InteractiveFly: GeneBrief

pale: Biological Overview | References


Gene name - pale

Synonyms -

Cytological map position - 65C3-65C3

Function - enzyme

Keywords - dopamine, catecholamine biosynthesis, sleep, arousal, visual attention, stress response, learning, sexual behavior, cuticle pigmentation

Symbol - ple

FlyBase ID: FBgn0005626

Genetic map position - 3L:6,706,455..6,712,624 [-]

Classification - Eukaryotic tyrosine hydroxylase

Cellular location - cytoplasmic



NCBI link: EntrezGene
pale orthologs: Biolitmine
Recent literature
Cichewicz, K., Garren, E. J., Adiele, C., Aso, Y., Wang, Z., Wu, M., Birman, S., Rubin, G. M. and Hirsh, J. (2016). A new brain dopamine deficient Drosophila and its pharmacological and genetic rescue. Genes Brain Behav [Epub ahead of print]. PubMed ID: 27762066
Summary:
Drosophila tyrosine hydroxylase (DTH) is the rate limiting enzyme for Dopamine (DA) biosynthesis. Viable brain DA deficient flies were previously generated using tissue selective GAL4-UAS binary expression rescue of a DTH null mutation and these flies show specific behavioral impairments. To circumvent the limitations of rescue via binary expression, this study achieved rescue utilizing genomically integrated mutant DTH. As expected, DA deficient flies have no detectable DTH or DA in the brain, and show reduced locomotor activity. This deficit can be rescued by L-DOPA/carbidopa feeding, similar to human Parkinson's disease treatment. Genetic rescue via GAL4/UAS-DTH was also successful, although this required the generation of a new UAS-DTH1 transgene devoid of most untranslated regions, since existing UAS-DTH transgenes express in the brain without a Gal4 driver via endogenous regulatory elements. A surprising finding of the newly constructed UAS-DTH1m is that it expresses DTH at an undetectable level when regulated by dopaminergic GAL4 drivers even when fully rescuing DA, indicating that DTH immunostaining is not necessarily a valid marker for DA expression. This finding necessitated optimizing DA immunohistochemistry, revealing details of DA innervation to the mushroom body and the central complex. When DA rescue is limited to specific DA neurons, DA does not diffuse beyond the DTH-expressing terminals, such that DA signaling can be limited to very specific brain regions.
Grover, S., Williams, M. E., Kaiser, R., Hughes, J. T., Gresham, L., Rebeiz, M. and Williams, T. M. (2018).. Augmentation of a wound response element accompanies the origin of a Hox-regulated Drosophila abdominal pigmentation trait. Dev Biol. PubMed ID: 29981311
Summary:
A challenge for evolutionary research is to uncover how new morphological traits evolve the coordinated spatial and temporal expression patterns of genes that govern their formation during development. Detailed studies are often limited to characterizing how one or a few genes contributed to a trait's emergence, and thus knowledge of how entire GRNs evolve their coordinated expression of each gene remains unresolved. The melanic color patterns decorating the male abdominal tergites of Drosophila melanogaster evolved in part by novel expression patterns for genes acting at the terminus of a pigment metabolic pathway, driven by cis-regulatory elements (CREs) with distinct mechanisms of Hox regulation. This study examined the expression and evolutionary histories of two important enzymes in this pathway, encoded by the pale and Ddc genes. While both genes exhibit dynamic patterns of expression, a robust pattern of Ddc expression specifically evolved in the lineage of fruit flies with pronounced melanic abdomens. Derived Ddc expression requires the activity of a CRE previously shown to activate expression in response to epidermal wounding. A binding site for the Grainy head transcription factor that promotes the ancestral wound healing function of this CRE was also shown to be required for abdominal activity. Together with previous findings in this system, this work shows how the GRN for a novel trait emerged by assembling unique yet similarly functioning CREs from heterogeneous starting points.
Poetini, M. R., Musachio, E. A. S., et al. (2021). Iron overload during the embryonic period develops hyperactive like behavior and dysregulation of biogenic amines in Drosophila melanogaster. Dev Biol 475: 80-90. PubMed ID: 33741348
Summary:
Iron (Fe) is used in various cellular functions, and a constant balance between its uptake, transport, storage, and use is necessary to maintain its homeostasis in the body. Changes in Fe metabolism with a consequent overload of this metal are related to neurological changes and cover a broad spectrum of diseases, mainly when these changes occur during the embryonic period. This work aimed to evaluate the effect of exposure to Fe overload during the embryonic period of Drosophila melanogaster. Progenitor flies (male and female) were exposed to ferrous sulfate (FeSO(4)) for ten days in concentrations of 0.5, 1, and 5 ​mM. After mating and oviposition, the progenitors were removed and the treatment bottles preserved, and the number of daily hatches and cumulative hatching of the first filial generation (F1) were counted. Subsequently, F1 flies (separated by sex) were subjected to behavioral tests such as negative geotaxis test, open field test, grooming, and aggression test. They have evaluated the levels of dopamine (DA), serotonin (5-HT), octopamine (OA), tryptophan and tyrosine hydroxylase (TH), acetylcholinesterase, reactive species, and the levels of Fe in the progenitor flies and F1. The Fe levels of F1 flies are directly proportional to what is incorporated during the period of embryonic development; a delay in hatching and a reduction in the number of the hatch of F1 flies exposed during the embryonic period to the 5mM Fe diet were observed, a fact that may be related to the reduction of the cell viability of the ovarian tissue of progenitor flies. The flies exposed to Fe (1 and 5 ​mM) showed an increase in locomotor activity (hyperactivity) and a significantly higher number of repetitive movements. In addition to a high number of aggressive encounters when compared to control flies. An increase was observed in the levels of biogenic amines DA and 5-HT and an increase in TH activity in flies exposed to Fe (1 and 5 ​mM) compared to the control group. It is concluded that the hyperactive-like behavior demonstrated in both sexes by F1 flies exposed to Fe may be associated with a dysregulation in the levels of DA and 5-HT since Fe is a cofactor of TH, which had its activity increased in this study. Therefore, more attention is needed during the embryonic development period for exposure to Fe overload.
Adedara, A. O., Babalola, A. D., Stephano, F., Awogbindin, I. O., Olopade, J. O., Rocha, J. B. T., Whitworth, A. J. and Abolaji, A. O. (2022). An assessment of the rescue action of resveratrol in parkin loss of function-induced oxidative stress in Drosophila melanogaster. Sci Rep 12(1): 3922. PubMed ID: 35273283
