This study describes the structural and functional properties of the promoter region of a dopamine receptor-gene (Dmdop1) from Drosophila. The transcriptional start site was identified by 5í-RACE (5í-rapid amplification of cDNA ends) cloning and primer-extension analysis. A consensus site for transcriptional initiation (INR element) is located 494 bp upstream of the ATG codon of the open reading-frame. The promoter neither contains TATA- nor CAAT boxes but several GC-rich elements. Relative promoter activity was monitored by CAT reporter-gene analysis in different neuronal cell lines. The Dmdop1 promoter contains one activating (-454/+125) and two silencing regions (-1481/-454 and +125/+495). Interestingly, one silencing region harbours a CRE (cAMP responsive element) site. Since the DmDOP1 receptor leads to cAMP production in cells, the CRE site might contribute to the receptorsí own expression by cAMP-dependent transcription factors (Kehren, 2005).
The circadian clock consists of a feedback loop in which clock genes are rhythmically expressed, giving rise to cycling levels of RNA and proteins. Four of the five circadian genes identified to date influence responsiveness to freebase cocaine in the fruit fly, Drosophila melanogaster. Sensitization to repeated cocaine exposures, a phenomenon also seen in humans and animal models and associated with enhanced drug craving, is eliminated in flies mutant for period, clock, cycle, and doubletime, but not in flies lacking the gene timeless. Flies that do not sensitize owing to lack of these genes do not show the induction of tyrosine decarboxylase normally seen after cocaine exposure. These findings indicate unexpected roles for these genes in regulating cocaine sensitization and indicate that they function as regulators of tyrosine decarboxylase (Andretic, 1999).
In response to exposure to volatilized freebase cocaine, Drosophila perform a set of reflexive behaviors similar to those observed in vertebrates, including grooming, proboscis extension, and unusual circling locomotor behaviors. Additionally, flies can show sensitization after even a single exposure to cocaine provided that the doses are separated by an interval of 6 to 24 hours. Sensitization, a process in which repeated exposure to low doses of a drug leads to increased severity of responses, has been linked to the addictive process in humans and is potentially involved in the enhanced craving and psychoses that occur after repeated psychostimulant administration. Circadian variation occurs in the agonist responsiveness of Drosophila nerve cord dopamine receptors functionally coupled to locomotor output. This variation is dependent on the normal functioning of the Drosophila period (per) gene, the founding member of the circadian gene family. Because changes in postsynaptic dopamine receptor responsiveness are also seen during cocaine sensitization in vertebrates, flies mutant in circadian functions were examined for alterations in responsiveness to cocaine. Wild-type (WT) flies as well as flies containing a per null mutation, pero, were exposed to 75 µg of cocaine four times over 2 days, and the fraction of flies showing severe responses was quantified after each exposure. Whereas WT flies show sensitization after the initial cocaine exposure, pero flies show no sensitization either to a normal or increased dose even after repeated exposures. As with WT flies, pero flies showed a dose-dependent increase in the severity of responses, and the normal cocaine-induced types of behaviors were observed. In other words, pero flies respond to cocaine but do not sensitize (Andretic, 1999).
per alleles that either shorten or lengthen the circadian periods show distinct patterns of cocaine responsiveness. The short-period mutants perS and perT both show increased responsiveness to the initial cocaine exposure and weak sensitization to a second 75-µg exposure, with only the sensitization of perS showing statistical significance. Sensitization is not observed in these lines when tested with other cocaine doses. The long-period mutant perL1 showes a normal initial cocaine response but no sensitization to a subsequent exposure (Andretic, 1999).
Similarly, other circadian genes appear to have effects on cocaine sensitization: Both clock and cycle mutants fail to sensitize when given two doses of cocaine. Because these mutants showed an increased sensitivity to the first exposure, cocaine doses were decreased to 50 µg. The inability of clock and cycle to sensitize is markedly similar to the behavior of pero mutants. The gene product of timeless (tim), Tim, is required for the nuclear translocation of Per and its stability in the cytoplasm; in timo mutants, cytoplasmic Per is degraded and per mRNA levels are constant (Andretic, 1999).
Recently, a doubletime (dbt) protein with homology to human casein kinase Iepsilon was identified and shown to be required for phosphorylation of Per. Cocaine responses were tested in two viable dbt mutants, dbtS and dbtL, which shorten and lengthen the circadian locomotor period, respectively. dbt mutants require a substantially higher cocaine dose to show behaviors normally observed at 75 µg, but even at these higher doses dbt flies do not show significant sensitization. If the role of dbt in cocaine responsiveness is analogous to its role in circadian behavior, then Per phosphorylation status may be important in regulating both initial cocaine responsiveness and sensitization (Andretic, 1999).
Modulation of dopamine receptor responsiveness is important in both the sensitization to cocaine in vertebrates and in the circadian modulation of locomotion in Drosophila. To see whether cocaine-sensitized flies would show an increase in the responsiveness of the nerve cord dopamine D2-like receptors, a test was performed using a preparation of behaviorally active decapitated flies that allows direct addition of drugs to the nerve cord. After decapitation, cocaine-sensitized WT flies locomote significantly more than sham-treated controls in response to the dopamine D2-like agonist quinpirole. However, there was no increase in quinpirole responsiveness of pero flies that did not sensitize to repeated cocaine exposures. Thus, similar to the inability of the pero mutant to modulate receptor responsiveness as a function of the time of day, pero is unable to modulate dopamine receptor responsiveness after cocaine exposure. The observation that cocaine sensitization is associated with increased responsiveness of postsynaptic dopamine receptors shows additional similarities between this system and that in higher vertebrates, where a similar relation holds (Andretic, 1999).
In Drosophila, sensitization requires the trace amine tyramine because the mutant inactive, which is defective in sensitization, shows both reduced tyramine and reduced levels of the enzyme involved in tyramine synthesis, tyrosine decarboxylase (TDC). An active role for tyramine in sensitization is indicated because TDC enzyme activity is induced after a single cocaine exposure, with a time course consistent with that for the development of sensitization. To test if the correlation between induction of TDC activity and behavioral sensitization holds for the circadian mutants, TDC activity was measured in the circadian mutant flies after a single exposure of cocaine. In contrast to WT flies, in which TDC activity is induced after cocaine exposure, the pero, cycle, and clock lines that are defective in sensitization show no such induction; only timo, which shows normal sensitization, induces TDC activity. It thus seems likely that the transcriptional regulator Per, presumably in conjunction with Clock and Cycle, is a direct or indirect regulator of TDC after exposure to cocaine (Andretic, 1999).
