulating sleep. In mapping the pathways downstream of PAM (protocerebral anterior medial) neurons innervating γ5 and β'2 MB compartments it was found that wakefulness is regulated by both DopR1 and DopR2 receptors in downstream Kenyon cells (KCs) and mushroom body output neurons (MBONs). Taken together, this study has identified and characterized a dopamine modulated sleep microcircuit within the mushroom body that has previously been shown to convey information about positive and negative valence critical for memory formation. These studies will pave way for understanding how flies balance sleep, wakefulness and arousal (Driscoll, 2021).

The mushroom body lobes are tiled by discrete anatomic compartments defined by the axons of a specific subset of DANs and the dendrites of one or two mushroom body output neurons (MBONs). This anatomical arrangement positions DANs to strategically convey positive and negative reinforced information by changing the synaptic weight of KC-MBONs in producing aversive and appetitive responses (Driscoll, 2021).

While, the most in-depth analysis of these synapses and distinct DAN-KC-MBON connectivity and behavioral output comes from studies of olfactory conditioning, there is evidence that these synapses play a critical role in innate behaviors like feeding and sleep. Although, role of DA on sleep has been extensively investigated in Drosophila, the commonly used TH-Gal4 driver line labels most dopamine neuron clusters, but is absent from the several PAM clusters that projects to MB (Driscoll, 2021).

This study specifically probed PAM subsets that project to γ5, γ4, and β'2 MB compartments. This study focused on this subset because KCs and MBONs downstream of these PAM neurons can be neuroanatomically resolved and have been shown to be required for wakefulness. Further, KCs and MBONs that form the γ5, γ4, and β'2 synaptic compartments alter their spontaneous neural activity in response to sleep need (induced by mechanical sleep-deprivation). The ability to use cell-specific split-GAL4 tools provides opportunity to resolve the precise circuit mechanisms by which PAM neurons regulate wakefulness (Driscoll, 2021).

GABA signaling also modulates sleep and wake microcircuits within MB. The key source of GABA in the MB is anterior paired lateral neurons, APL and dorsal paired medial neurons (DPM), which are electrically coupled and increase sleep by GABAergic inhibition of wake-promoting KCs. In the context of associative learning, there is strong evidence for interactions between KCs, APL, DPM and DANs but it is not clear if GABA and dopamine signaling represent opposing inputs to the KCs and MBONs in the regulation of sleep. This study found that the excitability of PAM DANs involved in wakefulness is blocked by sleep-promoting GABA signaling and mediated by ionotropic receptor subtype GABAA-Rdl (Driscoll, 2021).

A recent study showed that GABA inhibitory input to the presynaptic terminals of the PAM neurons regulates appetitive memory and that this interaction is mediated by GABA-B3 receptors that are clustered in PAM boutons localized to PAM-γ5 and -α1 compartments. These data are consistent with the findings that PAM-γ5 are GABA responsive and that multiple receptors are critical to this interaction. Since, no role was found for GABA-B3 in PAM mediated sleep regulation, it is likely that PAM γ5, γ4, and β'2 express multiple GABA receptors which are differentially recruited in sleep and learning. How and what regulates the expression of these receptors in PAM subsets presents a potential mechanism of presynaptic gating to MB core circuits. Transcriptomic analysis of PAM neurons reveals extremely high levels of Rdl expression followed by GABA-B3. Among the PAM subsets mean TPM or transcripts per million of Rdl receptor in PAM γ5, γ4, and β'2 are much higher as compared to other PAM subsets (Driscoll, 2021).

Simple connection query search of the recently released hemibrain data85 reveals there is significant bidirectional connectivity between APL, DPM, and PAM neurons. Further, a recent study showed that APL neurons express the inhibitory D2R receptor55. APL mediated GABAergic inhibition of the PAM neurons was recently shown to control the intensity and specificity of olfactory appetitive memory but previous results show that blocking GABA release from APL neurons only modestly affects sleep phenotypes (Driscoll, 2021).