Summary:
Loss-of-function mutations in parkin is associated with onset of juvenile Parkinson's disease (PD). Resveratrol is a polyphenolic stilbene with neuroprotective activity. This study evaluated the rescue action of resveratrol in parkin mutant D. melanogaster. The control flies (w1118) received diet-containing 2% ethanol (vehicle), while the PD flies received diets-containing resveratrol (15, 30 and 60 mg/kg diet) for 21 days to assess survival rate. Consequently, similar treatments were carried out for 10 days to evaluate locomotor activity, oxidative stress and antioxidant markers. mRNA levels were determined of Superoxide dismutase 1 (Sod1, an antioxidant gene) and ple, which encodes tyrosine hydroxylase, the rate-limiting step in dopamine synthesis. The data showed that resveratrol improved survival rate and climbing activity of PD flies compared to untreated PD flies. Additionally, resveratrol protected against decreased activities of acetylcholinesterase and catalase and levels of non-protein thiols and total thiols displayed by PD flies. Moreover, resveratrol mitigated against parkin mutant-induced accumulations of hydrogen peroxide, nitric oxide and malondialdehyde. Resveratrol attenuated downregulation of ple and Sod1 and reduction in mitochondrial fluorescence intensity displayed by PD flies. Overall, resveratrol alleviated oxidative stress and locomotor deficit associated with parkin loss-of-function mutation and therefore might be useful for the management of PD.
Trisal, S., Aranha, M., Chodankar, A., VijayRaghavan, K. and Ramaswami, M. (2022). A Drosophila Circuit for Habituation Override. J Neurosci 42(14): 2930-2941. PubMed ID: 35232763
Summary:
Habituated animals retain a latent capacity for robust engagement with familiar stimuli. In most instances, the ability to override habituation is best explained by postulating that habituation arises from the potentiation of inhibitory inputs onto stimulus-encoding assemblies and that habituation override occurs through disinhibition. Previous work has shown that inhibitory plasticity contributes to specific forms of olfactory and gustatory habituation in Drosophila. This study analyzed how exposure to a novel stimulus causes override of gustatory (proboscis extension reflex; PER) habituation. While brief sucrose contact with tarsal hairs causes naive Drosophila to extend their proboscis, persistent exposure reduces PER to subsequent sucrose stimuli. This study shows that in so habituated animals, either brief exposure of the proboscis to yeast or direct thermogenetic activation of sensory neurons restores PER response to tarsal sucrose stimulation. Similar override of PER habituation can also be induced by brief thermogenetic activation of a population of tyrosine hydroxylase (TH)-positive neurons, a subset of which send projections to the subesophageal zone (SEZ). Significantly, sensory-neuron induced habituation override requires transmitter release from these TH-positive cells. Treatments that cause override specifically influence the habituated state, with no effect on the naive sucrose response across a range of concentrations. Taken together with other findings, these observations in female flies are consistent with a model in which novel taste stimuli trigger activity in dopaminergic neurons which, directly or indirectly, inhibit GABAergic cells that drive PER habituation. The implications of these findings for general mechanisms of attentional and sensory override of habituation are discussed.
Ryu, T. H., Subramanian, M., Yeom, E. and Yu, K. (2022). The prominin-like Gene Expressed in a Subset of Dopaminergic Neurons Regulates Locomotion in Drosophila. Mol Cells 45(9): 640-648. PubMed ID: 35993164
Summary:
CD133, also known as prominin-1, was first identified as a biomarker of mammalian cancer and neural stem cells. Previous studies have shown that the prominin-like (promL) gene, an orthologue of mammalian CD133 in Drosophila, plays a role in glucose and lipid metabolism, body growth, and longevity. Because locomotion is required for food sourcing and ultimately the regulation of metabolism, this study examined the function of promL in Drosophila locomotion. Both promL mutants and pan-neuronal promL inhibition flies displayed reduced spontaneous locomotor activity. As dopamine is known to modulate locomotion, the effects of promL inhibition on the dopamine concentration and mRNA expression levels of tyrosine hydroxylase (TH) and DOPA decarboxylase (Ddc), the enzymes that are responsible for dopamine biosynthesis, were examined in the heads of flies. Compared with those in control flies, the levels of dopamine and the mRNAs encoding TH and Ddc were lower in promL mutant and pan-neuronal promL inhibition flies. In addition, an immunostaining analysis revealed that, compared with control flies, promL mutant and pan-neuronal promL inhibition flies had lower levels of the TH protein in protocerebral anterior medial (PAM) neurons, a subset of dopaminergic neurons. Inhibition of promL in these PAM neurons reduced the locomotor activity of the flies. Overall, these findings indicate that promL expressed in PAM dopaminergic neurons regulates locomotion by controlling dopamine synthesis in Drosophila.
Ryu, T. H., Subramanian, M., Yeom, E. and Yu, K. (2022). The prominin-like Gene Expressed in a Subset of Dopaminergic Neurons Regulates Locomotion in Drosophila. Mol Cells 45(9): 640-648. PubMed ID: 35993164
Summary:
CD133, also known as prominin-1, was first identified as a biomarker of mammalian cancer and neural stem cells. Previous studies have shown that the prominin-like (promL) gene, an orthologue of mammalian CD133 in Drosophila, plays a role in glucose and lipid metabolism, body growth, and longevity. Because locomotion is required for food sourcing and ultimately the regulation of metabolism, this study examined the function of promL in Drosophila locomotion. Both promL mutants and pan-neuronal promL inhibition flies displayed reduced spontaneous locomotor activity. As dopamine is known to modulate locomotion, the effects of promL inhibition on the dopamine concentration and mRNA expression levels of tyrosine hydroxylase (TH) and DOPA decarboxylase (Ddc), the enzymes that are responsible for dopamine biosynthesis, were examined in the heads of flies. Compared with those in control flies, the levels of dopamine and the mRNAs encoding TH and Ddc were lower in promL mutant and pan-neuronal promL inhibition flies. In addition, an immunostaining analysis revealed that, compared with control flies, promL mutant and pan-neuronal promL inhibition flies had lower levels of the TH protein in protocerebral anterior medial (PAM) neurons, a subset of dopaminergic neurons. Inhibition of promL in these PAM neurons reduced the locomotor activity of the flies. Overall, these findings indicate that promL expressed in PAM dopaminergic neurons regulates locomotion by controlling dopamine synthesis in Drosophila.
BIOLOGICAL OVERVIEW