The sensitization defects in inactive and per mutant flies can be distinguished by differences in the ability of tyramine feeding to restore sensitization. The locomotor and cocaine sensitization defects in inactive mutant flies can be rescued by feeding tyramine to adults, but the sensitization defect in pero flies is distinct, because feeding tyramine to pero adults does not rescue sensitization. It is presumed that tyramine from the food can enter tyramine nerve terminals in inactive flies, where it is still subject to a cocaine-stimulated release mechanism that mediates sensitization. The failure of tyramine feeding to rescue sensitization in pero flies is most readily understood if the per gene product is required for this regulated release (Andretic, 1999).
Similar to inactive , tyramine increases initial cocaine responsiveness in pero flies. Exposure of tyramine-fed pero flies to 35 µg of cocaine induces behaviors normally seen in control flies exposed to 75 µg. Thus, although long-term increase of tyramine levels can affect initial cocaine responsiveness, it is not sufficient for sensitization in flies lacking normal per function (Andretic, 1999).
A unifying feature of most genes that regulate circadian rhythmicity in Drosophila and vertebrates is the PAS dimerization domain, common to a subset of basic helix-loop-helix transcription factors. Within the circadian cycle, Clock/Cycle heterodimers activate per transcription, whereas Per/Tim heterodimers inhibit the activity of Clock/Cycle. Mutations in per, clock, and cycle share the same cocaine phenotype: a deficiency in the ability to sensitize after one or more drug exposures. This similarity leads to the suspicion that as in circadian behaviors, these genes are functioning in a common pathway. In contrast to the above mentioned genes, the timo mutant showed normal cocaine responses. The implication of this finding is twofold. (1) There must be an as yet unidentified Per binding partner that is specifically involved in regulation of drug responsiveness, and (2) drug responsiveness is likely regulated by per expression in a set of cells distinct from those involved in circadian function. In timo mutants, Per levels are constitutively low; if the same Tim-containing cells were involved in circadian and cocaine responses, timo flies should not sensitize (Andretic, 1999).
Drugs of abuse have a common property in mammals, which is their ability to facilitate the release of the neurotransmitter and neuromodulator dopamine in specific brain regions involved in reward and motivation. This increase in synaptic dopamine levels is believed to act as a positive reinforcer and to mediate some of the acute responses to drugs. The mechanisms by which dopamine regulates acute drug responses and addiction remain unknown. Evidence that dopamine plays a role in the responses of Drosophila to cocaine, nicotine or ethanol. A startle-induced negative geotaxis assay and a locomotor tracking system were used to measure the effect of psychostimulants on fly behavior. Using these assays, it was shown that acute responses to cocaine and nicotine are blunted by pharmacologically induced reductions in dopamine levels. Cocaine and nicotine showed a high degree of synergy in their effects, which is consistent with an action through convergent pathways. In addition, dopamine was found to beinvolved in the acute locomotor-activating effect, but not the sedating effect, of ethanol. This study shows that in Drosophila, as in mammals, dopaminergic pathways play a role in modulating specific behavioral responses to cocaine, nicotine or ethanol. It is therefore suggested that Drosophila can be used as a genetically tractable model system in which to study the mechanisms underlying behavioral responses to multiple drugs of abuse (Bainton, 2000).
The circadian function of Drosophila dopamine receptors was investigated by using a behaviorally active decapitated preparation that allows for direct application of drugs to the nerve cord. Quinpirole, a D2-like dopamine receptor agonist, induces reflexive locomotion in decapitated flies. The amount of locomotion induced changes as a function of the time of day, with the highest responsiveness to quinpirole during the subjective night. Furthermore, dopamine receptor responsiveness is under circadian control and depends on the normal function of the period gene. The head pacemaker is at least partly dispensable for the circadian modulation of quinpirole-induced locomotion, because changes in agonist responsiveness persist in decapitated flies that are aged for 12 h. This finding suggests a role for the period-dependent molecular oscillators in the body in the modulation of amine receptor responsiveness (Andretic, 2000).
The circadian variation in locomotor output of the Drosophila nerve cord in response to dopamine agonist stimulation shows two interesting differences from the pattern of locomotor responsiveness in living flies: (1) the rhythms of quinpirole-stimulated nerve cord responsiveness are in opposite phase with in vivo locomotor activity patterns, reaching peak levels during the subjective night, at the time when living flies are least active; (2) there are subtle differences in the activity profiles of the intact and decapitated flies during the light-to-dark transitions (Andretic, 2000).
The out-of-phase nature of in vivo vs. nerve cord locomotion is most readily explained if the nerve cord responses are modulated as compensatory postsynaptic effects, as has been observed in vertebrates and Drosophila. Postsynaptic dopamine receptors can compensate for differences in the amount of presynaptic release, decreasing sensitivity when dopamine release is high, and increasing when it is low. It has been shown that constitutive overexpression of a stimulatory G-alpha subunit in the dopamine and serotonin neurons, which is expected to increase amine release, results in a decrease in postsynaptic responsiveness to quinpirole. Reciprocally, overexpression of the inhibitory G-alpha subunit or tetanus toxin results in an increased responsiveness to quinpirole. It is speculated that increased dopamine release during the subjective day would stimulate locomotor behaviors, with decreased postsynaptic receptor sensitivity acting as a partially effective compensatory mechanism. At night when dopamine release is presumed to be lower, up-regulation of receptor responsiveness would mediate the enhanced response to quinpirole. Although this model postulates that dopamine release is under circadian control, dopamine synthesis is not, because no variation in brain dopamine content as a function of the 24-h day is found. If modulated dopamine release sets the responsiveness of the quinpirole-sensitive receptors, it could occur even in flies lacking brain input. In invertebrates, the ventral cord, unlike the higher vertebrate spinal cord, contains aminergic cell bodies. Thus, a rhythm of aminergic release under the control of body modulatory circuits could set the responsiveness of quinpirole-sensitive receptors. In intact flies, circadian behavior could be under coordinated control of both the body oscillators and the pacemaker in the brain, because additional dopamine input to the ventral cord originates in the brain and reaches the nerve cord through descending dopamine fibers. Alternatively, the observed circadian modulation of quinpirole sensitivity could be under more direct circadian control. By this scenario, modulation of dopamine receptor sensitivity or other signaling components downstream of the receptor would be modulated, independent of the magnitude of dopamine release (Andretic, 2000).
