The diverse physiological effects of dopamine are mediated by multiple receptor systems. The dDA1 represents one of the Drosophila dopamine receptors that activate the cAMP cascade. To gain insight into the role of dDA1, a polyclonal antibody was generated against the unique sequence in dDA1 and dDA1 distribution in the central nervous system (CNS) was investigaed. In both larval and adult CNS pronounced dDA1 immunoreactivity is present in the neuropil of the mushroom bodies, a brain structure crucial for learning and memory in insects, and four unpaired neurons in each thoracic segment. In addition, the larval abdominal ganglion contained two dDA1 cells in each segment. This expression pattern appears to be maintained in the condensed adult abdominal ganglion although the precise number and the intensity of staining were somewhat variable. The adult CNS also exhibits intense dDA1 immunoreactivity in the central complex, a structure controlling higher-order motor function, moderate expression in several neurosecretory cells, and weak staining in two unpaired neurons in the mesothoracic neuromere. The dDA1 expression in these areas was detected only in the adult, but not in third instar larval CNS (Kim, 2003).
A set of peptidergic neurons is conserved throughout all developmental stages in the Drosophila central nervous system. A small complement of 28 apterous-expressing cells (Ap-let neurons) in the ventral nerve cord (VNC) of Drosophila larvae co-express numerous gene products. The products include the neuroendocrine-specific bHLH regulator called Dimmed (Dimm), four neuropeptide biosynthetic enzymes (PC2, Fur1, PAL2, and PHM), and a specific dopamine receptor subtype (dDA1). For the PC2, Fur1, and PAL2 enzymes, and for the dDA1 receptor, this neuronal pattern represents the vast majority of their total expression in the VNC. In addition, while Dimm and PHM are present in the peritracheal Inka cells in larvae, pupae, and adults, Ap, PC2, Fur1, PAL2, and dDA1 are not. PC2, PAL2, and DA1 receptor expression are all controlled by both dimm and ap. Previous genetic analysis of animals deficient in PC2 revealed an abnormal larval ecdysis phenotype. Together, these data support the hypothesis that the small cohort of Ap-let interneurons regulates larval ecdysis behavior by secretion of an unidentified amidated peptide(s). This hypothesis further predicts that the production of the Ap-let neuropeptide(s) is dependent on each of four specific enzymes, and that a certain aspect(s) of its production and/or release is regulated by dopamine input (Park, 2004).
One of the Drosophila D1-like dopamine receptors, dDA1 (CG9562), is expressed in a subset of the larval and adult CNS neurons. In the VNC, dDA1 immunoreactivity is evident in a single Dorsal neuron in each thoracic and abdominal hemi-neuromere, and a single lateral neuron in each thoracic hemi-neuromere. This expression pattern is similar to that of the Ap-let group. To test whether dDA1 is expressed in Ap-let neurons, the larval CNSs of apGAL4/UAS-GFP or of c929/UAS-GFP were stained with the dDA1 antibody. All of the dDA1 cells were positive with apGAL4, and with c929, and they included the Dorsal chain and one of the two Tv neurons. This indicates the dDA1-positive T neuron is either Tv or Tvb. When the CNS was double-stained with anti-FMRFa antibody that stains the larval Tv neuron, the dDA1 and dFMRFa immunosignals were in distinct cells. Together, these data indicate that the dDA1-positive cell in the T cluster os the Tvb neuron. Similar to PHM, PC2, Fur1, and PAL2, none of the ap-positive ventral neuron pairs expressed dDA1 immunosignals (Park, 2004).
Neuronal specification is often seen as a multistep process: earlier regulators confer broad neuronal identity and are followed by combinatorial codes specifying neuronal properties unique to specific subtypes. However, it is still unclear whether early regulators are re-deployed in subtype-specific combinatorial codes, and whether early patterning events act to restrict the developmental potential of postmitotic cells. This study used the differential peptidergic fate of two lineage-related peptidergic neurons in the Drosophila ventral nerve cord to show how, in a feedforward mechanism, earlier determinants become critical players in later combinatorial codes. Among the progeny of neuroblast 5-6 are two peptidergic neurons: one expresses FMRFamide and the other one expresses Nplp1 and the dopamine receptor DopR. The HLH gene collier functions at three different levels to progressively restrict neuronal identity in the 5-6 lineage. At the final step, collier is the critical combinatorial factor that differentiates two partially overlapping combinatorial codes that define FMRFamide versus Nplp1/DopR identity. Misexpression experiments reveal that both codes can activate neuropeptide gene expression in vast numbers of neurons. Despite their partially overlapping composition, the codes are remarkably specific, with each code activating only the proper neuropeptide gene. These results indicate that a limited number of regulators may constitute a potent combinatorial code that dictates unique neuronal cell fate, and that such codes show a surprising disregard for many global instructive cues (Baumgardt, 2007).
In the developing Drosophila VNC, approximately 90 neurons express the LIM-homeodomain regulator Apterous (Ap), and these represent at least six different cell types. This study focuses on three of the Ap neurons: two cells of the Ap cluster, the Tv cells, which express FMRFa, and the Tvb cells, which together with the dAp cells express DopR. A number of regulators involved in Tv neuron specification have been identified, but to better understand specification of the related Tvb/dAp neurons, it was important to identify the putative neuropeptide gene expressed by Tvb/dAp neurons. The completion of the Drosophila genome led to the prediction of several additional neuropeptide genes, including the Neuropeptide like precursor protein 1-4 genes (Nplp1-4). The validity of these predictions has been confirmed by the identification of expressed sequence tags (ESTs) matching these genes, and by the detection of amidated and secreted peptides in circulation, and/or in brain extracts. Expression of gene products from one of these genes, Nplp1, was found in a set of cells in the VNC reminiscent of the Tvb/dAp neurons. In situ hybridization for Nplp1 verified that these cells indeed correspond to the dAp neurons and to one Ap cluster neuron. To further identify this Ap cluster neuron, antibodies were raised against pro-Nplp1 and against one of the processed and amidated peptides, IPNamide, and a similar pattern was detected. Markers were used for specific subsets of Ap neurons, and the Nplp1-expressing cell in the Ap cluster was identified as the Tvb neuron. In Tvb/dAp neurons, Nplp1 and DopR expression commences in the late embryo (18 h after egg laying [AEL]) and persists at least into the third larval stage. Nplp1 and DopR are thus specifically expressed by the 28 embryonic and larval Tvb/dAp neurons (Baumgardt, 2007).
Recent studies have revealed that ap and dimm are important for DopR expression in Tvb/dAp. It was asked whether these Ap neuron determinants, as well as eya, also affected Nplp1 expression. Nplp1 expression was found to depended on eya, ap, and dimm, and eya also regulates DopR. How is Tv versus Tvb/dAp cell fate then determined? Although both cell types express ap, dimm, and eya, only Tv neurons express dac and have activated the BMP pathway. Could the mere absence of dac and/or BMP activation be sufficient to specify the Tvb/dAp fate? To test this, expression of Nplp1 was analyzed in dac and BMP mutants, but no evidence was found of ectopic Nplp1 expression in Tv neurons. Thus, specification of Tvb/dAp neurons likely requires additional factors restricted to this cell type (Baumgardt, 2007).
The COE family of HLH regulators is highly evolutionary conserved, and is represented in Drosophila by a single member, col (Flybase, knot). COE genes play important roles during nervous system development in Caenorhabditis elegans and vertebrates, and col is expressed in the developing Drosophila central nervous system (CNS), although no function has yet been assigned to it there. The involvement of members of this gene family in nervous system development in other species, and the embryonic CNS expression of col, prompted an investigation of the possible role of col during Ap neuron specification. col has a dynamic expression pattern in the VNC, and focus was initially placed on its expression in mature Ap neurons, at 18 h AEL, and larval stages. At these stages, col is expressed specifically in Tvb/dAp neurons, and expression is maintained in these neurons at least into the third larval stage (see below). This raised the possibility that col plays a role in Tvb/dAp cell fate specification. This notion was supported by the complete loss of Nplp1 and DopR expression in col mutants (Baumgardt, 2007).
Previous studies have addressed the regulatory interactions between several of the Ap neuron determinants. However, these had not been addressed in the case of dac, eya, and dimm. As expected from the late onset of dimm expression, no evidence was found of dimm regulation of Dac or Eya. Similarly, as expected from the mild effect of dac upon FMRFa, dac does not regulate Dimm. In contrast, in eya mutants, a nearly complete loss of Dimm expression was found in the Tv, Tvb, and dAp neurons (Baumgardt, 2007).
With a more complete picture of how previously identified Ap neuron determinants interact genetically, whether col acts upstream, downstream, or in parallel to other Ap neuron determinants was determined. The expression of Col was examined in embryos mutant for these other regulators. In general, no severe effects on Col expression were found. The one exception was in sqz, in which a reproducible increase in the number of Col cells was found. This was, however, expected, since sqz affects the composition of Ap cluster cells, with an increase both in the number of Ap cluster cells (specifically in T1) and an increase in Tvb cells at the expense of Tv cells (in T1-T3). In line with this, an increase was also found in Nplp1 cells in sqz mutants (Baumgardt, 2007).
Does col act upstream of other Ap neuron determinants instead? To address this, the expression of these other regulators was examined in col mutant embryos. This analysis was facilitated by the fact that col2 and col3 mutants, which are both genetically strong alleles, are not protein null, thus allowing for detection of Col in col mutants. In addition, Col is expressed by all four Ap cluster neurons at stage 15. In col mutants, it was found that although sqz and Dac are largely unaffected, ap, Eya, and Dimm are all completely absent from Ap neurons. Because ap, eya, and dimm all regulate FMRFa, the loss of these regulators prompted a look at the expression of FMRFa as well, and as expected, in col mutants, a complete loss was found of FMRFa in the Tv neurons. However, FMRFa is still expressed in the SE2 neurons, a feature common to all identified FMRFa regulators except dimm. These results suggest that Ap cluster neurons are generated in col mutants, but are incompletely specified because they express part of their normal specification code such as dac and sqz, but not other elements of the code such as ap, Eya, Dimm, FMRFa, Nplp1, and DopR (Baumgardt, 2007).