Dopamine (DA) synthesis depends on the concerted action of the enzymes tyrosine hydroxylase (TH) and DOPA decarboxylase. TH catalyzes the first and rate-limiting step in catecholamine biosynthesis and mediates the oxidation of tyrosine to 3,4-dihydroxy-L-phenylalanine (L-DOPA). DOPA decarboxylase may then metabolize L-DOPA to DA. In Drosophila, DA plays a role in various complex neuronal processes such as sleep and arousal (Andretic, 2005; Ganguly-Fitzgerald, 2006; Kume, 2005) visual attention (Ye, 2004), stress response (Neckameyer, 2005), learning (Schwaerzel, 2003), and sexual behavior (Chang, 2006). In the larval and adult CNS, DA and TH immunoreactivity appear to localize to the same neurons (Budnik,1988; Nössel, 1992). Thus, TH-immunoreactive neurons are commonly referred to as dopaminergic neurons (Monastirioti, 1999). Central TH neurons specifically synthesize only one out of two possible TH splice variants (Birman, 1995; Vié, 1999) from a primary transcript encoded by the pale locus (Budnik, 1987, Neckameyer, 1993). The second TH splice variant locates to epidermal cells and serves a vital role in cuticle biosynthesis (Friggi-Grelin, 2003). Genetic as well as pharmacological inhibition of TH activity suggests that catecholamine loss decreases locomotor activity (Pendleton, 2002; Pendleton, 2005; Vömel, 2008).