These results are most simply consistent with a role for quinpirole-activated dopamine receptors acting in the output pathway from the brain circadian pacemaker. However, none of the results preclude a role for these as of yet unidentified receptors in modulating intercellular responses between cells of the brain circadian pacemaker. Biogenic amines have been implicated in the control of motor behaviors in vertebrates and invertebrates, both in the central and peripheral nervous system. In humans, the importance of dopamine in motor control is most evident in Parkinson's disease, where degeneration of dopamine cell bodies in substantia nigra results in movement disorders. Interestingly, some Parkinson's disease patients display variations in circadian activity patterns, whereas other studies show daily oscillations in the severity of the symptoms, indicating potential communication between the dopamine system and circadian clock. In spinal cats, where the neural axis has been transected, monoaminergic systems are involved in initiation and modulation of locomotion. In arthropod species, injections of dopamine, serotonin, or octopamine into the central nervous system evokes distinct motor postures, suggesting that they are released endogenously to mediate behavior (Andretic, 2000 and references therein).
Data indicate a role for modulation of dopamine receptor responsiveness in circadian behavior. Modulation of dopamine receptor sensitivity is involved in modulating responses to the indirect amine agonist cocaine, both in vertebrates and in flies. Cocaine functions as a stimulator of reflexive motor and locomotor behaviors both in flies and in vertebrates. It is thus not totally surprising that modulation of responsiveness to cocaine in Drosophila crucially depends on the normal function of a subset of the circadian genes. Given this overlap in functions, it seems likely that there will be altered circadian functions in other mutants showing altered cocaine responses (Andretic, 2000 and references therein).
Dopamine (DA) is the only catecholaminergic neurotransmitter in Drosophila. Dopaminergic neurons have been identified in the larval and adult central nervous system (CNS) in Drosophila and other insects, but no specific genetic tool was available to study their development, function, and degeneration in vivo. In Drosophila as in vertebrates, the rate-limiting step in DA biosynthesis is catalyzed by the enzyme tyrosine hydroxylase (TH). The Drosophila TH gene (DTH or pale) is specifically expressed in all dopaminergic cells and the corresponding mutant, pale (ple), is embryonic lethal. ple rescue experiments were performed with modified DTH transgenes. The results indicate that partially redundant regulatory elements located in DTH introns are required for proper expression of this gene in the CNS. Based on this study, a GAL4 driver transgene, TH-GAL4, was generated containing regulatory sequences from the DTH 5' flanking and downstream coding regions. TH-GAL4 specifically expresses in dopaminergic cells in embryos, larval CNS, and adult brain when introduced into the Drosophila genome. As a first application of this driver, it was observed that in vivo inhibition of DA release induces a striking hyperexcitability behavior in adult flies. It is proposed that TH-GAL4 will be useful for studies of the role of DA in behavior and disease models in Drosophila (Friggi-Grelin, 2003).
The catecholamines play a major role in the regulation of behavior. This study investigated the role of dopamine and octopamine (the presumed arthropod homolog of norepinephrine) during the formation of appetitive and aversive olfactory memories. For the formation of both types of memories, cAMP signaling is necessary and sufficient within the same subpopulation of mushroom-body intrinsic neurons. In contrast, memory formation can be distinguished by the requirement for different catecholamines, dopamine for aversive and octopamine for appetitive conditioning. These results suggest that in associative conditioning, different memories are formed of the same odor under different circumstances, and that they are linked to the respective motivational systems by their specific modulatory pathways (Schwaerzel, 2003; full text of article).
These results support three major conclusions: (1) during the association of an olfactory cue with either a sugar reward or an electric shock punishment, both forms of olfactory memories require cAMP signaling within the same 700 Kenyon cells of the MBs; (2) for memory retrieval but not acquisition with either of the two reinforcers, output from this same set of cells is required. Hence, the memory must be formed and stored upstream of this synaptic level. (3) Sugar and electric shock reinforcement are mediated by different modulatory neurotransmitters, DA in case of electric shock and octopamine (OA) in case of sugar reward. These findings confirm and extend previous work, concluding that output synapses of Kenyon cells are the site of olfactory memory. Appetitive and aversive olfactory memories are localized to the same neuropil Associative behavioral adaptations are mediated by the plasticity of synapses within neural circuits. But what are the smallest units of memory? Do they correspond to the modulation of a single synapse or to the concerted change of many or all synapses in a circuit? Attempts to localize olfactory memory in the Drosophila brain have provided partial answers to these questions (Schwaerzel, 2003).
In many species, including Aplysia, mouse, and Drosophila, the type-1 AC has been shown to be critical in synaptic plasticity. No cases of cAMP-independent associative synaptic or behavioral plasticity have yet been reported conclusively. In Drosophila, one of the corresponding mutants, rut, shows abnormal performance in every learning paradigm tested so far. By identifying the minimally sufficient set of neurons that in a rut mutant brain need to express a wild-type form of the RUT protein to restore a particular memory performance, one can localize the memory trace of the corresponding behavioral adaptation. This approach was successfully applied to two types of memory in Drosophila, heat box memory and olfactory memory (Schwaerzel, 2003).
Using the same approach in a side-by-side comparison between sugar and electric shock reinforcement, the current results show that wild-type rut-AC expression in 700 Kenyon cells (25%-30% of total) rescues memory performance for both kinds of reinforcement. Thus, aversive and appetitive olfactory memories are both mediated by synaptic plasticity in the same group of cells (Schwaerzel, 2003).
Attributing the rescue to an effect on synaptic plasticity in the adult Kenyon cells disregards the possibility that the genetic manipulation might rescue a developmental function of rut-AC, necessary later in the adult for memory. Several lines of evidence argue for an adult function, but only recently has a new genetic manipulation been designed that definitely rules out a developmental effect. Use of a temperature-sensitive Gal80, a suppressor of Gal4, ensured that wild-type rut-AC was expressed only during adulthood (Schwaerzel, 2003).
Although the current experiments do not specify where in the Kenyon cells cAMP signaling is required, the existing evidence suggests a presynaptic mechanism at Kenyon cell output synapses. At the Drosophila larval neuromuscular junction, cAMP signaling is presynaptically involved in plasticity. For the sensory-motor synapses mediating classical conditioning of the gill withdrawal reflex in Aplysia, it has been firmly established that the cAMP cascade is involved presynaptically. No conclusive example of a postsynaptic contribution of cAMP signaling has been reported (Schwaerzel, 2003).
In the Aplysia synapses, plasticity has a postsynaptic component based on a mechanism resembling the NMDA receptor and long-term potentiation in mammals. In Drosophila olfactory conditioning, a similar postsynaptic contribution is unlikely to play a role during the first 3 hr, because this effect would require neurotransmitter release from the presynapse, which can be blocked during acquisition and memory retention without a deleterious effect on memory, using shits1, a conditional blocker of synaptic transmission (Schwaerzel, 2003).