The severe effect of col upon ap, eya, and dimm within all Ap cluster neurons, and upon FMRFa within the Tv neuron, is at odds with the restricted expression of Col in Tvb/dAp neurons at late embryonic stages. Therefore whether Col is more widely expressed at earlier embryonic stages was analyzed, focusing on the Ap cluster neurons. This revealed that although col is restricted to Tvb neurons at stages 17 and 16, expression was observed in all four Ap cluster neurons at stage 15, the stage at which these neurons are first identifiable using ap, Eya, Dac, and sqz as specific markers (Baumgardt, 2007).
Is Col expressed even in the progenitor cells generating Ap cluster neurons? Because ap, Eya, Dac, and sqz are not expressed in Ap cluster neurons prior to stage 15, resolving this issue required the identification of the neuroblast lineage generating the Ap cluster. Extensive work during the last two decades has provided a detailed lineage map of most, if not all, of the 30 neuroblasts found in each hemisegment, and has identified regulatory genes expressed by different neuroblasts. These studies, together with the extreme lateral positioning of early Ap cluster neurons and their appearance only in thoracic hemisegments, allowed use a series of specific markers and determine that the Ap cluster is generated by NB 5-6 -- a lateral-most neuroblast that has been shown to generate larger lineages in the thoracic segments. These results, combined with previous detailed studies of NB 5-6 development, suggested a tentative model for the thoracic NB 5-6 lineage, and lead to placement Col expression within this lineage (Baumgardt, 2007).
Col is expressed by all four newly born Ap cluster neurons and is essential for Ap cluster specification, as evident from the complete loss of ap and Eya expression in col mutants. Col is rapidly down-regulated from three Ap cluster cells and maintained only in Tvb. Is the down-regulation of col important for proper Ap cluster differentiation? To test this, col was misexpressed using the apGAL4 driver, which is not expressed until stage 16, thus maintaining col expression in all four Ap cluster neurons at the time when Col is normally down-regulated. This experiment led to frequent activation of Nplp1 in one additional Ap cluster neuron, and staining for Dimm reveals that this cell is indeed the Tv neuron. FMRFa expression is frequently down-regulated in Tv, but no Tv cells were observed that co-express FMRFa and Nplp1. The finding that col misexpression in the Ap cluster only leads to one ectopic Nplp1 cell and no ectopic Dimm cells indicates that col cannot induce a peptidergic cell fate, at least not with this late driver. However, because the two unaffected cells already are expressing ap and Eya, it was predicted that co-misexpression of dimm, together with col, should trigger Nplp1 expression in all four Ap cluster neurons. This is indeed what was found. In summary, down-regulation of col in three Ap cluster neurons is essential for proper Ap cluster specification (Baumgardt, 2007).
Col is expressed prior to ap and Eya in the Ap cluster neurons, and it is essential for ap and Eya expression within these cells. To address whether col is also sufficient to activate ap and Eya, col was misexpressed in all neurons, using the elav-GAL4 driver. This led to ectopic activation of both ap and Eya. In addition, some activation of Dimm, Nplp1, and DopR expression was found. Although ectopic activation of ap or Eya alone was found in several regions, co-activation was largely confined to neuroblast row 5 -- the anterior region of Gsbn expression. Typically six to ten ap/Eya co-expressing cells were observed in the lateral-most part of row 5. Ectopic activation of ap/Eya, together with Dimm, Nplp1, and DopR, was also confined to lateral-most row 5, i.e., the posterior-most part of gsblacZ cells, and further confined to thoracic segments. Ectopic Nplp1/DopR expression is not overlapping with FMRFa, and there is clear evidence of ectopic Dimm expression, indicating that additional peptidergic neurons are being generated. Ectopic generation of ap/Eya double-expressing cells, i.e., ectopic 'Ap cluster' neurons, was observed already at stage 13, i.e., prior to when Ap cluster neurons are normally born (Baumgardt, 2007).
These results show that col can activate ap and Eya in a number of neurons, but can act to generate bona fide Ap cluster neurons only in a highly context-dependent manner: in lateral, thoracic, row 5 neurons. The appearance of six to ten Eya-expressing cells, but only three to five Nplp1/DopR-expressing cells, and no evidence of ectopic FMRFa expression, suggests that the generation of ectopic Ap cluster neurons is biased toward Tvb (Nplp1/DopR expressing) as opposed to Tv (FMRFa expressing) cell fate. In contrast, although col function depends upon these three positional cues, these results indicate that col is able to override the temporal coding within lateral row 5, and activate Nplp1 and DopR in earlier-born neurons (Baumgardt, 2007).
The loss- and gain-of-function studies place col clearly upstream of ap and eya. Does col act merely to regulate ap and eya in early postmitotic Ap cluster neurons, or does it play additional roles during Ap cluster formation? To further address this issue, attempts were made to 'cross-rescue' col with ap and eya, by expressing ap and eya in a col mutant background. First, as a positive control, attempts were made to rescue col by providing col activity using elav-GAL4/UAS-col. This led to a robust rescue, both of Ap cluster determinants (Eya, ap, and Dimm) and of terminal differentiation genes (Nplp1, DopR, and FMRFa). Similar to the col misexpression experiments, a clear increase was found in 'Ap cluster' neurons, primarily of the Tvb type, as evident from the finding of six to ten ap/Eya- and three to five Nplp1/DopR-expressing neurons per hemisegment. Next, attempts were made to 'cross-rescue' col mutants with ap and eya, and indeed a significant degree of rescue of Ap cluster formation was found, as evident both from Dimm and FMRFa expression. In contrast, no evidence of rescue of Nplp1 or DopR was found in these embryos. Because Col can be detected in the col2 and col3 mutant backgrounds, a Dimm/Col-expressing cell was identified adjacent to the Tv/FMRFa neuron. This indicates that ap/eya can partially rescue Tvb cell fate, but in the absence of col activity, these 'Tvb' neurons do not activate Nplp1. In summary, the finding that in col mutants, ap and eya can partially rescue the Tv cell fate, but not Tvb cell fate, suggests additional roles for col in Tvb specification (Baumgardt, 2007).
The results indicate that Tvb cell fate is not specified by a linear col-->ap/eya-->dimm-->Nplp1/DopR genetic cascade. To further address this issue, the sufficiency was examined of col, ap, and eya to activate Dimm when misexpressed both alone and in combination. These experiments reveal that although col can trigger some ectopic activation of Dimm, there is little effect upon Dimm when misexpressing ap, eya, or ap/eya. In contrast, co-misexpression of col with either ap or eya, and in particular, co-misexpression of all three genes, leads to striking ectopic Dimm expression (Baumgardt, 2007).
Does col play a role even at the final step of Tvb differentiation, i.e., in the activation of Nplp1? Attempts were made to address the possible late role of col by misexpressing it alone and together with other Ap neuron determinants, and then assay its potency in activating Nplp1. Importantly, if there is a simple linear col-->ap/eya-->dimmNplp1/DopR genetic cascade at work, the effect of triple co-misexpression of ap/eya/dimm should not be enhanced by addition of col to this code. However, a striking enhancement was found of ectopic Nplp1 expression when adding col to this code. One particular double co-misexpression combination, col/ap, was more potent than others in activating both Dimm and Nplp1. A likely explanation for this effect is that co-misexpression of col/ap activates significant ectopic eya expression (Baumgardt, 2007).
To further address the late role of col, a transgenic RNA-interference (RNAi) line, (UAS-col-dsRNA), was generated, and attempts were made to suppress col gene activity by crossing this line to apGAL4. Because apGAL4 also drives expression in the developing wing disc, the efficiency was examined of this novel tool in suppressing col gene activity in this tissue. This phenocopied the effects of col mutants on wing development, with a clear L3-L4 wing vein fusion, indicating that this RNAi transgene specifically blocks col gene activity. However, upon analyzing late larval (third instar) CNSs, no effect upon Col expression in Tvb neurons was found, and as expected, no effect was found upon Nplp1 expression. Recent studies reveal that RNAi can be efficiently enhanced by overexpression of components of the RNAi pathway, in particular of the Dicer-2 (Dcr-2) gene. Therefore Dcr-2 was co-expressed with col dsRNA (UAS-Dcr-2/+; apGAL4/UAS-col-dsRNA), and a clear effect was found not only upon Col, but importantly, also upon Nplp1 expression. No obvious effect was found in the first instar larvae, but in third instar larvae, Col expression is specifically and completely lost from all Tvb/dAp cells. This leads to a complete loss of Nplp1 in 44% of Tvb/dAp cells, and strongly reduced expression in the remaining expressing cells. Strikingly, this strong effect upon Nplp1 is not an indirect effect of down-regulation of ap, Eya, or Dimm. As anticipated, col RNAi has no effect upon FMRFa expression in the third instar larvae (Baumgardt, 2007).
Previous studies have identified several regulators acting to specify Tv fate and to control FMRFa expression. Although co-misexpression of parts of this code had been previously tested, all possible combinations had not. Similar to the combinatorial activation of Nplp1 and DopR, it was found that whereas co-misexpression of ap/sqz, ap/dac, or ap/dimm has limited effect upon FMRFa expression, triple co-misexpression of these regulators, and in particular of ap/dimm/dac, leads to a dramatic ectopic activation of FMRFa (Baumgardt, 2007).