Previous studies have described the morphology of TH-producing neurons (TH neurons) in the VG with immunocytochemistry (Lundell, 1994; Konrad, 1987; Friggi-Grelin, 2003). This study used the same Th-gal4 driver line as well as a commercially available monoclonal mouse-anti-TH antibody. In general, both approaches revealed identical neurons. Ventral midline neurons in a1-7, however, showed very weak or even lacked Th-gal4-driven mCD8-GFP expression (Vömel, 2008).

Inferred from Th-gal4-driven marker gene expression as well as TH immunostainings, the ventral ganglion (VG) contains two morphologically different TH neuron groups: The first group comprises three ventral median TH neurons (vmTH neurons) in t1, and a single vmTH neuron in each neuromere from t2 to a7. Their cell bodies locate to the midline beneath the VM tracts. The second TH neuron group consists of a bilateral pair of dorso-lateral TH neurons (dlTH neurons) with somata residing at the height of the DL (D: dorsal, C: central, V: ventral) and M: medial, I: intermedial, L: lateral) tracts in each neuromere from a1-7. Longitudinal TH projections are adjacent to the VL, beneath the CI, and close to the VM/DM tracts. Neurites of the vmTH neurons project dorsally and then appear to join longitudinal TH projections between the DM and VM tracts. The vmTH neurons of t1 also initially project dorsally until their neurites reach the height of the VM tracts. The neurites then diverge and build up a loop enclosing the DM/VM and CI tracts on each side of the neuromere. These neurite loops seem to establish a transversal connection between all longitudinal TH projections within the VG. As opposed to the vmTH neurites, the neurites of the dlTH neurons run ventrally and form fine longitudinal projections along the VL tracts. There, TH neurites divide and proceed in a loop to the median neuropil. Between the CI and the VM tracts, the TH neurites running beneath TP 4 join bilateral fine longitudinal projections somewhat ventro-laterally to the VM tracts. The TH neurites then proceed dorsally until they converge with the upper branch of the TH neurite loop in a prominent longitudinal projection between the DM and VM tracts. TH neurites ramify heavily in the neuropil between the DM/VM and the CI tracts (Vömel, 2008).

To identify the input and output compartments of TH neurons, the neuronal compartment markers neuronal synaptobrevin-GFP (SybGFP), and Drosophila Down syndrome adhesion molecule [17.1]-GFP (DscamGFP) were ectopically expressed. Th-gal4-driven SybGFP showed a dotted distribution within the VG and largely mimicked the mCD8GFP expression pattern. In t1-3, SybGFP uniformly labeled all TH neurites. The neuromeres a1-5 typically contained less SybGFP than t1-3 and a6-7, since labeling was restricted to TH neuron somata and longitudinal TH projections adjacent to the DM/VM and the VL tracts. Transversal TH neurites appeared to lack SybGFP in a1-5. In a6-7, high amounts of SybGFP accumulated around the DM/VM tracts and also located to transversal TH projections. Particularly, the dorsal branches of the bilateral transversal neurite loops showed intense SybGFP labeling. In contrast to SybGFP, Th-gal4-driven SytGFP strongly labeled segmentally reiterated neurite arborizations next to the VM tracts. These median arborizations appeared to belong to the transversal TH neurites connecting both neuropil hemispheres. SytGFP further located to longitudinal projections running along the VL tracts and to the neuropil between the CI and VM tracts. Compared to other neuromeres, a6-7 seemed to contain the highest concentration of SytGFP. There, SytGFP particularly accumulated around the longitudinal TH projections adjacent to the VL tracts and in the ventral branches of the transversal TH neurite loops. Th-gal4-driven DscamGFP mainly labeled the somata and primary neurites of the dlTH neurons and the longitudinal TH projections adjacent to the VL tracts. Furthermore, in a6-7, DscamGFP located to longitudinal TH projections next to the DM tracts and to the ventral parts of the transversal neurite loops. The neuropil between the CI and VM tracts, however, showed only faint DscamGFP labeling (Vömel, 2008).