The tentative presynaptic effect of cAMP signaling locates the synaptic plasticity underlying olfactory memory to the synapses connecting Kenyon cells to MB output neurons. These are found in the MB lobes, including the rostral peduncle and spur. Additional support for cAMP signaling to occur in the lobes rather than calyx is derived from the 'memory' gene amn, which has been shown to be involved in cAMP regulation. The putative AMN neuropeptide is required exclusively in two prominent neurons, the so-called dorsal paired medial neurons, that profusely innervate the MB lobes. Other components of the cAMP pathway such as rut and receptors for DA and OA, all are predominantly expressed in the adult MB lobes (Schwaerzel, 2003).
Associative synaptic plasticity depends on the convergence between impulses from two signals, the CS and the US. Considering the proposed role of rut-AC as a molecular coincidence detector, one can assume that the MB input neurons carrying the US for sugar and electric shock should also connect to the lobes, although their direct anatomical identification is pending (Schwaerzel, 2003).
The current results show that acquisition of an olfactory memory with electric shock is dependent on the dopaminergic system, whereas acquisition with sugar depends on the octopaminergic system. OA as neurotransmitter in sugar learning seems to be conserved between Drosophila and the honeybee. The bee VUMmx1 neuron, an unpaired neuron localized in the subesophageal ganglion, appears to be octopaminergic and has been shown to carry some of the reinforcing properties of the US. It innervates the calices, antennal lobes, and lateral protocerebrum but not the MB lobes. Nevertheless, the learning paradigms used [individual conditioning of the proboscis extension reflex in bees vs the population-based conditioned osmotaxis in Drosophila] are different; therefore, it might be too early to compare the sugar memories in the bee and Drosophila with respect to its organization on a circuit level. Unfortunately, the role of the monoamines in aversive conditioning has not been tested in bees (Schwaerzel, 2003).
These findings raise the question of whether the effects of the two catecholamines on electric shock and sugar learning can be generalized to other appetitive and aversive reinforcers and to positive and negative behavioral modulation in general. In the monkey, midbrain dopaminergic neurons have been described that carry the reinforcing properties of a US in appetitive but not aversive conditioning. It will be interesting to see whether a similar dissociation between modulatory systems for appetitive and aversive conditioning, with the contingency between good-bad and monoamines exchanged, also applies to the monkey, and, potentially, to humans (Schwaerzel, 2003).
Separate memory traces for electric shock and sugar conditioning had been suggested previously, because these have different kinetics of consolidation and decay. The distinct effects of the two catecholamines in the reinforcement pathways discovered in this study underline this notion. Surprisingly, however, localization experiments assign the two memories to the same neuropil structure, a subset of 700 Kenyon cells (Schwaerzel, 2003).
Based on the functional anatomy of the olfactory pathway, odors are assumed to be represented in the MBs by specific sets of Kenyon cells. For an odorant to become predictive of a given reinforcing event (e.g., sugar or electric shock), the output synapses of this set of Kenyon cells should be modified to drive an MB output neuron mediating the conditioned response (e.g., approach or avoidance). MB input neurons representing the appropriate reinforcers (e.g., sugar or electric shock) should provide the modulatory input. At present it is still not known whether the identified monoamines, OA and DA, are the modulatory neurotransmitters at the site of synaptic plasticity or act further upstream in the respective US pathway. The former alternative is supported by the observation that the MB lobes are equipped with DA and OA receptors that can couple to AC of the rut type via Gs protein. In addition, the neurons relevant for electric shock learning send TH-Gal4-positive fibers to the MB neuropil at the level of the spur and the vertical lobes (Schwaerzel, 2003).
The respective output neurons are prespecified to announce sugar or electric shock and so are the modulatory neurons. Hence, these form specific pairs (US-CR pairs) that are functionally linked to adapt the CR neuron to one of many odors in the conditioning events. Two schemes can be proposed of how the US-CR pairs and sets of Kenyon cells might be interconnected. The two diagrams differ with respect to the organization of odor representations in the MBs. If the same odors were represented by several sets of Kenyon cells, each set could be connected with just one US-CR pair. In this case, sugar and electric shock memories could be formed in different sets, both specifically responding to the same odorant, but one being modulated by OA, the other by DA. Both these sets would be contained within the set of 700 Kenyon cells of the 247-Gal4 driver line. Alternatively, if each odor is represented by only one set, the Kenyon cells should be responsive to multiple modulatory inputs. In this case, both memories would be formed within the same cells. The synapses of the US-CR pairs with the Kenyon cells should be closely associated, and these synaptic domains would have to be independently modulated by cAMP signaling. Because Drosophila can associate many events (US) with odors, Kenyon cells may accommodate many such US-CR pairs. A requirement for space at the Kenyon cells may then explain the stalk-like structure of MBs. At present it is not possible to distinguish between these two alternatives (Schwaerzel, 2003).
Sleep and arousal are known to be regulated by both homeostatic and circadian processes, but the underlying molecular mechanisms are not well understood. It has been reported that the Drosophila rest/activity cycle has features in common with the mammalian sleep/wake cycle, and it is expected that use of the fly genetic model will facilitate a molecular understanding of sleep and arousal. This study reports the phenotypic characterization of a Drosophila rest/activity mutant known as fumin (fmn). fmn mutants have abnormally high levels of activity and reduced rest (sleep); genetic mapping, molecular analyses, and phenotypic rescue experiments demonstrate that these phenotypes result from mutation of the Drosophila Dopamine transporter gene. Consistent with the rest phenotype, fmn mutants show enhanced sensitivity to mechanical stimuli and a prolonged arousal once active, indicating a decreased arousal threshold. Strikingly, fmn mutants do not show significant rebound in response to rest deprivation as is typical for wild-type flies, nor do they show decreased life span. These results provide direct evidence that dopaminergic signaling has a critical function in the regulation of insect arousal (Kume, 2005).
Although fmn flies exhibit such rest/activity phenotypes, there is apparently no effect of the mutation on development or longevity. This contrasts markedly with the results observed for mutations of two other genes that have been implicated in the regulation of Drosophila rest: cyc and Shaker (Sh). Mutations in cyc or Sh reduce life span, relative to genetic background controls, although complete life-span curves were not reported for Sh and there appears to be only a small effect on longevity for the Sh102 allele. The different effects of these mutations on longevity may reflect the relatively selective effect of fmn on arousal. Alternatively, the effects of Sh and cyc mutations on life span might reflect requirements for these genes in developmental or physiological processes other than rest. Cyc protein is a broadly expressed basic helix-loop-helix transcription factor, whereas Sh is a voltage-activated potassium channel with a broad localization in the nervous system. In contrast, DAT deficits only affect dopaminergic neuromodulation and therefore might have a less general impact on development and physiological processes (Kume, 2005).
