DopEcR, a G-protein coupled receptor for ecdysteroids, is involved in activity- and experience-dependent plasticity of the adult central nervous system. Remarkably, a courtship memory defect in rutabaga (Ca²⁺/calmodulin-responsive adenylate cyclase) mutants is rescued by DopEcR overexpression or acute 20E feeding, whereas a memory defect in dunce (cAMP-specific phosphodiestrase) mutants is counteracted when a loss-of-function DopEcR mutation is introduced. A memory defect caused by suppressing dopamine synthesis is also restored through enhanced DopEcR-mediated ecdysone signaling, and rescue and phenocopy experiments revealed that the mushroom body (MB) - a brain region central to learning and memory in Drosophila - is critical for the DopEcR-dependent processing of courtship memory. Consistent with this finding, acute 20E feeding induced a rapid, DopEcR-dependent increase in cAMP levels in the MB. The multidisciplinary approach demonstrates that DopEcR mediates the non-canonical actions of 20E and rapidly modulates adult conditioned behavior through cAMP signaling, which is universally important for neural plasticity. This study provides novel insights into non-genomic actions of steroids, and opens a new avenue for genetic investigation into an underappreciated mechanism critical to behavioral control by steroids (Ishimoto, 2013).

Steroid hormones are essential modulators of a broad range of biological processes in a diversity of organisms across phyla. In the adult nervous system, the functions of steroids such as estrogens and glucocorticoids are of particular interest because they have significant effects on the resilience and adaptability of the brain, playing essential roles in endocrine regulation of behavior. Reflecting their importance in neural functions, steroid hormones are implicated in the etiology and pathophysiology of various neurological and psychiatric disorders, and are thus often targeted in therapies. The biological actions of steroids are mediated mainly by nuclear hormone receptors - a unique class of transcription factors that activate or repress target genes in a steroid-dependent manner. Substantial evidence suggests, however, that steroid hormones can also exert biological effects quickly and independently of transcriptional regulation, by modulating intracellular signaling pathways. Such 'non-genomic' effects might be induced by direct allosteric regulation of ion channels, including receptors for GABA and NMDA. Alternatively, in certain contexts, non-genomic steroid signaling could be mediated by classical nuclear hormone receptors acting as effector molecules in the cytosol (Ishimoto, 2013).

Behavior cannot be predicted from a 'connectome' because the brain contains a chemical 'map' of neuromodulation superimposed upon its synaptic connectivity map. Neuromodulation changes how neural circuits process information in different states, such as hunger or arousal. This study describes a genetically based method to map, in an unbiased and brain-wide manner, sites of neuromodulation under different conditions in the Drosophila brain. This method, and genetic perturbations, reveal that the well-known effect of hunger to enhance behavioral sensitivity to sugar is mediated, at least in part, by the release of dopamine onto primary gustatory sensory neurons, which enhances sugar-evoked calcium influx. These data reinforce the concept that sensory neurons constitute an important locus for state-dependent gain control of behavior and introduce a methodology that can be extended to other neuromodulators and model organisms (Inagaki, 2012).

The Tango system transforms a transient ligand/receptor interaction into a stable, anatomical readout of reporter gene expression. The reporter gene is activated by a 'private,' synthetic signal transduction pathway, using a bacterial transcription factor (lexA) that is covalently coupled (via a specific tobacco etch virus [TEV] protease-sensitive cleavage site) to the exogenous DA receptor expressed in the cells of interest. The transcription factor is cleaved from the DA receptor following ligand binding, by recruitment of an arrestin-TEV protease fusion protein, and translocates to the nucleus where it activates a lexAop-driven reporter. This system was originally developed to detect receptor activation in cultured mammalian cell lines, but it used to detect receptor activation in this system (Inagaki, 2012).

The data presented here provide proof-of-principle for the utility of a new method, called TANGO-map, to identify, in a brain-wide and relatively unbiased manner, circuit-level substrates of neuromodulation relevant to a particular state-dependent influence on behavior (Inagaki, 2012).

Sweet taste sensitivity in the labellum is enhanced with increasing duration of food deprivation in Drosophila. This observation confirms and extends previous reports in Drosophila and is consistent with observations in many other animal species. This phenomenon was used as a prototypic case of a state-dependent change in behavior to investigate the ability of TANGO-map to identify underlying neuromodulatory mechanisms (Inagaki, 2012).