Increased dopamine level enhances male-male courtship in Drosophila

Sexual behavior between males is observed in many species, but the biological factors involved are poorly known. In mammals, manipulation of dopamine has revealed the role of this neuromodulator on male sexual behavior. This study used genetic and pharmacological approaches to manipulate the dopamine level in dopaminergic cells in Drosophila and investigated the consequence of this manipulation on male-male courtship behavior. Enhanced dopamine (DA) levels was achieved by overexpressing pale (ple) in DA neurons using the GAL4-UAS system. pale encodes Tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis. Males with increased dopamine level showed enhanced propensity to court other males but did not change their courtship toward virgin females, general olfactory response, general gustatory response, or locomotor activity. The results indicate that the high intensity of male-male interaction shown by these manipulated males was related to their altered sensory perception of other males (Liu, 2008).

In nature, animals use multiple sensory cues to recognize conspecifics and to choose a potentially suitable mate for reproduction. In Drosophila melanogaster, courtship behavior, which precedes mating, mostly depends on visual, acoustic, and chemosensory signals exchanged by the two partner flies that alternatively and reciprocally act as the courter and as the courtee. Some of these sensory signals can stimulate male or female courtship, whereas others can inhibit sexual behavior. Although wild-type male flies rarely show male-male courtship, the frequency and intensity of this behavior can be strongly increased by genetic manipulation (Greenspan, 2000). For example, substantial male-male courtship has been found in flies with mutation of fruitless (fru), prospero, or quick-to-court genes. The ectopic expression of a female-dominant form of the transformer gene (traF) and the presence of the mini-white transgene (mw) (Zhang, 1995; Hing, 1996) are also associated with male-male courtship. Several brain regions involved in male-male courtship behavior have been identified by targeted expression of traF and fru in male brains under the control of specific galactosidase-4 (GAL4) lines. For example, when traF is expressed in either antennal lobes or in mushroom bodies, feminized male flies showed high male-male courtship behavior (Ferveur, 1995; O'Dell, 1995). The genetic alteration of either a subset of peripheral taste neurons or glial cells located in the olfactory centers of the brain can also affect male-male courtship without altering male-female courtship (Lacaille, 2007; Grosjean, 2008; Liu, 2008 and references therein).

In mammals, male sexual behavior is regulated by several neuromodulators, including dopamine (DA) and serotonin (5-HT). Pharmacological manipulation of DA or 5-HT systems in mammalian can alter their sexual behavior. These two substances seem to exert reciprocal effects, with DA facilitating and 5-HT inhibiting male sexual behavior (Melis, 1995; Hull, 2004). Although the possible effect of 5-HT on Drosophila male sexual orientation was discussed (Zhang, 1995) and DA was shown to modulate male arousal and visual perception during heterosexual courtship (Andretic, 2005; Kume, 2005), locomotor activity (Pendleton, 2002), female sexual receptivity (Neckameyer, 1998a), male courtship conditioning (Neckameyer, 1998b), and ethanol-induced courtship disinhibition (Lee, 2008), the effect of DA on male-male courtship behavior remains poorly known (Liu, 2008).

This study used genetic and pharmacological tools to modulate DA level in DA cells. The effect was evaluated of these manipulations on both the DA level in male brain and the intensity of Drosophila male courtship behavior in relation with sensory perception. The results showed that increased DA level was correlated with a more intense male-male courtship toward other mature males (Liu, 2008).

Increased DA amount in the brain of genetically and/or pharmacologically manipulated males correlates with increased propensity to court other wild-type males. This effect is clearly related to the targeting of the UAS-TH transgene (overexpression of tyrosine hydroxylase) in DA cells. Although TH-GAL4 driver is not active in all DA cells, it was efficient enough to strongly increase both the amount of DA in the brain and the intensity of male-male courtship behavior of manipulated males. A recent paper also reported that dopamine is crucial for the ethanol-induced male-male courtship (Lee, 2008). Given that TH-GAL4 can drive TH overexpression in both nervous system and the hypoderm during all developmental stages, elav-GAL4 was used to overexpress TH only in the nervous system: this was sufficient to strongly enhance male-male courtship. Because DA synthesized within the nervous system can be secreted out of nervous system to function elsewhere, the alteration of male-male courtship behavior may possibly result from the DA secreted out of the nervous system (Liu, 2008).