Fmn flies carry a mutation in the Drosophila dopamine transporter gene, indicating that alterations of dopamine signaling are responsible for the observed phenotypes. dDAT functions in the dopaminergic pathway, as shown by (1) dDAT protein has significant sequence similarity to comparable mammalian and invertebrate transporters, (2) dDAT gene expression is restricted to dopaminergic neurons (as expected for a presynaptic transporter), (3) this transporter has a substrate specificity paralleling that of the mammalian DATs, with dopamine and tyramine being the preferred substrates, and (4) the dDAT transporter mediates uptake of dopamine in cell-based assays and responds to dopamine when expressed in Xenopus oocytes (Kume, 2005).
Dopamine is cleared from the synaptic cleft via presynaptic DAT, and DAT mutant mice exhibit altered presynaptic autoreceptor function, dopamine clearance, and biosynthetic rate in addition to behavioral alterations including spontaneous hyperlocomotion and hyperactivity. These phenotypes are presumably caused by the elevated persistence of released dopamine in these mice. Similarly, it seems likely that increased dopamine signaling in fmn is responsible for the observed hyperactivity and shortening of the rest phase, regarded as sleep in Drosophila (Kume, 2005).
Previous studies in Drosophila implicate biogenic amines in the modulation of activity. In larval Drosophila, 5-HT, OA, TA, and DA regulate locomotion or the sensory-motor circuitry on which such behavior depends. In adult flies, evidence suggests that DA and 5-HT function to regulate locomotor activity and flight, respectively. The study by Lima (2005) is particularly informative in that it demonstrates state-dependent effects of dopamine neuronal stimulation in behaving flies. In flies with low basal activity, the response to targeted (and transient) stimulation of dopamine neurons is an increased probability of locomotor bouts, whereas a similar stimulation of flies showing high basal activity leads to an inhibition. These results are most consistent with a biphasic role for modulation of locomotor activity by released dopamine, with the highest levels of dopamine release leading to locomotor inhibition. This is in agreement with studies that have examined the responses of flies to the psycho-stimulant cocaine, an inhibitor of aminergic transporters. Cocaine stimulation of flies results in transient stereotypies and hyperactivity that are strikingly similar to those seen after cocaine exposure to vertebrate animals. However, the most severely affected flies become akinesic, consistent with a biphasic effect of high extracellular dopamine (Kume, 2005).
Based on the decreased arousal threshold and the prolonged responses of fmn flies to mechanical stimulation, it is suggested that this mutant is characterized by an arousal state with enhanced alertness associated with the expected increase in extracellular dopamine. The absence of significant rest rebound in fmn supports this conclusion, along with the finding that activity level during each arousal period is normal. This is the first direct evidence that altered arousal threshold and decreased rebound can result from perturbations of dopaminergic signaling. Previous results have indirectly implicated DA in arousal. Those studies examined animals with lesions of dopaminergic neuronal populations, changes in firing rates of dopaminergic neurons as a function of sleep states and arousal, or the actions of wake-promoting drugs, some of which modulate DAT or DA receptor activity. The analysis of fmn shows directly that a selective lesion of DAT, presumably with accompanying increased DA levels, results in an alteration of arousal threshold (Kume, 2005).
It is of interest that mouse DAT mutants, like fly fmn, have abnormal sleep, although arousal sensitivity has not been explicitly examined. DAT mutant mice show enhanced spontaneous locomotor hyperactivity that is greatly enhanced by the stimulating effects of novel environment. More importantly, mouse DAT mutants have significantly increased wake bout duration and moderately increased activity levels during the latter half of the active (night) phase of the diurnal cycle. The extension of the active phase in these mice mimics the phenotype of fmn flies that have a lengthened active period and shortened rest phase during the diurnal cycle. DAT knock-out mice also exhibit altered responses to wake-promoting drugs such as GBR12909, modafinil, and caffeine. Such mice show increased sensitivity to caffeine and decreased sensitivity to GBR12909 and modafinil, suggesting that the latter two drugs act on DAT to promote wakefulness. Modafinil has wake-promoting properties in Drosophila, and it will be of interest to determine whether modafinil action is altered in fmn as it is in DAT mutant mice. A role for DAT and dopaminergic signaling in regulating wakefulness in flies and mice provides additional evidence for a similarity between mammalian sleep and insect rest (Kume, 2005).
Dopamine is probably also important for the regulation of arousal in humans. Parkinson's disease patients, who have reduced dopamine levels, often complain of sleepiness. When treated with L-3,4-dihydroxyphenylalanine (L-DOPA), which increases dopamine levels, they recover from sleepiness, but with excessive L-DOPA, they exhibit insomnia. Moreover, patients and animals with narcolepsy, which show increased wakefulness and excessive sleepiness, show a compensational increase in brain D2-like receptors, suggesting a reduced level of dopamine (Kume, 2005).
Although fmn mutants have higher basal levels of activity, they nonetheless exhibit diurnal and circadian rhythms in locomotor activity. However, rhythmicity is less evident compared with that observed in wild-type flies, presumably because there is a higher baseline of activity at all times of day and therefore a corresponding decrease in the amplitude of rhythmicity (Kume, 2005).
Part of the evidence that fmn is a mutation in dDAT comes from transgenic rescue using pan-neurally driven expression of dDAT. It is notable that this rescue is only partial. One obvious explanation for this partial rescue is that expression of ELAV-GAL4 in the dopamine neurons, the sole site of dDAT localization, is weak. However, even stronger dopamine neuron expression of dDAT, under the control of TH-GAL4, yields even weaker rescue. This counterintuitive result may indicate that the precise level of dDAT in dopamine neurons is critical for normal dopamine homeostasis. It is possible that homeostatic mechanisms potentially overcompensate for high DAT levels and reduced synaptic DA by hyperactivating postsynaptic receptors, as has been seen after inhibition of dopamine and serotonin neurons. Thus, dDAT may be a component of a precisely regulated dopamine homeostatic mechanism that controls arousal threshold and overall activity levels (Kume, 2005).
This study of a Drosophila DAT mutant indicates another striking parallel between Drosophila and vertebrates with regard to the functions of biogenic amine systems. It demonstrates that the regulated reuptake of dopamine by DAT is important for setting arousal threshold. Equally important, the identification of a mutation in the pharmacologically important dopamine transporter opens new avenues for use of this genetically tractable model in pharmacological and behavioral studies (Kume, 2005).