The identification of two partly overlapping and highly potent combinatorial codes allowed lead to asking of an important question: Does combinatorial misexpression of these regulators merely lead to a general confusion with a mixed neuronal identity, or are these codes truly instructive and specific? To address this issue, the expression of Nplp1 and FMRFa was examined in the various misexpression backgrounds. Not surprisingly, when common and partial components of these codes are misexpressed, such as ap/dimm (common to both Tv and Tvb/dAp neurons), ectopic activation of both Nplp1 and FMRFa was found in different subsets of cells. However, as a third, and cell-type specific, regulator is added, not only does the amount of ectopic, terminal differentiation gene expression increase dramatically, but less evidence of cross-activation of the inappropriate downstream gene was found. This surprising finding reveals that combinatorial misexpression may act in a highly specific and instructive manner, and that these combinatorial codes may be viewed as potent binary switches for cell fate specification. In addition, for both codes, ectopic activation of FMRFa and Nplp1 is observed in neurons throughout the VNC and brain, and traverses many developmental boundaries, such as anteroposterior, dorsoventral, and mediolateral boundaries (Baumgardt, 2007).
This study has identified a sequential regulatory cascade of combinatorial coding that acts to specify two unique neuronal cell fates during Drosophila CNS development. Combined with previous studies, the findings provide the following model for Ap cluster generation and specification. Neuroblast 5-6 forms in the first wave of neuroblast delamination, at late stage 8, and generates a mixed lineage of glia and neurons. At stage 13, Col expression is turned on specifically in thoracic NB 5-6, in two subsequent ganglion mother cells (GMCs) at stages 13/14, and in the four Ap cluster neurons generated from these GMCs, during stages 14/15. The birth order of the four Ap cluster neurons has not been resolved, and the sibling relationship of Tv and Tvb is thus unclear. When Ap neurons are born, col activates ap and eya, whereas sqz and dac are activated by unknown regulator(s). sqz appears to play an early postmitotic role, apparently acting in the Notch pathway, to ensure proper Ap cluster composition, and sqz mutants display both additional Ap cluster cells (in T1) and additional Tvb cells (in T1-T3). col is rapidly down-regulated from three Ap neurons, but remains expressed in the Tvb, where it acts with ap and eya to activate dimm at stage 16. At late embryogenesis, col acts with ap, eya, and dimm to activate Nplp1 and DopR in Tvb. In the Tv neuron, ap and eya act, apparently independently of col, to activate dimm expression. In the Tv neuron, eya furthermore plays a role in setting up competence to respond to the BMP signal. At stage 17, the Tv axon reaches the dorsal neurohemal organ (DNH) and receives the BMP ligand Gbb that activates the Wit receptor, and then triggers activation of the BMP pathway in the Tv neuron. At 18 h AEL, ap, eya, sqz, dac, dimm, and BMP signaling cooperate to activate FMRFa in the Tv neuron. In addition to their roles in neuropeptide regulation and BMP signaling, both ap and eya act to ensure proper axon pathfinding of Tv neurons (eya), as well as Tvb and dAp neurons (ap and eya). The role that col may play more directly in axon pathfinding has not been resolved due to the fact that the expression of the appropriate axonal markers (apGAL4, Nplp1, DopR, and FMRFa) is completely absent in col mutants. Given the complexity of axon pathfinding, it is anticipated that several other regulators are yet to be identified before a combinatorial code for 'Tv-type' or 'Tvb-type' axonal pathfinding is deciphered. Indeed, no evidence was found that combinatorial misexpression of the abovementioned regulators can dictate axonal projections, because ectopic Nplp1 or FMRFa axons are following many different routes in the VNC. Finally, dimm also plays additional roles to those described above and is necessary for the expression of neuropeptide-processing enzymes in peptidergic neurons. Importantly, dimm acts independently to control expression of the neuroamidase gene PHM in the Tv and Tvb neurons, and dimm is sufficient to activate PHM in most, if not all, VNC neurons. Thus, during the specification and differentiation of the Ap neurons, there exists a remarkable diversity in the division of labor between the identified regulators, with most of them participating in more than one, but never all, of the identified events (Baumgardt, 2007).
This study has identified a multistep process for specifying the Tv and Tvb cell fates. What would be the purpose of this type of sequential combinatorial coding? In other model systems with higher genetic resolution, such as Escherichia coli and yeast, extensive genetic analysis has revealed that this type of sequential gene regulation is quite common, and a recent study in C. elegans suggests it may also function during neuronal specification. These regulatory nodes, also known as feedforward loops (FFL), have been shown to ensure fidelity in gene regulation. For instance, in a simple FFL in which gene A regulates gene B, and A/B then co-operate to regulate C, activation of C depends upon prolonged A expression such that A/B will have time to activate C. If A is only active in a short burst, B may be activated, but C is not, because A/B never co-express for a sufficiently prolonged period of time. The role of col during Ap cluster specification provides an excellent example of a FFL used during neuronal cell fate specification. col is expressed in all four Ap cluster cells and plays an early role in activating ap and Eya, but is only maintained in Tvb, where it plays a later role in activating first dimm, then Nplp1. Maintained expression of col in all four Ap cluster neurons, by driving col expression from apGAL4, leads to activation of the Tvb program also in the Tv neuron. Thus, a burst of col expression has a different informational value than persistent col expression -- general Ap cluster specification versus Tvb specification (Baumgardt, 2007).
Misexpression of each of the two identified combinatorial codes leads to striking ectopic activation of the Nplp1 and FMRFa genes, and two particular aspects of these findings were surprising. First, the global potency of these codes: co-misexpression triggers ectopic FMRFa of Nplp1 in a number of neurons, located in many different anteroposterior, dorsoventral, and mediolateral positions. Thus, it would appear that early regulators mainly act to ensure proper combinatorial coding in each neuron, and play a minor role in restricting cell fate by limiting the cell's competence. Once the proper code is in place, the cell fate specification program will be carried out irrespective of the history of the cell. Second, the striking binary effect of these codes is noteworthy: the change of one single player in a code completely alters target gene choice. For instance, misexpression of ap/dimm/dac leads almost exclusively to strong FMRFa activation, but simply replacing dac with col leads to almost exclusively Nplp1 activation. Thus, it appears that more complete codes not only have great potency, but also have great specificity (Baumgardt, 2007).
Col shows a very dynamic expression pattern in the VNC, exemplified in NB 5-6 by the expression in the neuroblast, in two GMCs, in all four Ap cluster neurons, and finally only in Tvb. This poses three obvious questions: what activates col in the neuroblast, what shuts it down in three of the Ap cluster neurons, and finally, what maintains col in Tvb? As for the activation of col in the late 5-6 neuroblast, row 5 neuroblast determinants, thoracic determinants, and late temporal determinants are obvious candidates. Indeed, current work has identified input from a number of such upstream regulators. It is less clear why col expression is lost from three Ap cluster cells and maintained in Tvb. It is possible that the initial expression in all four Ap cluster cells merely reflects residual expression, as an effect of the activation by earlier determinants acting in the neuroblast. But why is col then maintained in Tvb, and similarly, what maintains eya and ap in all four Ap cluster cells? One simple solution would be autoregulation of each gene. But surprisingly, there is no evidence of autoregulation of col. In addition, no clear evidence was found of cross-regulation between col, ap, or eya, at least not during embryonic stages. Thus, it seems likely that other mechanisms, either unidentified regulators or, perhaps, epigenetic mechanisms, act to ensure the continual expression of these regulators during larval (and perhaps adult) life (Baumgardt, 2007).
Arousal is fundamental to many behaviors, but whether it is unitary or whether there are different types of behavior-specific arousal has not been clear. In Drosophila, dopamine promotes sleep-wake arousal. However, there is conflicting evidence regarding its influence on environmentally stimulated arousal. This study shows that loss-of-function mutations in the D1 dopamine receptor DopR enhance repetitive startle-induced arousal while decreasing sleep-wake arousal (i.e., increasing sleep). These two types of arousal are also inversely influenced by cocaine, whose effects in each case are opposite to, and abrogated by, the DopR mutation. Selective restoration of DopR function in the central complex rescues the enhanced stimulated arousal but not the increased sleep phenotype of DopR mutants. These data provide evidence for at least two different forms of arousal, which are independently regulated by dopamine in opposite directions, via distinct neural circuits (Lebestky, 2009).
'Arousal', a state characterized by increased activity, sensitivity to sensory stimuli, and certain patterns of brain activity, accompanies many different behaviors, including circadian rhythms, escape, aggression, courtship, and emotional responses in higher vertebrates. A key unanswered question is whether arousal is a unidimensional, generalized state. Biogenic amines, such as dopamine (DA), norepinephrine (NE), serotonin (5-HT), and histamine, as well as cholinergic systems, have all been implicated in arousal in numerous behavioral settings. However, it is not clear whether these different neuromodulators act on a common 'generalized arousal' pathway or rather control distinct arousal pathways or circuits that independently regulate different behaviors. Resolving this issue requires identifying the receptors and circuits on which these neuromodulators act, in different behavioral settings of arousal (Lebestky, 2009).
Most studies of arousal in Drosophila have focused on locomotor activity reflecting sleep-wake transitions, a form of 'endogenously generated' arousal. Several lines of evidence point to a role for DA in enhancing this form of arousal in Drosophila. Drug-feeding experiments, as well as genetic silencing of dopaminergic neurons, have indicated that DA promotes waking during the subjective night phase of the circadian cycle. Similar conclusions were drawn from studying mutations in the Drosophila DA transporter (dDAT). Consistent with these data, overexpression of the vesicular monoamine transporter (dVMAT-A), promoted hyperactivity in this species, as did activation of DA neurons in quiescent flies (Lebestky, 2009).