In TH-GAL4/UAS-TH males, as in ebony1 (e1) mutant males, DA was kept at a high level during most developmental stages. ebony encodes an enzyme, beta-alanyl-dopamine synthase, that regulates β-alanyl conjugation of dopamine and histamine, thus 'trapping' these biogenic amines preventing their further function. These males showed much higher Chaining indices (ChIs) than that of males fed with L-DOPA only during their adult life. This behavioral difference may be attributable to the effect of DA on the development of adult sensory organ. Similarly, the pharmacological attempt to decrease the level of DA and of male-male courtship only induced a partial effect: TH-GAL4/UAS-TH adult males fed with drugs only during adulthood showed a strongly decreased but not completely abolished male-male courtship . This indicates that increased DA level during preimaginal development also affects adult male-male courtship behavior. Therefore, the high amplitude of male-male courtship behavior shown by TH-GAL4/UAS-TH and e1 males could result from the cumulative effects of DA elevation during both preimaginal and adult developmental stages. (1) The 'preimaginal' effect could alter the development of adult sensory systems. This supports previous studies showing that that manipulation of DA during late larval developmental stage affects the formation of adult sensory nervous system (in particular, the visual system) (Neckameyer, 1996; Neckameyer, 2001). (2) The 'adult' effect could alter the signaling role of DA in the male nervous system (Liu, 2008).

The data indicate that increased DA synthesis during development has no general debilitating effect on behavior. Increased DA level in male brain is correlated with relatively specific behavioral defects: male-male courtship behavior is enhanced, whereas male-female courtship, general olfactory/gustatory response, and spontaneous locomotor activity remained unaltered. Moreover, although manipulated males showed reduced ability of discrimination between the sexes, they still clearly preferred females when they had the chance to choose between the sexes. This indicates that increased levels of DA strongly alter male perception of other male's sensory signals but have a weaken effect on male perception of female's sensory signals, which results in a courtship toward both females and males without drastic loss of sexual discrimination. A similar behavioral phenotype was induced by manipulating the level of extracellular glutamate in the synapses of the brain regions involved in pheromonal perception (Grosjean, 2008). In this case, males showed a stimulation instead of an avoidance toward 7-tricosene, the principal male contact pheromone. Given that the agonist of DA receptors affect the threshold of sex arousal (Andretic, 2005), the apparently unaffected male-female courting index (CI) shown by TH-GAL4/UAS-TH males may be a ceiling effect because this level may be at its maximum. However, thisis not the case, because TH-GAL4/UAS-TH males tested under red light showed a much lower CI of control females than wild-type males. These results are similar to those of Andretic (2005), which showed that males fed with agonist of DA receptor, methamphetamine, had an altered processing of visual signals (Liu, 2008).

The effect of DA on Drosophila locomotor activity was shown by pale mutant (Pendleton, 2002) and by fly fed with methamphetamine (Andretic, 2005), whereas DA function in fumin mutant did not alter spontaneous short-term locomotor activity (Kume, 2005). The results showed that males with increased DA level displayed a normal spontaneous short-term locomotor activity, which was somewhat in accordance with the study of Kume. The contradiction of locomotor activity between the current result and that of Andretic may be attributable to different experimental manipulation. Two other studies demonstrated that DA depletion can affect two aspects of courtship behavior: female sexual receptivity and male courtship conditioning toward immature males (Neckameyer, 1998a; Neckameyer, 1998b). The fact that DA depletion does not change male-female courtship (Neckameyer, 1998a) but affects male-male courtship indicates that this manipulation induces sex-specific effect on courtship behavior (Liu, 2008).

Courtship behavior results from the coordination of a series of motor activities evoked in response to multiple sensory cues exchanged during courtship. Dopamine concentration and receptor activation have important roles in many behavioral situations (Schultz, 2002; Andretic, 2005). Dopamine can modulate neurotransmitter action on target neurons and coordinate the output of neuronal ensembles to generate behavioral patterns of varying complexity (Nusbaum, 2001; LeBeau, 2005). Dopamine is an important neuromodulator for Drosophila, and DA neurons have enormous fields of innervation covering essentially most neuropil regions of the fly brain (Monastirioti, 1999). This supports the current results that DA neurons play a crucial role in integrating information from multiple sensory modalities (Liu, 2008).