The temporal pairing of a neutral stimulus with a reinforcer (reward or punishment) can lead to classical conditioning, a simple form of learning in which the animal assigns a value (positive or negative) to the formerly neutral stimulus. Olfactory classical conditioning in Drosophila is a prime model for the analysis of the molecular and neuronal substrate of this type of learning and memory. Neuronal correlates of associative plasticity have been identified in several regions of the insect brain. In particular, the mushroom bodies have been shown to be necessary for aversive olfactory memory formation. However, little is known about which neurons mediate the reinforcing stimulus. Using functional optical imaging, it has been shown that dopaminergic projections to the mushroom-body lobes are weakly activated by odor stimuli but respond strongly to electric shocks. However, after one of two odors is paired several times with an electric shock, odor-evoked activity is significantly prolonged only for the 'punished' odor. Whereas dopaminergic neurons mediate rewarding reinforcement in mammals, these data suggest a role for aversive reinforcement in Drosophila. However, the dopaminergic neurons' capability of mediating and predicting a reinforcing stimulus appears to be conserved between Drosophila and mammals (Riemensperger, 2005; full text of article).
How do these results fit into the current model of associative olfactory learning in Drosophila? Most data are in favor of a coincidence of CS and US onto the presynapses of Kenyon cells and a resultant strengthening of the synaptic efficacy from Kenyon cells to mushroom-body-extrinsic neurons that ultimately influence the flyís behavior. The data suggest an extension of that model by pointing to an as-yet-anatomically-unidentified excitatory feedback loop from Kenyon-cell output neurons onto DA neurons. As a consequence of this proposed model, during the course of CS-US association an odor stimulus of little relevance gains a relevance that is subsequently represented in a prolonged activation of the DA neurons mediating aversive reinforcement. Of course, the possibility cannot be excluded that other brain regions innervated by DA neurons exhibit similar predictive features. However, these predictive features do not seem to be necessary for the initial formation of an aversive memory because mushroom-body output is needed only for retrieval, not for the formation of an aversive olfactory memory (Riemensperger, 2005).
Interestingly, the phenomenon of predictive features of DA neurons resembles the predictive properties of vertebrate DA neurons. The most obvious difference, however, is the fact that DA neurons mainly mediate rewarding reinforcement in vertebrates, whereas in Drosophila DA release is dispensable for appetitive learning (Schwaerzel, 2003). In contrast, octopamine is involved in appetitive olfactory learning in Drosophila and honeybees. In bees, US mediating and predicting properties in the context of appetitive olfactory conditioning have been observed in an identified octopaminergic neuron. It will be an interesting future experiment to test whether DA neurons in Drosophila do indeed respond exclusively to aversive stimuli or whether they respond to reinforcing stimuli in general (Riemensperger, 2005).
In this context it is conspicuous that calcium activities evoked by an electric shock outlast the stimulus. If the calcium activities revealed by these experiments reflect effective time windows for CS-US coincidence, one would expect that backward pairing of US and CS with short interstimulus intervals should also lead to aversive learning. Indeed, This has indeed shown to be the case. However, when backward pairing is performed with longer US-CS intervals (30 s), an approach behavior toward the backward-conditioned odor can be observed, with the functional implication that the odor may now indicate the absence of the punishment. It will be of interest in the future to test whether such a conditioned approach behavior involves also DA neurons (Riemensperger, 2005).
A second difference with observations in vertebrates concerns the fact that in mammals the responsiveness of DA neurons to the increasingly predicted US diminishes during the course of the training and shifts toward the predicting CS. There is no evidence for such a phenomenon in Drosophila because the responses to the first US presentation and US in the last training trial are indistinguishable in amplitude. In this respect, the reinforcement system in insects might be significantly simpler than in vertebrates. Alternatively, the training procedure used in Drosophila could be too short to detect such a phenomenon, or the US used could be too strong. Despite those differences, these data suggest that a reinforcing and US-predicting role of DA neurons represents a common principle of complex brains rather than a specific feature of the mammalian central nervous system (Riemensperger, 2005).
During classical conditioning, a positive or negative value is assigned to a previously neutral stimulus, thereby changing its significance for behavior. If an odor is associated with a negative stimulus, it can become repulsive. Conversely, an odor associated with a reward can become attractive. By using Drosophila larvae as a model system with minimal brain complexity, the question of which neurons attribute these values to odor stimuli was addressed. In insects, dopaminergic neurons are required for aversive learning, whereas octopaminergic neurons are necessary and sufficient for appetitive learning. However, it remains unclear whether two independent neuronal populations are sufficient to mediate such antagonistic values. Transgenically expressed channelrhodopsin-2, a light-activated cation channel, was used as a tool for optophysiological stimulation of genetically defined neuronal populations in Drosophila larvae. Distinct neuronal populations can be activated simply by illuminating the animals with blue light. Light-induced activation of dopaminergic neurons paired with an odor stimulus induces aversive memory formation, whereas activation of octopaminergic/tyraminergic neurons induces appetitive memory formation. These findings demonstrate that antagonistic modulatory subsystems are sufficient to substitute for aversive and appetitive reinforcement during classical conditioning (Schroll, 2006; full text of article).
This study addressed a question of central interest in neuroscience: are there distinct neuronal subsystems mediating opposing types of reinforcement? In vertebrates, the activity of dopaminergic neurons reflects reinforcing properties of rewarding stimuli. Conversely, serotonergic neurons have been proposed to mediate aversive reinforcement, but clear evidence is still lacking. In addition, the sufficiency for any neuronal population's activity to substitute for reinforcing stimuli in vertebrates has not been demonstrated. A more informative basis for opposing reinforcement systems is provided from experiments on diverse insect species. In adult Drosophila, dopaminergic neurons respond to a punishing electric shock stimulus, and blocking synaptic transmission from dopaminergic neurons during olfactory learning impairs aversive but not appetitive memory formation. In accordance with these findings, dopamine receptor antagonists disrupt aversive but not appetitive olfactory learning in crickets. In contrast, octopamine has been shown to be a necessary transmitter for appetitive olfactory learning in adult Drosophila and crickets. The sufficiency of a neuronal cell type for mediating a reinforcing stimulus has been demonstrated for honey bees: electrophysiological stimulation of a single neuron substitutes for an appetitive reinforcing stimulus in olfactory conditioning. This neuron most likely belongs to a cluster of octopaminergic neurons that has been described in honeybees, adult Drosophila, and other insect species. In accordance with these findings, local injection of octopamine also substitutes for the reinforcing stimulus in appetitive olfactory conditioning of honey bees. Therefore, several lines of evidence have led to the idea of two modulatory systems being causative for opposite types of learning in insects. These data provide a direct proof of this concept. It will be of interest to see whether opposing modulatory transmitter systems can be identified in vertebrates as well, a task for which ChR2 might provide a valuable tool (Schroll, 2006).