Evidence regarding the nature of DA effects on 'exogenously generated' or environmentally stimulated arousal, such as that elicited by startle, is less consistent. Classical genetic studies and quantitative trait locus (QTL) analyses have suggested that differences in DA levels may underlie genetic variation in startle-induced locomotor activity (see Carbone, 2006 and Jordan, 2006). Fmn (dDAT; Dopamine transporter) mutants displayed hyperactivity in response to mechanical shocks, implying a positive-acting role for DA in controlling environmentally induced arousal (Kume, 2005). In contrast, other data imply a negative-acting role for DA in controlling stimulated arousal. Mutants in Tyr-1, which exhibit a reduction in dopamine levels, show an increase in stimulated but not spontaneous levels of locomotor activity. Genetic inhibition of tyrosine hydroxylase-expressing neurons caused hyperactivity in response to mechanical startle (Friggi-Grelin, 2003). Finally, transient activation of DA neurons in hyperactive flies inhibited locomotion (Lima, 2005). Whether these differing results reflect differences in behavioral assays, the involvement of different types of DA receptors, or an 'inverted U'-like dosage sensitivity to DA (Birman, 2005), is unclear (Lebestky, 2009).
This investigation has developed a novel behavioral paradigm for environmentally stimulated arousal, using repetitive mechanical startle as a stimulus, and a screen was carried out for mutations that potentiate this response. One such mutation is a hypomorphic allele of the D1 receptor ortholog, DopR. This same mutation caused decreased spontaneous activity during the night phase of the circadian cycle, due to increased rest bout duration. In both assays, cocaine influenced behavior in the opposite direction as the DopR mutation, and the effect of cocaine was abolished in DopR mutant flies, supporting the idea that DA inversely regulates these two forms of arousal. Genetic rescue experiments, using Gal4 drivers with restricted CNS expression, indicate that these independent and opposite influences of DopR are exerted in different neural circuits. These data suggest the existence of different types of arousal states mediated by distinct neural circuits in Drosophila, which can be oppositely regulated by DA acting via the same receptor subtype (Lebestky, 2009).
Previous studies of arousal in Drosophila have focused on sleep-wake transitions, a form of 'endogenous' arousal. This study has introduced and characterized a quantitative behavioral assay for repetitive startle-induced hyperactivity, which displays properties consistent with an environmentally triggered ('exogenous') arousal state. A screen was conducted for mutations affecting this behavior, the phenotype of one such mutation (DopR) was analyzed, and the neural substrates of its action was mapped by cell-specific genetic rescue experiments. The results reveal that DopR independently regulates Repetitive Startle-induced Hyperactivity (ReSH) and sleep in opposite directions by acting on distinct neural substrates. Negative regulation of the ReSH response requires DopR function in the ellipsoid body (EB) of the central complex (CC), while positive regulation of waking reflects a function in other populations of neurons, including PDF-expressing circadian pacemaker cells. Both of these functions, moreover, are independent of the function of DopR in learning and memory, which is required in the mushroom body. These data suggest that ReSH behavior and sleep-wake transitions reflect distinct forms of arousal that are genetically, anatomically, and behaviorally separable. This conclusion is consistent with earlier suggestions, based on classical genetic studies, that spontaneous and environmentally stimulated locomotor activity reflect 'distinct behavioral systems' in Drosophila (Lebestky, 2009).
Several lines of evidence suggest that ReSH behavior represents a form of environmentally stimulated arousal. First, hyperactivity is an evolutionarily conserved expression of increased arousal. Although not all arousal is necessarily expressed as hyperactivity, electrophysiological studies indicate that mechanical startle, the type of stimulus used in this study, evokes increases in 20-30 Hz and 80-90 Hz brain activity, which have been suggested to reflect a neural correlate of arousal in flies (Nitz, 2002; van Swinderen, 2004). Second, ReSH does not immediately dissipate following termination of the stimulus, as would be expected for a simple reflexive stimulus-response behavior, but rather persists for an extended period of time, suggesting that it reflects a change in internal state. Third, this state, like arousal, is scalable: more puffs, or more intense puffs, produce a stronger and/or longer-lasting state of hyperactivity. Fourth, this state exhibits sensitization: even after overt locomotor activity has recovered to prepuff levels, flies remain hypersensitive to a single puff for several minutes. Fifth, this sensitization state generalizes to a startle stimulus of at least one other sensory modality (olfactory). In Aplysia, sensitization of the gill/siphon withdrawal reflex has been likened to behavioral arousal. Taken together, these features strongly suggest that ReSH represents an example of environmentally stimulated ('exogenous') arousal in Drosophila (Lebestky, 2009).
DopR mutant flies exhibited longer rest periods during their subjective night phase, suggesting that DopR normally promotes sleep-wake transitions. These data are consistent with earlier studies indicating that DA promotes arousal by inhibiting sleep (Andretic, 2005, Kume, 2005; Wu, 2008). In contrast, prior evidence regarding the role of DA in startle-induced arousal is conflicting. Some studies have suggested that DA negatively regulates locomotor reactivity to environmental stimuli, consistent with the current observations, while others have suggested that it positively regulates this response. Even within the same study, light-stimulated activation of TH+ neurons produced opposite effects on locomotion, depending on the prestimulus level of locomotor activity (Lima, 2005; Lebestky, 2009 and references therein).
This study has found that DA and DopR negatively regulate environmentally stimulated arousal: the DopR mutation enhanced the ReSH response, while cocaine suppressed it. Furthermore, the effect of cocaine in the ReSH assay was eliminated in the DopR mutant but could be rescued by Gal4-driven DopR expression, confirming that the effect of the drug is mediated by DA. Taken together, these results reconcile apparently conflicting data on the role of DA in 'arousal' in Drosophila by identifying two different forms of arousal -- repetitive startle-induced arousal and sleep-wake arousal -- that are regulated by DA in an inverse manner (Lebestky, 2009).
The finding that DopR negatively regulates one form of environmentally stimulated arousal leaves open the question of whether this is true for all types of exogenous arousing stimuli. The 'sign' of the influence of DA on exogenously generated arousal states may vary depending on the type or strength of the stimulus used, the initial state of the system prior to exposure to the arousing stimulus (Birman, 2005; Lima and Miesenbock, 2005), or the precise neural circuitry that is engaged. Future studies using arousing stimuli of different sensory modalities or associated with different behaviors should shed light on this question (Lebestky, 2009).
Several lines of evidence suggest that endogenous DopR likely acts in the ellipsoid body (EB) of the central complex (CC) to regulate repetitive startle-induced arousal. First, multiple Gal4 lines that drive expression in the EB rescued the ReSH phenotype of DopR mutants. Second, endogenous DopR is expressed in EB neurons, including those in which the rescuing Gal4 drivers are expressed. Third, the domain of DopR expression in the EB overlaps the varicosities of TH+ fibers. In an independent study of dopaminergic inputs required for regulating EtOH-stimulated hyperactivity TH+ neurons were identified that are a likely source of these projections to the EB. Fourth, rescue of the ReSH phenotype is associated with re-expression of DopR in EB neurons. Finally, rescue is observed using conditional DopR expression in adults. Taken together, these data argue that rescue of the ReSH phenotype by the Gal4 lines tested reflects their common expression in the EB and that this is a normal site of DopR action in adult flies (Lebestky, 2009).
A requirement for DopR in the EB in regulating ReSH behavior is consistent with the fact that the CC is involved in the control of walking activity. However, the mushroom body has also been implicated in the control of locomotor behavior, and DopR is strongly expressed in this structure as well. Rescue data argue against the MB and in favor of the CC as a neural substrate for the ReSH phenotype of DopR mutants. Unexpectedly, the nocturnal hypoactivity phenotype of DopR mutants was not rescued by restoration of DopR expression to the CC. Thus, not all locomotor activity phenotypes of the DopR mutant necessarily reflect a function for the gene in the CC (Lebestky, 2009).
Interestingly, Gal4 line c547 expresses in R2/R4m neurons of the EB, while lines 189y and c761 express in R3 neurons, yet both rescued the ReSH phenotype of DopR mutants. Similar results have been obtained in experiments to rescue the deficit in ethanol-induced behavior exhibited by the DopR mutant. Double-labeling experiments suggest that endogenous DopR is expressed in all of these EB neuronal subpopulations. Perhaps the receptor functions in parallel or in series in R4m and R3 neurons, so that restoration of DopR expression in either population can rescue the ReSH phenotype. Whether these DopR-expressing EB subpopulations are synaptically interconnected is an interesting question for future investigation (Lebestky, 2009).
Despite its power as a system for studying neural development, function, and behavior, Drosophila has not been extensively used in affective neuroscience, in part due to uncertainty about whether this insect exhibits emotion-like states or behaviors. Increased arousal is a key component of many emotional or affective behaviors. The data presented in this study indicate that Drosophila can express a persistent arousal state in response to repetitive stress. ReSH behavior exhibits several features that distinguish it from simple, reflexive stimulus-response behaviors: scalability, persistence following stimulus termination, and sensitization. In addition, the observation that mechanical trauma promotes release from Drosophila of an odorant that repels other flies suggests that the arousal state underlying ReSH behavior may have a negative 'affective valence' as well. These considerations, taken together with the fact that ReSH is influenced by genetic and pharmacologic manipulations of DA, a biogenic amine implicated in emotional behavior in humans, support the idea that the ReSH response may represent a primitive 'emotion-like' behavior in Drosophila (Lebestky, 2009).
The phenotype of DopR flies is reminiscent of attention-deficit hyperactivity disorder (ADHD), an affective disorder linked to dopamine, whose symptoms include hyper-reactivity to environmental stimuli. If humans, like flies, have distinct circuits for different forms of arousal, then the current data suggest that ADHD may specifically involve dopaminergic dysfunction in those circuits mediating environmentally stimulated, rather than endogenous (sleep-wake), arousal. Given that DA negatively regulates environmentally stimulated arousal circuits in Drosophila, such a view would be consistent with the fact that treatment with drugs that increase synaptic levels of DA, such as methylphenidate (ritalin), can ameliorate symptoms of ADHD (Lebestky, 2009).