The results indicate that starvation enhances endogenous DA release onto primary gustatory receptor neurons (GRNs) in the sub-esophageal ganglion, as detected by increased expression of the DopR-Tango reporter in vivo. In contrast, starvation did not increase the DopR-Tango reporter in the MB or AL, although L-dopa feeding did so. These data indicate that DopR-Tango is capable of revealing selective sites of endogenous DA release in a brain-wide manner, under specific behavioral conditions (Inagaki, 2012).

The results indicate that a mutation in the DA receptor DopEcR, as well as specific knockdown of this receptor in sugar-sensing GRNs, eliminates the effect of starvation to enhance the sucrose sensitivity of the proboscis extension reflex (PER). However, this phenotype was only observed at 6 hr of starvation; after 24 hr of food deprivation, these genetic manipulations no longer had an effect. This is not because these manipulations themselves became ineffective at later times, as the same manipulations did attenuate the increased PER sensitivity caused by L-dopa feeding for 24 hr. This suggests that at an early stage of starvation, DA is necessary to enhance the sugar sensitivity of the PER, whereas at later stages additional factors come into play. The slow kinetics of Tango reporter accumulation preclude the detection of statistically significant increases in signal as early as 6 hr following an experimental manipulation. However, the level of reporter expression detected in animals examined after 48 hr of treatment likely reflects the integration of increases in dopaminergic signaling occurring throughout the first 12-24 hr of the treatment period. Thus, although an increase was detected in DopR-Tango signal at a starvation time point when genetic reduction of DopEcR levels no longer impaired the behavioral effect of starvation and observed a behavioral phenotype at a time point too early to be evaluated directly by the TANGO-map method, this should not be taken to imply that no DA release occurred after 6 hr of starvation. Importantly, given the kinetics of the system, the DopR-Tango signals detected in vivo are likely to reflect primarily changes in tonic levels of DA signaling, rather than brief episodes of phasic DA release. Further improvements of the TANGO-map method are required to increase its temporal resolution. Nevertheless, the present methodology provides a powerful method to identify sites where dopaminergic modulation of a given behavior may occur, even if it cannot reveal precisely how quickly such regulation is exerted (Inagaki, 2012).

Several lines of evidence suggest that the dopaminergic modulation of sugar-sensing GRNs revealed in this study may involve an enhancement of Ca2+ influx at the nerve terminal. Both starvation and L-dopa feeding increased sucrose-evoked Ca2+ influx, without changing the frequency of action potentials measured extracellularly at GRN somata. Furthermore, it was found that direct exposure of the brain to DA increased Ca2+ influx at the presynaptic terminals of sugar-sensing GRNs in a DopEcR-dependent manner. A model consistent with these data is that starvation leads to increased DA release, which increases calcium influx into sugar-sensing GRNs via DopEcR, leading to increased neurotransmitter release. The fact that DopEcR signals via the cAMP/PKA pathway, and that this pathway has been reported to increase Ca2+ channel currents in Drosophila, is also consistent with this scenario. Nevertheless, the genetic data suggest that there are additional pathways through which starvation modulates feeding behavior in this system. The finding that DA modulates primary GRNs to control starvation-dependent changes in behavioral sensitivity to sugar echoes the observation of a similar influence of food deprivation on odorant sensitivity in Drosophila. Such neuromodulatory gain control at the level of primary sensory neurons has also been reported in a variety of other invertebrate as well as vertebrate species. Although the possibility that hunger also influences PER behavior at higher-order synapses in the circuit cannot be excluded, the data add to a growing body of information indicating that modulation of primary sensory neurons is a general mechanism for implementing state-dependent changes in behavioral responses to the stimuli detected by these neurons (Inagaki, 2012).