The synaptic machinery for neurotransmitter storage is cell-type specific. Although most elements of biosynthesis and transport have been identified, it remains unclear whether additional factors may be required to maintain this specificity. The Drosophila serotonin transporter (dSERT) is normally expressed exclusively in serotonin (5-HT) neurons in the CNS. This study examine the effects of ectopic transcriptional expression of dSERT in the Drosophila larval CNS. A surprising limitation on 5-HT storage was found following ectopic expression of dSERT and green fluorescence protein-tagged dSERT (GFP-dSERT). When dSERT transcription is driven ectopically in the CNS, 5-HT is detectable only in 5-HT, dopamine (DA), and a very limited number of additional neurons. Addition of exogenous 5-HT does not dramatically broaden neuronal storage sites, so this limitation is only partly due to restricted intercellular diffusion of 5-HT. Furthermore, this limitation is not due to gross mislocalization of dSERT, because cells lacking or containing 5-HT show similar levels and subcellular distribution of GFP-dSERT protein, nor is it due to lack of the vesicular transporter, dVMAT. These data suggest that a small number of neurons selectively express factor(s) required for 5-HT storage, and potentially for function of dSERT (Park, 2006).
The simplest explanation for this inability to detect intracellular 5-HT is that there is a cell-type-specific limitation on dSERT function. Arguing for this interpretation is the finding that most if not all neurons will nonspecifically take up 5-HT if incubated at a sufficiently high concentration of 5-HT (10 mM), that lack of expression of the vesicular monoamine transporter dVMAT does not affect the storage ability of neurons, and that no increased levels of the expected 5-HT metabolite N-Ac-5-HT is detected in CNS with ectopic dSERT expression. Part but not all of the limitation is due to limited intracellular diffusion of 5-HT, because incubation in a more moderate concentration of 5-HT, 1 mM, leads to a moderate expansion in the number of cells that can uptake 5-HT, but in a dSERT-dependent manner (Park, 2006).
Nevertheless, this interpretation would stand in contrast to conventional wisdom regarding vertebrate amine transporters. Vertebrate amine transporters can be readily expressed in several types of non-neural transformed cell lines as well as in Xenopus oocytes, with only small differences in their functional properties as a function of cellular host, leading to the general view that the nature of the host cell is not critical. Because dSERT can also be expressed in heterologous cell types including Drosophila S2 cells, it seems that the critical difference between this study versus previous studies is the site of expression, the nerve cord of living flies. There are no analogous amine transporter overexpression studies in the vertebrate nervous system, and it is possible that similar mechanisms might act to limit expression of these transporters to appropriate cell types (Park, 2006).
There is much evidence indicating that amine transmitter transporters are subject to post-translational modifications, including glycosylation and phosphorylation. In particular, roles for protein kinase C (PKC) and phosphatase 2A (PP2A) have become apparent through studies with enzyme activators and inhibitors in SERT-transfected cells, where SERT proteins are rapidly phosphorylated in parallel with transporter internalization and loss of functional uptake capacity. In addition, p38 MAPK activation can stimulate hSERT activity in CHO cells without affecting trafficking to the cell surface (Park, 2006).
Multiprotein complexes that include the transporters, scaffolding proteins, kinases, and phosphatases also can affect their activity and propensity to undergo membrane internalization. Any of these proteins could be candidates for a factor limiting dSERT function to specialized types of neurons in the fly CNS. The only limitation on this factor(s) is that it would not appear to limit the membrane appearance of dSERT, that is, that it does not appear to function at an early step in dSERT Golgi maturation, because GFP-linked dSERT shows similar membrane association at the level of light microscopy in all cell types. In rodents, transient expression of SERT and VMAT2 allows the storage and release of 5-HT in developing thalamocortical projections, despite the absence of serotonin biosynthesis in these cells. It has been assumed that VMAT2 and SERT expression are sufficient to allow uptake of 5-HT synthesized at exogenous sites, and storage in the thalamic neurons. It is possible that the additional 5-HT uptake/accumulation that that was observed following incubation in 1 mM 5-HT in the presumptive adult neuroblasts and their progeny reflects a similar phenomenon, transient expression of dSERT in a subset of developing neurons. More importantly, however, the vast majority of neurons do not accumulate 5-HT even in the presence of exogenous 5-HT. Thus, these data suggest that regulated expression of additional factors may be needed to accomplish a change in neurochemical identity. Cell-type-specific ectopic expression of dSERT in Drosophila will be a favorable tool by which to search genetically for factor(s) limiting 5-HT storage activity (Park, 2006).
While studying the developmental functions of the Drosophila dopamine synthesis pathway genes, interesting and unexpected mutant phenotypes were noticed in the developing trachea, a tubule network that has been studied as a model for branching morphogenesis. Specifically, Punch (Pu) and pale (ple) mutants with reduced dopamine synthesis show ectopic/aberrant migration, while Catecholamines up (Catsup) mutants that over-express dopamine show a characteristic loss of migration phenotype. Expression of Punch, Ple, Catsup and dopamine was seen in tracheal cells. The dopamine pathway mutant phenotypes can be reproduced by pharmacological treatments of dopamine and a pathway inhibitor 3-iodotyrosine (3-IT), implicating dopamine as a direct mediator of the regulatory function. Furthermore, these mutants genetically interact with components of the endocytic pathway, namely shibire/dynamin and awd/nm23, that promote endocytosis of the chemotactic signaling receptor Btl/FGFR. Consistent with the genetic results, the surface and total cellular levels of a Btl-GFP fusion protein in the tracheal cells and in cultured S2 cells are reduced upon dopamine treatment, and increased in the presence of 3-IT. Moreover, the transducer of Btl signaling, MAP kinase, is hyper-activated throughout the tracheal tube in the Pu mutant. Finally it was shown that dopamine regulates endocytosis via controlling the dynamin protein level (Hsouna, 2007).
This report demonstrates that genes involved in DA biosynthesis also regulate tracheal cell migration, and that this function is mediated by DA. This is unexpected since DA is normally associated with neuronal function. However, this novel developmental function is not fortuitous because of the strong and highly specific expression of Punch in tracheal cells during the migratory phase of tracheal development. In addition, the ectopic migration tracheal phenotypes in Pu/GTPCH mutants can be rescued by expressing a Pu/GTPCH transgene in developing trachea using a btl promoter, demonstrating the trachea-specific function of the DA pathway. Moreover, the expression of this transgene shows a dosage-dependent progression of phenotypic outcomes. That is, mild expression rescues ectopic migration, while over-expression tips the balance to blocked migration (Hsouna, 2007).