In further support of this suggestion, in mammals, dopamine D1 receptors in the prefrontal cortex (PFC) have been proposed to negatively regulate activity, while D1 receptors in the nucleus accumbens are thought to promote sleep-wake transitions. Numerous studies have linked dopaminergic dysfunction in the PFC to ADHD. While most research has focused on the role of the PFC in attention and cognition, rather than in environmentally stimulated arousal per se, dysfunction of PFC circuits mediating phasic DA release has been invoked to explain behavioral hypersensitivity to environmental stimuli in ADHD (Sikstrom, 2007). This view of ADHD as a disorder of circuits mediating environmentally stimulated arousal suggests that further study of such circuits in humans and in vertebrate animal models, as well as in Drosophila, may improve understanding of this disorder and ultimately lead to improved therapeutics (Lebestky, 2009).
To identify dDA1 mutants, multiple fly lines with lesions that are known to map at the chromosomal location 88A where the dDA1 gene resides were surveyed. Two lines showed abnormal dDA1 immunoreactivities in the brain. One of them is the inversion line In(3LR)234, which has the break points at 67D and 88A-88B. The other is f02676 containing the transposable element piggyBac inserted at the fimmunoreactivitiest intron in the dDA1 locus. dDA1 is highly enriched in the MB lobes, the central complex, a few scattered cells in the brain, and the Apterous-positive cells in the thoracico-abdominal ganglion (Kim, 2003; Park, 2004). Both In(3LR)234 and f02676 have negligible dDA1 immunoreactivities in the MB and the central complex but intact immunoreactivities in the scattered and Apterous-positive cells. Consistently, full-length dDA1 transcripts were detected in both lines by RT-PCR. Thus, In(3LR)234 and f02676 appear to have lesions in the regulatory sequence for tissue-specific dDA1 expression, representing hypomorphic dDA1 alleles, and are designated as dumb1 [D1(uno) in mushroom bodies] and dumb2, respectively (Kim, 2007b).
The observations that dDA1 is concentrated in the MB neuropil and can activate the cAMP pathway (Sugamori, 1995; Kim, 2003) prompted an investigation of the role of dDA1 in olfactory conditioning. When subjected to aversive conditioning using odorants octanol (OCT) and benzaldehyde (BA) as conditioned stimuli and electric shock as a US, dumb1 homozygous mutants showed severely impaired performance immediately after training. Performance of dumb1 did not decline at 1 h after training, suggesting that dumb1 is defective in learning rather than memory. dumb1 has two break points caused by inversion. Thus, to investigate the lesion accountable for poor performance of dumb1, two deficiency lines were used, Df(3L)AC1 and Df(3R)su(Hw)7, having deletion between chromosomes 67A2 and 67D13 and chromosomes 88A9 and 88B2, respectively, which include each break point. Similar to dumb1, dumb1/Df(3R)su(Hw)7 trans-heterozygous mutants exhibited poor performance immediately or 1 h after training. In contrast, performance of dumb1/Df(3L)AC1 was comparable to that of Canton-S and dumb1/+ or Df(3R)su(Hw)7/+ heterozygous flies immediately after training. These data indicate that the lesion in chromosome 88A is responsible for poor learning of dumb1 mutants. The flies heterozygous for both deficiency chromosomes had slightly lower performance scores compared with those of Canton-S and dumb1/+ at 1 h after training. This could be attributable to putative memory genes in the deleted chromosomes (Kim, 2007b).
It was next asked whether the dumb1 phenotype is linked to the lesion in dDA1 by examining the independent dumb allele dumb2 and dumb1/dumb2 trans-heterozygous mutants in aversive conditioning. Like dumb1, both genotypes had negligible performance scores immediately after or 1 h after training, supporting the potential role of dDA1 in punishment-mediated olfactory learning. dumb1 and dumb2 heterozygous flies exhibited normal performance; thus, a single copy of dDA1 may be sufficient for mediating this process (Kim, 2007b).
To test whether dumb mutants could learn better with different conditioned stimuli, other odorants were used in electric shock-mediated olfactory conditioning. When trained with ethyl acetate (EA) and isoamyl acetate (IAA) as conditioned stimuli, dumb1 mutants also displayed severely impaired learning. This suggests that dDA1 is involved in aversive learning induced by diverse odor inputs. The impaired performance of dumb mutants is not attributable to anomalous sensory modalities because all dumb alleles and the control Canton-S and w1118 flies showed comparable avoidance of the CS odors and electric shock presented at two different concentrations or intensities, respectively. Thus, poor learning of dumb mutants is likely attributable to their inability to associate CS+ with US (Kim, 2007b).
Synaptic output of dopamine neurons has been shown to be required during training for aversive learning (Schwaerzel, 2003), implicating the similar requirement of dDA1 at the time of learning. To test this, the pan-neuronal driver Elav-GAL4 and GAL80ts, which allows the temporal control of GAL4 activities, was used. GAL80 binds to GAL4 to sequester it from activating upstream activating sequence (UAS). The temperature-sensitive GAL80ts can no longer bind to GAL4 at 30°C, allowing it to act on UAS to induce downstream gene expression. The piggyBac inserted at the first intron of the dDA1 gene in dumb2 has UAS. Although the piggyBac insertion itself interferes with endogenous dDA1 expression in dumb2, UAS in piggyBac, after binding to GAL4, may induce dDA1 transcription from the second exon containing the 5' untranslated sequence and the start codon. Thus, dumb2 was crossed with dumb1 carrying Elav-GAL4 and GAL80ts to generate Elav-GAL4,GAL80ts/+;dumb1/dumb2 flies. The Elav-GAL4,GAL80ts/+;dumb1/dumb2 kept at room temperature did not have any detectable dDA1 induction; however, when the flies were reared at 30°C for 3 d, conspicuous dDA1 IR was visible in the MB lobes and pedunculi, the central complex, and other brain areas including antennal lobes. Whereas Elav-GAL4 is expressed in all neurons, membrane-bound GFP reporters driven by Elav-GAL4 are enriched in certain brain areas including the aforementioned structures. Therefore, the temporal manipulation of GAL80ts and Elav-GAL4 activities was effective in restricting dDA1 expression at the adult stage in dumb mutants (Kim, 2007b).
When Elav-GAL4,GAL80ts/+;dumb1/dumb2 flies reared at room temperature were subjected to electric shock-mediated conditioning, they showed poor learning; however, their performance was dramatically improved after temperature shift to 30°C. The performance score of Elav-GAL4,GAL80ts/+;dumb1/dumb2 with the restored dDA1 expression was slightly lower than that of Canton-S; nonetheless, it was not significantly different from that of Canton-S treated with the same temperature shift but was different from that of uninduced Elav-GAL4,GAL80ts/+;dumb1/dumb2 (p = 0.0009). Therefore, dDA1 is required in the adult neurons, presumably at the time of training, for aversive memory formation. Notably, the same manipulation in the dumb2 heterozygous background (Elav-GAL4,GAL80ts/+;dumb2/+) did not alter the performance scores after brief training (2 pulses of electric shock) or regular training (12 pulses of electric shock). This indicates that the ectopically expressed dDA1 has a negligible effect on normal learning of the heterozygous flies and thus unlikely contribute to the reinstated performance of dumb1/dumb2 mutants (Kim, 2007b).
It was next asked whether the learning phenotype of dumb mutants is attributable to deficient dDA1 function in the MB rather than in the central complex or other neurons. MB247-GAL4 contains 247 bp of dMEF2 regulatory sequence that allows GAL4 expression rather specifically in a subset of the MB neurons projecting to the α/ß lobes and the gamma lobes, but not the α'/ß' lobes. When MB247-GAL4/UAS-GFP in the wild-type background was stained with the dDA1 antibody, the GFP-labeled (thus MB247-GAL4-expressing) MB neurons were positive for dDA1 immunoreactivities although the relative intensities of GFP and dDA1 signals varied in the different MB lobes. Thus, MB247-GAL4 was used to reinstate dDA1 expression in the MB of dumb mutants. After staining with anti-dDA1 antibody, dDA1 expression was apparent in the MB lobes and pedunculi but not in other neural structures of MB247-GAL4/+;dumb1/dumb2. When subjected to electric shock-mediated conditioning, MB247-GAL4/+;dumb1/dumb2 or MB247-GAL4/+;dumb2/dumb2 had the learning scores comparable to those of Canton-S. Moreover, fully reinstated performance was observed in MB247-GAL4/+;GAL80ts,dumb2/dumb1 reared at 30°C for 3 d before training but not in MB247-GAL4/+;GAL80ts,dumb2/dumb1 reared at room temperature. Therefore, dDA1 expressed only in the subset of the adult MB neurons is necessary and sufficient to rescue the dumb mutant's impaired learning, indicating the indispensable role of the MB dDA1 in aversive memory formation (Kim, 2007b).
Dopamine is crucial in appetitive learning in mammals; however, the previous study (Schwaerzel, 2003) of TH-GAL4/UAS-Shits flies suggests that this is not the case in Drosophila. To investigate this further, dumb mutants were tested in sugar-mediated olfactory conditioning. Surprisingly, both dumb1 and dumb2 mutants exhibited poor performance immediately after training. Although dumb mutants' performance in appetitive learning was not as severely impaired as in aversive conditioning, it was significantly different from that of Canton-S. As in electric shock-mediated conditioning, dumb mutants' performance did not decline at 1 h after training, indicating a crucial role of dDA1 in acquisition, as opposed to short-term memory, of appetitive conditioning. Moreover, dumb2 homozygous or dumb1/dumb2 trans-heterozygous mutants carrying MB247-GAL4 displayed fully reinstated learning in sugar-mediated conditioning. These data indicate that dDA1 is required in the same subset of the MB neurons for aversive and appetitive learning (Kim, 2007b).