While, the role of APL in GABA signaling to PAM γ5, γ4, and β'2 cannot be completely ruled out, other inputs to wake-regulating PAM DANs could also be GABAergic and critical for promoting sleep. A recent study using EM dataset of a Full Adult Female Fly Brain (FAFB) mapped the inputs and outputs of the PAMγ5 DANs and identified that this cell type is highly heterogenous and in addition to recurrent feedback from MBON01 γ5β'2a, it receives extensive input from other MBONs, sub-esophageal output neurons (SEZONs) and lateral horn output neurons86. The EM data also reveals that octopaminergic neurons synapse onto PAM γ5, γ4, and β'2 DANs. Whether, these inputs play a role in wakefulness is unknown but suggests that the PAMγ5 could serve as a key link between sensory inputs, wake-promoting octopamine signal and core sleep regulating circuitry within the MB. Each of these inputs could modulate PAM-DAN activity and dopamine release in regulating wakefulness via the MB (Driscoll, 2021).

In addition to probing the release and activity of these PAM-DANs the dopamine receptors and their location within the MB in signaling wakefulness were also explored. To this end validated RNAi lines were expressed in subsets of KCs and MBONs; DopR1 and DopR2 were found to be critical in mediating the wakefulness signal via KCs and γ5β'2 MBONs. Knocking down the receptor consistently increased total sleep and bout length. Furthermore, specific manipulations of DopR receptors within the MB did not directly alter locomotor activity as observed by manipulation of these receptors in CX. Although, loss-of-function mutations of D1 dopamine receptor DopR are shown to enhance repetitive air puff startle-induced arousal and increase sleep. Expression and restoration of DopR in the mutant background specifically in the central complex rescues the startle response, while, the sleep phenotype is rescued via a broad MB driver. The current data extends these findings by showing that the DopR receptors regulate sleep via the MB γ5 and β'2 compartment. Although, targeted RNAi experiments show that DopR's are required for sleep regulation by KCs and MBONs, the lack of a sleep phenotype in DopR2 mutant could be a result of global loss of receptor in the mutant as opposed to targeted loss of receptor function within MB. Dopamine signals wakefulness by activation of wake-promoting neurons of MB via DopR1 and DopR2 and within. the central complex, neurons of dFB are inhibited by dopamine via DopR2. Hence, DopR2 has opposing effects within MB and CX (Driscoll, 2021).

In vitro characterization indicates that DopR's signal through distinct G-proteins, with DopR1 via Gαs to stimulate cAMP production and DopR2 coupling to Gαq via increased calcium. These receptors are thought to have differential sensitivity to dopamine and could be potentially recruited by varying DA release or DAN activity. In the context of sleep regulation, this work reveals that both DopR1 and DopR2 induce wakefulness via the γ5 β'2 MB compartment but not γ4 compartment. Although, chronic activation of PAM γ4 induces wakefulness, the glutamatergic MBON γ4 < γ1,2 projects to multiple compartments and could potentially activate or inhibit MBONs and PAMs projecting to γ1 and γ2 compartment. The interaction between compartments is not well understood in the context of sleep and wake regulation and requires further investigation to better understand the role of DopR2 in regulating the γ4 compartment. The neuroanatomical specificity obtained from split-Gal4 lines combined with EM data has paved way for more detailed analysis of the role of dopamine signaling to MB in the context of sleep and other behaviors (Driscoll, 2021).

The sleep-regulating PAM DANs and associated KCs and MBONs identified in this study are also involved in mediating satiety, novelty, caffeine induced arousal, punishment and reward associated experiences suggesting that the activity of these neurons is tuned to several wake and arousal associated behaviors. This is further supported by the EM connectome data showing that MB receives extensive gustatory, auditory and visual input in addition to olfactory input (Driscoll, 2021).

Current models of sleep regulation rely on two main processes, the circadian clock and the sleep homeostat and don't completely account for multiple external and internal factors that influence wakefulness. The ability to sleep, however, is influenced by motivational or cognitive stimuli. It is therefore envisioned that sleep, wakefulness and arousal within MB are not located in distinct circuits, but rather mediated by distinct processes within a common circuit (Driscoll, 2021).

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: 23 June 2023

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