The comparison between visually deprived or olfactory/auditory-deprived TH-GAL4/UAS-TH males with control sibling males suggests that the strongly enhanced male-male courtship results from the altered perception of these sensory signals. Although male visual and olfactory stimuli seem to induce a strong effect on TH-GAL4/UAS-TH male-male courtship, acoustic signals (not emitted by decapitated targets) also play a role, yet with less importance compared with the effect induced by the two former sensory modalities. It is not known whether the intense male-male courtship behavior shown by manipulated flies is stimulated by male visual, olfactory, and auditory signals that are normally aversive (Greenspan, 2000) or whether these males have defective perception of their inhibitory effect. If the latter hypothesis is true, it means that, in the absence of these sensory signals, other yet unknown male sensory stimuli may be able to stimulate male courtship. Because NAM-operated TH-GAL4/UAS-TH males showed significantly decreased male-male courtship behavior in chaining and paired courtship assays, the strong male-male courtship behavior shown by intact TH-GAL4/UAS-TH males may not simply be attributable to the poor perception of aversive signals but rather to the perception of male olfactory/auditory signals that they find stimulating as in the case of genderblind mutant males toward the aversive male pheromone (Grosjean, 2008; Liu, 2008).

Although the data clearly allow ruling out of the effect of the mini-white gene (mw) on the high male-male courtship behavior observed in this study, it is worth drawing a parallel with the high level of intermale courtship shown by pairs of mw males, which tended to be also drastically reduced in the absence of visual cues (Hing, 1996; Liu, 2008).

This study reveals the intriguing effect of DA on Drosophila male-male courtship behavior. At the moment, it is not known whether the results can be generalized to other species. It is not known how the perturbation of DA precisely affects male-male courtship in Drosophila. Additional dissection of Drosophila male-male courtship may help in understanding the fine mechanisms underlying sensory communication regulating interindividual behavior (Liu, 2008).

Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis via regulation of Breathless turnover

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).

The DA biosynthesis machinery is highly conserved in mammalian and Drosophila systems. DA production requires the tightly regulated collaboration of two enzymatic pathways. The DA pathway itself is initiated by the conversion of tyrosine to L-3-4-dihydroxyl-phenylalanine (L-Dopa), which is subsequently converted to DA. The rate-limiting step in the pathway, conversion of tyrosine to L-Dopa, is catalyzed by the enzyme tyrosine hydroxylase (TH), which is encoded by pale (ple) in Drosophila (Neckameyer, 1993). Human and Drosophila TH share 60% amino acid similarity (Neckameyer, 2005a). TH catalytic activity requires and is regulated by the cofactor, tetrahydrobiopterin (BH4), synthesized via the pteridine pathway (Krishnakumar, 2000). The initiating and limiting component of BH4 biosynthesis, and therefore, of DA production, is the activity of the enzyme GTP cyclohydrolase I (GTPCH). The human and Drosophila GTPCH proteins share 80% similarity (McLean, 1993). Mutations in the Drosophila GTPCH gene Punch (Pu) result in dose-dependent deficiencies in DA pools. Similarly, mutations in the human GTPCH locus lead to the hereditary diseases hyperphenylalaninemia and Dopa-responsive dystonia. Both Pu and ple mutations are homozygous lethal during embryogenesis. A third regulatory gene of DA synthesis, Catecholamines up (Catsup), acts as a negative regulator of both GTPCH and TH. The protein encoded by Catsup contains seven predicted transmembrane domains and functions in post-translational modification of both enzymes. Homozygous loss-of-function alleles of Catsup also show developmental defects and die in an allele-dependent fashion, from embryogenesis to pupariation (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).

Mapping of serotonin, dopamine, and histamine in relation to different clock neurons in the brain of Drosophila

Several sets of clock neurons cooperate to generate circadian activity rhythms in Drosophila. To extend the knowledge on neurotransmitters in the clock circuitry, this study analyzed the distribution of some biogenic amines in relation to identified clock neurons. This was accomplished by employing clock neuron-specific GAL4 lines driving green fluorescent protein (GFP) expression, combined with immunocytochemistry with antisera against serotonin, histamine, and tyrosine hydroxylase (for dopamine). In the larval and adult brain, serotonin-immunoreactive (-IR) neuron processes are in close proximity of both the dendrites and the dorsal terminals of the major clock neurons, the s-LN(v)s. Additionally, the terminals of the l-LN(v) clock neurons and serotonergic processes converge in the distal medulla. No histamine (HA)-IR processes contact the s-LN(v)s in the larval brain, but possibly impinge on the dorsal clock neurons, DN2. In the adult brain, HA-IR axons of the extraocular eyelet photoreceptors terminate on the dendritic branches of the LN(v)s. A few tyrosine hydroxylase (TH)-IR processes were seen close to the dorsal terminals of the s-LN(v)s, but not their dendrites, in the larval and adult brain. TH-IR processes also converge with the distal medulla branches of the l-LN(v)s in adults. None of the monoamines was detectable in the different clock neurons. By using an imaging system to monitor intracellular Ca(2+) levels in dissociated GFP-labeled larval s-LN(v)s, loaded with Fura-2, it was demonstrated that application of serotonin induced dose-dependent decreases in Ca(2+). Thus, serotonergic neurons form functional inputs on the s-LN(v)s in the larval brain and possibly also in adults (Hamasaka, 2006).