The arousing and motor-activating effects of psychostimulants are mediated by multiple systems. In Drosophila, dopaminergic transmission is involved in mediating the arousing effects of methamphetamine, although the neuronal mechanisms of caffeine (CAFF)-induced wakefulness remain unexplored. This study shows that in Drosophila, as in mammals, the wake-promoting effect of CAFF involves both the adenosinergic and dopaminergic systems. By measuring behavioral responses in mutant and transgenic flies exposed to different drug-feeding regimens, it was shown that CAFF-induced wakefulness requires the Drosophila D1 dopamine receptor (dDA1) in the mushroom bodies. In WT flies, CAFF exposure leads to downregulation of dDA1 expression, whereas the transgenic overexpression of dDA1 leads to CAFF resistance. The wake-promoting effects of methamphetamine require a functional dopamine transporter as well as the dDA1, and they engage brain areas in addition to the mushroom bodies (Andretic, 2008).
In Drosophila, the wake-promoting action of the adenosinergic antagonist CAFF is mediated through the dDA1 receptor. Genetic manipulations of the dDA1 receptor, as in dumb1 mutants, or overexpression of dDA1 in the MBs of transgenic flies both lead to resistance to the arousing effects of CAFF. These apparently paradoxical findings can be reconciled if the CAFF response requires downregulation of the dDA1 receptor in the MBs within a certain range. In support of this model, the dDA1 mRNA transcript in WT flies is downregulated in response to either short-term exposure (STE) or long-term exposure (LTE) to CAFF, the dDA1 product is already reduced to a negligible level in the MBs (and most other regions) of the dumb mutant, and excess expression of the dDA1 receptor in the MBs produces CAFF resistance, suggesting that levels in these flies cannot be sufficiently downregulated (Andretic, 2008).
MBs are thought to play a role in the control of arousal. MBs have an inhibitory effect on locomotor activity but a stimulatory effect toward sleep. Genetic and transgenic manipulations of MBs, which lead to decreasing amounts of sleep, are often accompanied by a shortening of sleep episodes, and can thus be explained by a premature arousing signal (Andretic, 2008).
The observation that the doses of CAFF that decrease sleep also increase motor activity is similar to the effect of CAFF in vertebrates. In mammals, the antagonistic effect of CAFF on adenosine receptors located on dopaminergic neurons leads to increased release (see Solinas, 2002). A similar mechanism might be operating in flies, based on the correlation between CAFF responsiveness and functional dDA1 receptors in MBs as well as on the motor-activating effects of dopamine (Andretic, 2008).
Although CAFF and methamphetamine (METH) lead to similar wake-promoting and motor-activating effects, the neuronal mechanisms underlying responses to these drugs are only partially overlapping. Both responses require a functional dDA1 receptor, particularly in the MBs, but METH does not lead to uniform downregulation of dDA1 in the brain, although it is conceivable that downregulation might occur in a limited area of the brain outside of the MB. Although CAFF-induced wakefulness involves dDA1 downregulation in MBs, METH-induced wakefulness could involve a selective increase of dDA1 in MBs, whereas dDA1 expression might be unchanged or even decreased in other brain areas. Such an interpretation is supported by the lack of significant modulation of dDA1 transcript in samples obtained from the entire brain of METH-fed flies as well as weaker rescue of METH response when dDA1 was expressed in the entire brain vs. the MBs. When dDA1 expression is restricted only to areas outside of the MBs, METH response is at least as great as in panneural (elav) expression, further suggesting the possibility of antagonism between MBs and other areas for this effect. Another DA receptor, damb, which is specific to the MBs, is not relevant to these responses. It does not show altered regulation in response to CAFF or METH in WT or dumb mutants, and dDA1 expression alone or in combination with CAFF or METH is not altered in damb mutants (Andretic, 2008).
Altogether, these findings suggest a model in which the arousing and motor-activating effects of CAFF are a consequence of its neuromodulatory action on dopaminergic signaling. This is based on similar behavioral responses to CAFF and CPT, a stimulant drug, in Drosophila, which implies that the arousing properties of CAFF involve close interaction between the adenosine and dopamine systems, as they do in mammals. Presynaptically, CAFF can increase dopamine release by antagonizing adenosine receptors on dopaminergic neurons. Resistance to the wake-promoting effect of the A1R antagonist in dumb1 mutants and decreased expression of dDA1 in WT flies after CAFF exposure support a model in which the adenosinergic system acts as a neuromodulator of dopaminergic signaling. CAFF acting through AdoR on dopaminergic neurons could stimulate dopamine synthesis or release through protein kinase A dependent mechanisms similar to the A2A receptor in mammals. Postsynaptically, dDA1 receptors located on MB neurons respond homeostatically by downregulating their expression, a common adaptive mechanism in response to excessive stimulation. A related mechanism involving A1-D1 receptor interaction was observed in the rodent brain and implicated in the psychostimulant properties of CAFF. Furthermore, a recent Drosophila report shows increased dopaminergic content concomitant with decreased dDA1 expression in the brains of sleep-deprived flies (Andretic, 2008).
Although the function of sleep still remains a mystery, one line of evidence suggests that synaptic plasticity underlying memory consolidation might occur during sleep. That such a conserved function of sleep might be present in Drosophila has been sparked by a number of recent reports showing overlap between genes [dunce, rutabaga, Clock, Shaker, 5HT1A, and GABA(A)] and anatomical regions (MBs), which regulate sleep as well as learning and memory. The current findings show that dDA1, a receptor with a role in neuronal plasticity in MB-dependent learning tasks, has only a moderate role in regulation of baseline sleep, although it is important in conditions of elevated arousal, such as those induced by stimulants (Andretic, 2008).
Optimal behavioral performance, such as learning, is dependent on adequate levels of arousal. Although psychostimulant exposure increases dopaminergic transmission and increases general arousal, it also influences specific functions related to reward. These multiple roles are preserved in Drosophila, in which mechanisms for arousal and learning converge on the dDA1 receptor, thus ensuring that learning associated with survival occurs in an attentive and awake organism. CAFF and METH effects on dDA1 receptors in MBs could be mimicking, albeit at an elevated level, the increased dopaminergic signaling that otherwise occurs during learning and memory, reflecting the role that dDA1 receptors play in that process (Andretic, 2008).
Extended wakefulness disrupts acquisition of short-term memories in mammals. However, the underlying molecular mechanisms triggered by extended waking and restored by sleep are unknown. Moreover, the neuronal circuits that depend on sleep for optimal learning remain unidentified. In this study learning was evaluated with aversive phototaxic suppression. In this task, flies learn to avoid light that is paired with an aversive stimulus (quinine-humidity). Extensive homology is demonstrated in sleep-deprivation-induced learning impairment between flies and humans. Both 6 hr and 12 hr of sleep deprivation are sufficient to impair learning in Canton-S (Cs) flies. Moreover, learning is impaired at the end of the normal waking day in direct correlation with time spent awake. Mechanistic studies indicate that this task requires intact mushroom bodies (MBs) and requires the dopamine D1-like receptor (dDA1). Importantly, sleep-deprivation-induced learning impairments could be rescued by targeted gene expression of the dDA1 receptor to the MBs. These data provide direct evidence that extended wakefulness disrupts learning in Drosophila. These results demonstrate that it is possible to prevent the effects of sleep deprivation by targeting a single neuronal structure and identify cellular and molecular targets adversely affected by extended waking in a genetically tractable model organism (Seugnet, 2008).
Sleep-deprivation-induced learning impairments were evaluated via an assay that requires flies to inhibit a prepotent attraction toward light. In this task, flies are placed in a T maze and allowed to choose between a lighted and a dark chamber. Filter paper is wetted with 10-1M quinine hydrochloride solution and placed into the lighted chamber such that the quinine and the humidity provide an aversive stimulus. The percentage of times the fly visits the dark vial is tabulated during 16 trials. Flies learn to select the dark alley more frequently over the course of the 16 trials. Learning reaches a maximum during the last four trials of the test and does not improve with additional training. Thus, the performance index is calculated as the percentage of times the fly chooses the dark vial during the last four trials. The assay will be referred to as aversive phototaxic suppression (APS) (Seugnet, 2008).
Flies, like humans, are awake during the day and consolidate their sleep during the night. Canton-S (Cs) flies exhibit a sleep rebound after 3 hr, 6 hr, and 12 hr of sleep deprivation. This study shows that 6 hr and 12 hr of sleep deprivation disrupt learning. Low motivation is an unlikely explanation for the impairment because the time to complete the 16 trials (TCT) was not significantly different from that of controls. Similarly, after sleep deprivation, male flies maintained motivation to court virgin females, another prepotent response, and were not different from controls. Sleep deprivation does not alter the photosensitivity index (PI; percentage of photopositive choices in the T maze in ten trials in the absence of quinine-humidity) nor the quinine-sensitivity index (QSI; time in seconds flies reside on the nonquinine side of a chamber), indicating that the learning impairment is due to sleep loss and not due to sleep-deprivation-induced alterations in sensory thresholds. Indeed, sleep deprivation does not alter photosensitivity when measured over a range of light intensities, nor does it change performance with a fast phototaxis assay. Because flies must climb upward to enter either chamber, the effects of sleep deprivation on geotaxis were evaluated; it was found to be unaffected by sleep deprivation. Importantly, flies that have been selected to prefer climbing downward with gravity learn as well as flies that have been selected to prefer climbing upward against gravity, indicating that geotaxis is not required in this assay. Together, these data indicate that the effects of extended waking are not due to changes in sensory thresholds (Seugnet, 2008).