TANGO-map affords a number of unique advantages to study neuronal modulation in the brain. First, and most importantly, it permits the detection of increases in endogenous neuromodulator release in vivo, in an organism in which the application of conventional methods is not feasible. Second, it provides an anatomical readout of neuromodulation at the neural circuit level. The use of a pan-neuronal GAL4 driver to express the sensor permits, in principle, an unbiased survey of potential sites of neuromodulatory activity throughout the brain. Third, the sensor has ligand specificity. The modular design of the Tango system affords the ability to develop in vivo Tango reporters for other biogenic amines and neuropeptides that work via G protein-coupled receptors (GPCRs). Importantly, because the method employs a synthetic, 'private' signal transduction pathway, the readout of the reporter should be relatively insensitive to interference from conventional signal transduction pathways activated by other endogenous receptors. Systematic and comprehensive application of this approach could, in principle, provide an overview of anatomic patterns of neuromodulation in the brain in a given behavioral setting. Finally, because the Tango system is transcriptionally based, in principle it permits the expression not only of neutral reporters but also of effectors such as RNAi’s or ion channels in the neurons receiving neuromodulatory input. Although the TANGO-map system can certainly benefit from improvements in its kinetics and SNR, it affords a means of identifying points-of-entry for studying circuit-level mechanisms of behaviorally relevant neuromodulation that are currently difficult to access in any otherway. The extension of thismethodology to other neuromodulators and model organisms should further understanding of state-dependent control of neural activity and behavior (Inagaki, 2012).

Nongenomic response pathways mediate many of the rapid actions of steroid hormones, but the mechanisms underlying such responses remain controversial. In some cases, cell-surface expression of classical nuclear steroid receptors has been suggested to mediate these effects, but, in a few instances, specific G-protein-coupled receptors (GPCRs) have been reported to be responsible. This study describes the activation of a novel, neuronally expressed Drosophila GPCR by the insect ecdysteroids ecdysone (E) and 20-hydroxyecdysone (20E). This is the first report of an identified insect GPCR interacting with steroids. The Drosophila melanogaster dopamine/ecdysteroid receptor (DmDopEcR) shows sequence homology with vertebrate beta-adrenergic receptors and is activated by dopamine (DA) to increase cAMP levels and to activate the phosphoinositide 3-kinase pathway. Conversely, E and 20E show high affinity for the receptor in binding studies and can inhibit the effects of DA, as well as coupling the receptor to a rapid activation of the mitogen-activated protein kinase pathway. The receptor may thus represent the Drosophila homolog of the vertebrate 'gamma-adrenergic receptors,' which are responsible for the modulation of various activities in brain, blood vessels, and pancreas. Thus, DmDopEcR can function as a cell-surface GPCR that may be responsible for some of the rapid, nongenomic actions of ecdysteroids, during both development and signaling in the mature adult nervous system (Srivastava, 2005).

It is unlikely that the effects described in this study are mediated by cell-surface expression of nuclear EcRs because, in both Sf9 and CHO cells, they only occur in transfected cells expressing DmDopEcR and not in nontransfected control cells. Although intact Sf9 cells contain cytoplasmic EcRs that can be detected by [3H]PoA binding, these receptors exhibit a different ecdysteroid-binding pharmacology. This contrasts with the current results. In addition, the bisacylhydrazine ecdysteroid agonists RH-5849 and RH-0345 (halofenozide), which bind effectively to EcRs from Lepidoptera and Drosophila, do not displace [3H]PoA binding to DmDopEcR at concentrations up to 1 μM. Thus, DmDopEcR shows a different pharmacology to insect nuclear EcRs. This conclusion is supported by the observation that [3H]PoA binds to plasma membranes from the anterior silk gland of the silkworm Bombyx mori (Srivastava, 2005).

Expression studies suggest that DmDopEcR can be activated by both E and 20E. In most cases, the physiological actions of insect ecdysteroids are thought to involve 20E rather than E. The latter is usually considered to be a metabolic precursor of 20E. It is synthesized and released by the Drosophila ring glands, along with 20-deoxymakisterone A, and these steroids are metabolized in peripheral tissues to 20E and MaA, respectively . However, there are few known physiological actions of E and MaA. An exception is the stimulatory role of E in cell proliferation in the optic lobe of Manduca sexta. In addition, many arthropod rapid gustatory sensilla responses to ecdysteroids show selectivity in their responses to E and 20E (Srivastava, 2005).

An unusual property of the DmDopEcR receptor is its ability to respond to both catecholamines and to ecdysteroids. These ligands could bind competitively to overlapping topographical sites on the outer surface of the receptor or noncompetitively to two distinct nonoverlapping sites that could interact allosterically. It is extremely difficult to distinguish between these two possible forms of ligand interaction experimentally for GPCRs. However, it would appear in the present case that the interactions described in this study are not likely to be the result of a classical allosteric action of the ecdysteroids on the dopamine-binding site, because the ecdysteroids themselves are capable of activating the MAPK pathway through the receptor. The phenomenon of allosteric agonism appears to be very rare for GPCRs, and most allosteric ligands are either antagonists or modulators. However, a recent exception has been described for the chemokine receptor CXCR4. Additionally, allosteric modulation has been defined recently as requiring a reciprocal interaction between the sites, but this study could detect no effect of DA on PoA binding up to a concentration of 100 μM. Although it seems likely that the ecdysteroids and dopamine bind to overlapping sites, a definitive description of the extent of the overlap of these binding sites will require a combination of extensive in vitro mutagenesis studies, together with x-ray diffraction studies on purified receptor protein, which are beyond the scope of the present study (Srivastava, 2005).