The product of GTPCH enzymatic activity, BH4, is also a stabilizing cofactor of nitric oxide synthase (NOS), which is needed for hypoxia-induced outgrowth of terminal tracheal branches. However, the Pu mutant phenotypes reported in this study are most likely unrelated to this developmental process because of the following reasons: (1) Terminal branching occurs near the end of embryogenesis and during larval development whereas the defects in primary and secondary branch migration occur during mid-embryogenesis. (2) Mutations in ple, which has no role in NOS function, also results in ectopic migration phenotypes. (3) DA and the DA pathway inhibitor can phenocopy the genetic mutants. (4) DA treatment can rescue Pu/GTPCH mutant phenotypes, implicating a direct role of DA in primary and secondary branch migration. Interestingly, DA is enriched in the trachea (Hsouna, 2007).
An inhibitory role for DA in the regulation of cell migration has been reported for a number of cell types over recent years. DA is capable of blocking the migration of vascular smooth muscle cells, a factor in the formation of atherosclerotic lesions, in a process mediated through the D1 class of DA receptors (Yasunari, 2003). DA also was reported to interfere with activated neutrophil transendothelial migration. Similarly, DA, acting through D1 receptors, is reported to reduce the migration of regulatory T cells in damaged neural regions, thereby providing protection against T cell-mediated neurodegeneration (Kipnis, 2004). Finally, DA acting through the D2 class of DA receptors, can inhibit tumor angiogenesis in highly vascularized gastric and ovarian tumors. It would be interesting to determine whether the mammalian GTPCH-TH-DA enzymatic pathways also have a non-neurological function in modulating tubulogenesis during development (Hsouna, 2007).
DA pathway mutants show tracheal abnormalities that are strikingly similar to the perturbations of tracheal cell migration in embryos with abnormalities in FGF signaling. Diminution of DA expression in Drosophila embryos, either by mutations (Pu and ple) or by pharmacological depletion (3-IT treatment), has dramatic effects on the stereotyped migratory behavior of tracheal cells and patterning of the resulting branched structure. Entire branches are misdirected and individual cells move away from the tracheal branches, a phenotype that has been termed 'run-away cells'. Under conditions of excess DA, via mutations (Catsup) or pharmacological modification (DA treatment), the converse phenotype, one of blocked migration, is evident. This phenotype is often associated with clustering of tracheal cells near the stunted ends of tracheal tubes. These opposing phenotypes are correlated with the increased or decreased levels of MAPK activation, the mediator of FGFR signaling (Hsouna, 2007).
The data further demonstrate that DA regulates FGF receptor turnover. Btl;;GFP fusion protein is down-regulated in the presence of DA and up-regulated in the presence of 3-IT, either in trachea or in cultured S2 cells. These results are consistent with the model that DA promotes internalization of Btl/FGFR, leading to its degradation through the endocytic pathway. The role of DA in internalization of FGFR is further suggested by the genetic interaction between the DA synthesis and endocytic pathway genes. Mutations in the human tumor suppressor nucleoside diphosphate kinase (NDK) gene (nm23) are strongly associated with tumor metastatic activity. Its functional homolog in Drosophila, abnormal wing discs (awd), has been shown to genetically interact with a temperature-sensitive allele of the shibire gene (shits), which encodes dynamin, a large GTPase required for the formation of clathrin-coated endocytic vesicles. A function for awd/nm23 in the migratory phase of tracheal development has been demonstrated and functional interactions occur with shi/dynamin that are required for the modulation of FGFR/Btl levels in tracheal cells. This report shows that Pu and ple phenotypes are exacerbated by awd and rescued by btl, while the Catsup phenotypes are rescued by awd but exacerbated by btl. These results indicate that Pu/GTPCH and Ple/TH, and by extension, DA, are positive regulators of endocytosis (Hsouna, 2007).
This analysis was extended by implicating direct involvement of DA in regulating the Shi/dynamin protein itself. Mutations in Pu/GTPCH, which regulates DA pools, result in reduction of the Shi/dynamin levels and, in consequence, the subsequent accumulation of FGFR/Btl in the plasma membranes of tracheal cells, thus accounting for ectopic migratory behavior of these mutant cells. Importantly, down-regulation of Shi/dynamin level in the Pu mutants can be rescued by treatment with DA. A direct correlation between DA and dynamin levels is also demonstrated in cultured S2 cells. Thus, the genetic and pharmacological evidence in this report supports the hypothesis that the diminished DA pools that accompany loss-of-function mutations in the Pu/GTPCH and ple/TH genes result in deficits of DA-mediated signaling necessary for Shi/dynamin accumulation (Hsouna, 2007).
Evidence of a role of DA in receptor endocytosis has emerged recently. For instance, DA can promote VEGFR endocytosis in cultured human endothelial cells. DA D3 receptor-mediated modulation of GABAA receptor and DA-regulated endocytosis of the renal cell Na+, K+-ATPase are similarly dynamin-mediated events. Why is a neurohormone also a mediator of endocytosis in other cell types? It is interesting to consider the possibility that the endocytic activity of DA may in fact be its ancestral function, which was adopted by the neurons and tubular cells later. Indeed, primitive, nerve-less multicellular organisms such as sponge can produce dopamine. The precise mechanism(s) by which DA regulates dynamin assembly is not yet clear. However, DA signaling promotes dynamin stabilization and assembly at the plasma membrane in cultured human kidney cells, and thus endocytosis, by activating protein phosphatase 2A which dephosphorylates dynamin-2. It is possible that a similar mechanism of action occurs in the tracheal cells and future experiments will help to address this possibility (Hsouna, 2007).
The effects of increased level of dopamine (DA) (feeding flies with DA precursor, L-dihydroxyphenylalanine, L-DOPA) on the level of 20-hydroxyecdysone (20E) and on juvenile hormone (JH) metabolism in young (2-day-old) wild type females (the strain wt) of Drosophila virilis have been studied. Feeding the flies with L-DOPA increased DA content by a factor of 2.5, and led to a considerable increase in 20E level and a decrease of JH degradation (an increase in JH level). The levels of 20E were measured in the young (1-day-old) octopamineless females of the strain Tbetah(nM18) and in wild type females, Canton S, of D. melanogaster. The absence of OA led to a considerable decrease in 20E level (earlier it was shown that in the Tbetah(nM18) females, JH degradation was sharply increased). The effects were studied of JH application on 20E level in 2-day-old wt females of D. virilis; an increase in JH titre results in a steep increase of 20E level. The supposition that biogenic amines act as intermediary between JH and 20E in the control of Drosophila reproduction is discussed (Rauschenbach, 2007).
Drosophila melanogaster can make appropriate choices among alternative flight options on the basis of the relative salience of competing visual cues. This choice behavior consists of early and late phases; the former requires activation of the dopaminergic system and mushroom bodies, whereas the latter is independent of these activities. Immunohistological analysis showed that mushroom bodies are densely innervated by dopaminergic axons. Thus, the circuit from the dopamine system to mushroom bodies is crucial for choice behavior in Drosophila (Zhang, 2007).
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