To determine whether the decrement in performance was the consequence of the stimulus used to keep the animal awake rather than sleep loss per se, several control experiments were conducted. First, flies wee exposed to the perturbations induced by the apparatus for 6 hr between zeitgeber time ZT0 and ZT5:59. Keeping flies awake during this time does not result in subsequent changes in sleep. As expected, exposure to the stimulus in the absence of sleep loss did not result in an additional learning deficit. Currently, all studies that have kept flies awake, including sleep deprivation by gentle handling, have used methods that share common features. To exclude the possibility that these methods impair performance, a novel sleep-deprivation apparatus was invented. The sleep-interrupting device (SLIDE) consists of a thin plastic floor inserted into the tubes underneath flies that can be manipulated like a treadmill. When flies are kept awake with this approach, learning is impaired (Seugnet, 2008).
Sleep fragmentation in humans and rodents is associated with learning impairments. To determine whether sleep fragmentation also deteriorates learning in flies, advantage was taken of the observation that ~10%-15% of Cs flies spontaneously exhibit fragmented sleep while maintaining normal total sleep time. Learning was impaired in flies with fragmented sleep compared to their siblings with consolidated sleep. Thus, even in the absence of mechanical stimulation, sleep fragmentation is associated with learning impairments. Flies with consolidated and fragmented sleep displayed similar control metrics, indicating that they did not differ in sensory thresholds or motoric ability. Importantly, experimentally induced sleep fragmentation impairs learning in otherwise sleep-consolidated flies indicating that sleep fragmentation impairs learning in flies as it does in humans (Seugnet, 2008).
Performance decrements observed in sleep-deprived humans have, at times, been attributed to the intrusion of sleep into periods of waking rather than cognitive impairment per se. Are the learning impairments in flies simply due to high sleep drive? To test this hypothesis, a protocol was designed that allowed separation of the effects of extended wakefulness from increased sleepiness. When flies are deprived of sleep for 22 hr and released into recovery in the evening, sleep rebound is only observed the following morning. If sleep drive impairs performance, flies released into recovery at night should show a deficit when tested the next morning. Flies with high sleep drive exhibit normal performance, indicating that the amount of prior waking rather than interference due to sleepiness is responsible for learning deficits (Seugnet, 2008).
Is a full night of sleep required to restore learning? Performance after sleep deprivation was restored to the baseline level when flies were allowed to nap for 2 hr. In contrast to spontaneous daytime sleep, which is characterized by short sleep bouts, the naps following sleep deprivation resemble nighttime sleep. Thus, as in humans, naps improve learning in flies. Environmental and social factors can alter motivation and temporarily reduce the negative impact of sleep deprivation on performance. For example, sleep-deprived subjects who were given a monetary reward for correct responses were able to maintain performance longer than controls. To evaluate this relationship in flies, the assay was modified by placing a piece of dry filter paper previously soaked in a sucrose solution in the dark vial. Under baseline conditions, the presence of sucrose did not alter performance. However, after 12 hr of sleep deprivation, flies tested with dry sucrose in the dark alley performed as well as flies that had obtained a full nights' sleep. These beneficial effects were lost when sleep deprivation was extended to 36 hr, indicating that deficits cannot be entirely compensated by motivational factors (Seugnet, 2008).
It has been hypothesized that in humans, neurobehavioral deficits accrue when wake time extends beyond a minimal interval measured in hours. In flies, daytime sleep is characterized by short bouts. Interestingly, learning is highest in the morning and declines as the amount of waking accrues during the biological day. Control metrics are similar over the course of the day, indicating that the decrements in performance cannot be explained by circadian modulation of sensory thresholds. However, circadian factors have been shown to influence learning. Thus sleep deprivation was combined with the napping protocols described above to vary the duration of waking at a given circadian time. Three experimental conditions were used, and in each instance performance was evaluated at ZT4. Performance at ZT4 was dependent upon prior wake duration, suggesting that learning is impaired as a function of time spent awake. Interestingly, daytime sleep appears to be less restorative than consolidated sleep observed during the nap. Because performance is reduced by the end of the day, these data suggest that consolidated sleep is required after each waking day to restore optimal learning. Indeed, learning is restored in the evening after 3 hr of spontaneous sleep (ZT12-ZT15) but remains impaired in circadian matched siblings that were kept awake until ZT15 (Seugnet, 2008).
No neural substrate has been identified for APS. A likely candidate is the mushroom bodies (MBs), given their role in many but not all learning and memory tests. MBs play a role in olfactory memory acquisition and play a role in decision making under conflicting situations. The MBs have recently been shown to regulate sleep and inhibitory control. They can be ablated in the fly by feeding larvae hyroxyurea (HU). Although ablation of the MBs disrupts sleep, a minority of HU flies exhibit normal sleep, thereby allowing determination of whether performance is influenced by the MBs independently of sleep time. Learning is impaired in the absence of MBs in all short- and long-sleeping flies; control metrics were unaffected. HU also results in a reduction of antennal lobe size, raising the possibility that the learning impairment may be due to deficits in olfactory processing. However, smell-blind (sbl-1) flies that are olfactory defective perform as well as Cs flies, indicating that olfactory input is not required in this assay (Seugnet, 2008).
To determine whether sleep-deprivation-induced impairments in learning can be explained through alterations in DA signaling, DA levels were evaluated. Whole-head DA levels are significantly elevated after sleep deprivation and are associated with the transcriptional downregulation of the Drosophila dopamine 1-like receptor (dDA1). Downregulation of dDA1 transcripts is also seen in flies with spontaneously fragmented sleep. The pharmacology of DA agonists has been characterized, and these drugs are known to be biologically active in flies. Ritalin, methamphetamine, L-DOPA, and the D1 agonist SKF82958 rescued performance after sleep deprivation; none of these treatments enhanced learning in baseline conditions. Control metrics were unaffected by pharmacologic manipulations. Thus, global enhancement of dopamine signaling overcomes deficits in learning in flies as it does in humans (Seugnet, 2008).
To determine the extent to which DA is involved in this learning assay, additional genetic and pharmacological experiments were conducted. DA levels were reduced by feeding flies the tyrosine hydroxylase (TH) inhibitor 3-iodo L-tyrosine (3IY). Performance was impaired in flies fed 3IY, and this impairment could be rescued by coadministration of L-DOPA. Consistent with previous reports, 3IY consolidated sleep without reducing the intensity of locomotor activity. PI and QSI were unaffected by drug treatment, whereas flies fed 3IY took significantly longer to complete 16 trials. Although TCT was increased in flies fed 3IY, it was not outside the range seen in Cs flies and thus cannot explain the deficit. In addition, disruption of synaptic output from dopaminergic neurons by expression of a temperature-sensitive allele of shibire (UAS-shits1) also impairs learning. Thus, reduction of DA signaling, with either pharmacology or genetics, impairs performance in APS (Seugnet, 2008).
Although a recent study has shown that the dDA1 receptor is important for Pavlovian conditioning, its role in other learning paradigms is unknown. Because the pharmacology of the four Drosophila DA receptors has been investigated, either a D1 (SCH23390) or D2 (eticlopride) antagonist were administered for 2 hr before evaluating learning. SCH23390 and eticlopride have been shown to activate separate behaviors in flies, and although SCH23390 blocks both D1-like receptors (dDA1 and dopamine receptor in mushroom bodies [DAMB]), eticlopride does not. Both D1 and D2 antagonists modified sleep at this dose, indicating they are biologically active. However, only the D1 antagonist disrupted performance. Importantly, the induction of G?s in flies fed the D1 agonist SKF 82958 was blocked by coadministration of the D1 antagonist (Seugnet, 2008).
Because the D1 agonist and D1 antagonist are active at both the dDA1 and the DAMB receptors, learning was evaluated in flies mutant for dDA1. The dDA1 receptor is heavily expressed in MB neuropile and is required for olfactory learning. dumb2 is a hypomorphic allele that reduces dDA1 expression in the mushroom bodies. The P element insertion PL00420 (dumb3) removes most of dDA1 expression in the MBs while inducing ectopic expression in glia and the optic lobes. Both alleles have reduced learning. dumb2 and dumb3 mutants exhibited normal sleep, PI, and QSI, but dumb3 flies had 12% longer TCT. To confirm that this phenotype maps to the dDA1 locus, mutant flies were crossed with flies carrying a deficiency (Df) of the dDA1 locus, Df(3R)red1. Learning was significantly reduced in the resulting dumb2/Df and dumb3/Df flies, indicating that the impairments were due to disruption of dDA1 expression. Finally, the D1 agonist SKF 82958 was administered to mutant flies, and learning was assessed. Performance could not be rescued by the D1 agonist. Because the D1 agonist did not restore learning in either the mutants, it is unlikely that the previous improvement in learning after sleep deprivation was due to nonspecific effects of SKF82958 at other receptors (Seugnet, 2008).
Imaging studies in humans suggest that performance decrements after waking may not be due to global brain impairments and thus may reflect a molecular vulnerability in specific neuronal circuits. To determine whether waking impairs learning by modifying dDA1 globally or in specific circuits, dDA1 was manipulated only in the MBs. The piggyBac inserted into the first intron of the dDA1 gene in the dumb2 mutants contains a UAS that can be used to induce functional dDA1 receptor. The gene-switch system (MBSwitch) was used to avoid potential developmental defects. MB-Switch/+; dumb2/+ flies fed RU486 maintained learning after extended wakefulness, whereas their vehicle-fed siblings were impaired. Interestingly, RU486-treated MB-Switch/+; dumb2/+ had no effect on baseline learning in the absence of sleep loss, and baseline sleep was not altered. As expected, the parental lines learn normally when exposed to RU486 and are impaired after extended waking (Figure 6G). Furthermore, RU486 has no effect on learning in Cs flies, either under baseline condition or during extended waking (Seugnet, 2008).