The DmDopEcR receptor appears to demonstrate agonist-specific coupling. DA couples the receptor to the stimulation of adenylyl cyclase activity and activates PI3K, as assessed via Akt phosphorylation, whereas the ecdysteroids couple the receptor to the activation of the MAPK pathway, as assessed by ERK2 phosphorylation, presumably by each agonist inducing a different conformation of the receptor. Both the PI3K pathway and catecholamines are known to have important roles in the control of Drosophila development, and, in addition, the activation of the MAPK pathway is an important regulator of cellular differentiation in Drosophila. A wide range of other Drosophila GPCRs, including the OA/TYR receptor, the DopR99B D1-like DA receptor , and the short neuropeptide F receptor, have also been shown to exhibit various forms of agonist-specific coupling, as have several vertebrate adrenergic and neuropeptide GPCRs. It remains to be determined whether both DA and the ecdysteroids can signal or modulate each other's actions through DmDopEcR in vivo. Nevertheless, it is interesting to speculate that the many surges in ecdysteroid levels observed during insect development, which can reach concentrations as high as 1.35 μM, could serve to rapidly turn off DA-mediated signaling through this receptor. Such a suggestion would be consistent with the rapid ecdysteroid-mediated inhibition of nitric oxide signaling in Manduca optic lobe neurogenesis. It would allow the neural precursor cells to proceed into mitosis, because ecdysteroids induce a conformation of DmDopEcR that does not couple to the PI3K pathway activating nitric oxide synthesis. Equally, it is interesting to speculate that the expression of DmDopEcR in the developing eye imaginal disk may relate to the progression of the morphogenetic furrow, because this requires E, but not EcR, to function (Srivastava, 2005).

The in situ hybridization and the PCR expression studies on DmDopEcR suggest that the expression of the receptor is tightly controlled during development and is likely to have a number of well defined, stage-specific, functional roles. Thus, it is likely to be involved with the control of cell proliferation and differentiation in the salivary ducts and the midgut, although its specific role in these regions remains to be elucidated. The strong expression of DmDopEcR during the early development of the nervous system, when neuroblasts are dividing, suggests that the receptor could be involved with the control of cell proliferation. In addition, the strong expression of the receptor in adult heads suggests that the receptor may also be involved in the modulation of neuronal signaling because ecdysteroids are known to have rapid effects on neural activity in insect brains, paralleling the actions of vertebrate steroids on the brain (Srivastava, 2005).

Olfactory information mediating sexual behavior is crucial for reproduction in many animals, including insects. In male moths, the macroglomerular complex (MGC) of the primary olfactory center, the antennal lobe (AL) is specialized in the treatment of information on the female-emitted sex pheromone. Evidence is accumulating that modulation of behavioral pheromone responses occurs through neuronal plasticity via the action of hormones and/or catecholamines. A G-protein-coupled receptor (GPCR), AipsDopEcR, with its homologue known in Drosophila for its double affinity to the main insect steroid hormone 20-hydroxyecdysone (20E), and dopamine (DA), has been shown to be present in the ALs, and is involved in the behavioral response to pheromone in the moth, Agrotis ipsilon. This study tested the role of AipsDopEcR as compared to nuclear 20E receptors in central pheromone processing, combining receptor inhibition with intracellular recordings of AL neurons. The sensitivity of AL neurons for the pheromone in males decreases strongly after AipsDopEcR-dsRNA injection but also after inhibition of nuclear 20E receptors. Moreover the involvement of 20E and DA was tested in the receptor-mediated behavioral modulation in wind tunnel experiments, using ligand applications and receptor inhibition treatments. Both ligands are necessary and act on AipsDopEcR-mediated behavior. Altogether these results indicate that the GPCR membrane receptor, AipsDopEcR, controls sex pheromone perception through the action of both 20E and DA in the central nervous system, probably in concert with 20E action through nuclear receptors (Abrieux, 2004).