These data provide direct evidence that extended waking disrupts learning and is amenable to genetic dissection in Drosophila. Importantly, manipulation of dDA1 only in the MBs, which represent ~2% of the total number of neurons in the Drosophila central nervous system, was sufficient to prevent the learning deficits associated with extended waking. These data support the hypothesis that extended waking can deteriorate the function of specific brain areas that are critical for adaptive behavior (Seugnet, 2008). Sleep-deprivation experiments are inherently problematic in that it is frequently difficult to determine whether an observed outcome is because of the lack of sleep or the methods used to keep the organism awake. Thus, several control experiments were conducted to evaluate potential confounding variables. It was found that although learning is disrupted when extended waking is achieved by mechanical stimulation, mechanical stimulation in the absence of sleep loss produced no deficits in learning. Importantly, spontaneous waking and sleep fragmentation impair learning without mechanical stimulation. Together, these data indicate that it is the extended waking per se that disrupts learning (Seugnet, 2008).
In Drosophila, dopaminergic neurons project arborizations to the MB neuropile, where they influence aversive learning. Although a recent study has shown that the dDA1 receptor is important for olfactory conditioning, its role in other learning paradigms is unknown. The current results extend the role of dDA1 receptor beyond olfactory learning. It is worth noting that a role for D1 receptor in short-term memory and response inhibition has been reported in humans, nonhuman primates, and rodents. Previous studies have shown that DA in the MBs plays a role in decision making under conflicting situations and may signal the aversive stimulus to the MBs in olfactory conditioning. Interestingly, flies in the APS also face a conflicting choice between their prepotent attraction toward light and the aversive stimulus. Thus, the modulation of DA signaling observed during extended waking may disrupt performance by multiple mechanisms. Interestingly, children with Attention Deficit Hyperactivity Disorder exhibit both disorganized DA signaling and difficulty with response inhibition. Moreover, sleep problems are highly prevalent in ADHD and, when present, are associated with poorer child outcomes (Seugnet, 2008).
In conclusion, sleep deprivation impairs short-term memory and response inhibition in Drosophila. The data demonstrate that waking is particularly deleterious for DA circuits that are crucial for maintaining adaptive behavior. Because optimal performance can only occur within a narrow range of DA signaling and DA signaling is easily disrupted by waking, it is proposed that an important role of sleep may be to restore DA homeostasis. Nonetheless, it is likely that sleep loss impacts the brain by altering a number of molecular pathways. Together, these experiments pave the way for the identification of the underlying molecular mechanisms (Seugnet, 2008).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila Dopamine receptor
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Andretic, R. and Hirsh, J. (2000). Circadian modulation of dopamine receptor responsiveness in Drosophila melanogaster. Proc. Natl. Acad. Sci. 97: 1873-1878. Medline abstract: 10677549
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Baumgardt, M., Miguel-Aliaga, I., Karlsson, D., Ekman, H. and Thor, S. (2007). Specification of neuronal identities by feedforward combinatorial coding. PLoS Biol. 5(2): e37. Medline abstract: 17298176
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Budnik, V. and White, K. (1987) Genetic dissection of dopamine and serotonin synthesis in the nervous system of Drosophila melanogaster. J. Neurogenet. 4: 309-314. Medline abstract: 3126282
Cannon, C. M., Scannell, C. A. and Palmiter, R. D (2005). Mice lacking dopamine D1 receptors express normal lithium chloride-induced conditioned taste aversion for salt but not sucrose. Eur. J. Neurosci. 21: 2600-2604. Medline abstract: 15932618
Carbone, M.A., Jordan, K.W., Lyman, R .F., Harbison, S.T., Leips, J., Morgan, T. J., DeLuca, M., Awadalla, P. and Mackay,T. F., (2006). Phenotypic variation and natural selection at catsup, a pleiotropic quantitative trait gene in Drosophila. Curr. Biol. 16: 912-919. PubMed Citation: 16682353
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Dalley, J. W., et al. (2005). Time-limited modulation of appetitive Pavlovian memory by D1 and NMDA receptors in the nucleus accumbens. Proc. Natl. Acad. Sci. 102: 6189-6194. Medline abstract: 15833811
El-Ghundi, M., O'Dowd, B. F. and George, S. R. (2001). Prolonged fear responses in mice lacking dopamine D1 receptor. Brain Res. 892: 86-93. Medline abstract: 11172752
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Gotzes, F. and Baumann, A. (1996). Functional properties of Drosophila dopamine D1-receptors are not altered by the size of the N-terminus. Biochem. biophys. Res. Commun. 222(1): 121-126. Medline abstract: 8630055
Guarraci, F. A., Frohardt, R. J. and Kapp, B. S. (1999). Amygdaloid D1 dopamine receptor involvement in Pavlovian fear conditioning. Brain Res 827: 28-40. Medline abstract: 10320690
Han, K. A., Millar, N. S., Grotewiel, M. S. and Davis, R. L. (1996). DAMB, a novel dopamine receptor expressed specifically in Drosophila mushroom bodies. Neuron 16: 1127-1135. Medline abstract: 8663989
Han, K.-A., Millar, N. S. and Davis, R. L. (1998) A novel octopamine receptor with preferential expression in Drosophila mushroom bodies. J Neurosci 18: 3650-3658. Medline abstract: 9570796
Holmes, A., et al. (2001). Behavioral characterization of dopamine D5 receptor null mutant mice. Behav Neurosci 115: 1129-1144. Medline abstract: 11584926
Hsouna, A., Lawal, H. O., Izevbaye, I., Hsu, T. and O'Donnell, J. M. (2007). Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis. Dev. Biol. 308(1): 30-43. Medline abstract: 17585895
Jordan, K.W., Morgan, T. J. and Mackay, T. F., (2006). Quantitative trait loci for locomotor behavior in Drosophila melanogaster. Genetics 174: 271-284. PubMed Citation: 16783013
Kehren, V. and Baumann, A. (2005). Characterization of the 5' regulatory region of the Drosophila Dmdop1 dopamine receptor-gene. Arch. Insect Biochem. Physiol. 59(3): 118-31 . Medline abstract: 15986377
Kim, O. J., et al. (2004). The role of phosphorylation in D1 dopamine receptor desensitization: evidence for a novel mechanism of arrestin association. J. Biol. Chem. 279(9): 7999-8010. Medline abstract: 14660631
Kim, Y. C., Lee, H. G., Seong, C. S. and Han, K. A. (2003). Expression of a D1 dopamine receptor dDA1/DmDOP1 in the central nervous system of Drosophila melanogaster. Gene Expr. Patterns 3: 237-245. Medline abstract: 12711555
Kim, Y. C., Lee, H. G. and Han, K. A. (2007a). Classical reward conditioning in Drosophila melanogaster. Genes Brain Behav. 6: 201-207. Medline abstract: 16740144
Kim, Y. C., Lee, H. G. and Han, K. A. (2007b). D1 dopamine receptor dDA1 is required in the mushroom body neurons for aversive and appetitive learning in Drosophila. J. Neurosci. 27(29): 7640-7647. Medline abstract: 17634358
Kindt, K. S., et al. (2007). Dopamine mediates context-dependent modulation of sensory plasticity in C. elegans. Neuron 55(4): 662-76. Medline abstract: 17698017
Kipnis, J., et al. (2004). Dopamine, through the extracellular signal-regulated kinase pathway, downregulates CD4+CD25+ regulatory T-cell activity: implications for neurodegeneration. J. Neurosci. 24(27): 6133-43. Medline abstract: 15240805
Kong, M., et al. (2007). Regulation of D1 dopamine receptor trafficking and signaling by caveolin-1. Mol Pharmacol. [Epub ahead of print]. Medline abstract: 17699686
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Lebestky, T., et al. (2009). Two different forms of arousal in Drosophila are oppositely regulated by the dopamine D1 receptor ortholog DopR via distinct neural circuits. Neuron 64(4): 522-36. PubMed Citation: 19945394
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Lima, S. Q. and Miesenbock, G., (2005). Remote control of behavior through genetically targeted photostimulation of neurons. Cell 121: 141-152. PubMed Citation: 15820685
Mao, Z, Roman, G., Zong, L. and Davis, R. L (2004). Pharmacogenetic rescue in time and space of the rutabaga memory impairment by using Gene-Switch. Proc. Natl. Acad. Sci. 101: 198-203. Medline abstract: 14684832
McGuire, S. E., Le, P. T., Osborn, A. J., Matsumoto, K. and Davis, R. L. (2003). Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302: 1765-1768. Medline abstract: 14657498
Nitz, D. A., van Swinderen, B., Tononi G. and Greenspan, R. J., (2002). Electrophysiological correlates of rest and activity in Drosophila melanogaster. Curr. Biol. 12: 1934-1940. PubMed Citation: 12445387
Park, D., Han, M., Kim, Y.-C., Han, K.-A. and Taghert, P. H. (2004). Ap-let neurons-a peptidergic circuit potentially controlling ecdysial behavior in Drosophila. Dev. Biol. 269: 95-108. Medline abstract: 15081360
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Reyes, F. D., Mozzachiodi, R., Baxter, D. A. and Byrne, J. H. (2005). Reinforcement in an in vitro analog of appetitive classical conditioning of feeding behavior in Aplysia: blockade by a dopamine antagonist. Learn Mem 12: 216-220. Medline abstract: 15930499
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Schwaerzel, M., Monastirioti, M., Scholz, H., Friggi-Grelin, F., Birman, S. and Heisenberg, M. (2003). Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila. J. Neurosci. 23: 10495-10502. Medline abstract: 14627633
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date revised: 10 June 2009
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