Eight different types of dopaminergic neurons innervate the Drosophila mushroom body neuropil: anatomical and physiological heterogeneity

Tyrosine hydroxylase (TH-GAL4) expression and anti-TH immunoreactivity were examined in the Drosophila protocerebrum and single cell clones of the TH-GAL4 neurons were characterized. Eight clusters of putative dopaminergic neurons were characterized. Neurons in three of the clusters project to the mushroom body neuropil: PAM neurons project to the medial portion of the horizontal lobes; PPL1 neurons project to the vertical lobes, the junction area, the heel and distal peduncle; and PPL2ab neurons project to the calyx. Five types of PPL1 neurons were discovered that innervate different zones of the mushroom body lobes. Functional imaging experiments showed that the dopaminergic processes in four of the zones differ in response properties to odor, electric shock, or following the pairing of odor and electric shock. These results indicate that distinct dopaminergic neurons define separate zones of the mushroom body lobes and are probably involved in different functions. Differences in functional response properties of these neurons suggest that they are involved in different behavioral processes (Mao, 2009).

One of the more surprising discoveries is the complexity of DA innervation of the mushroom body neuropil. Past models had envisioned DA inputs as providing uniform innervation of the mushroom bodies to convey unconditioned stimulus (US) information. The current results revealed three components of putative DA innervation of the mushroom bodies, the PAM DA neuron-horizontal lobe system, the PPL1 DA neuron-vertical lobe-junction-heel system, and the PPL2ab DA neuron-calyx system. The complexity layers further upon separating the PPL1 system into the subcomponents that innervate different zones of the vertical lobes. The PPL1 neurons that project towards the mushroom body lobes displayed five distinct paths, each involving a specific sub-area of the vertical lobes, the lower stalk/junction, the heel and distal peduncle. In a screen for GAL4 enhancer trap strains that label specific subsets of mushroom body intrinsic and extrinsic neurons (MBINs and MBENs, respectively), it has been found that MB extrinsic neurons (MBENs) arborize in only specific zones of the lobes and form two to five zones in each lobe. It was proposed that the lobes are divided into smaller units for carrying out different functional activities. The current findings on the projection patterns of PPL1 neurons, as well as the functional imaging, support the idea of lobe subcompartmentation. TH-GAL4 expression in the horizontal lobes was noticeably less widespread and intense than TH expression in the same area. Coincidentally, significantly fewer PAM neurons, located in the most anterior part of the protocerebrum, expressed TH-GAL4 compared to TH. Although some PAM neurons express TH-GAL4 and innervate the distal tips of the horizontal lobes, the coverage is incomplete. Therefore, past studies of DA function using TH-GAL4 may have underestimated the role of the PAM neuron-horizontal lobe component (Mao, 2009).

Another unexpected finding that was made is that the calyx is innervated by putative DA terminals. Two types of neurons that are members of the PPL2ab cluster were found that innervate the calyx and are putatively DA. Previous models suggest that DA neurons influence mushroom body output neurons through synapses in the lobes. The current results suggest that another set of interactions occurs at synapses in the calyx (Mao, 2009).

Overall, these neuroanatomical results indicate that the circuitry of DA influence on the mushroom bodies is enormously complex. Many of the neurites from the three cell body clusters (PAM, PPL1, PPL2ab) extending into the mushroom body neuropil are varicose and contain boutons that may reflect a presynaptic function. This predicts that the neurotransmitter DA is released onto discrete zones of the mushroom body neuropil from the putative presynaptic terminals from different types of TH-GAL4 neurons. If so, then where are the postsynaptic regions of the putative DA neurons? Three broad possibilities are considered for input regions. First, some TH-GAL4 neuron cell bodies exhibit short and fine processes. It is possible that these processes provide some synaptic or non-synaptic input to the neurons. Second, the primary neurite from the PAM, PPL1, and PPL2ab neurons exhibit branching into fine processes en route to the mushroom body neuropil. These fine processes could provide a dendritic function to the neurons. If so, then large, multiple regions of the adult brain, including the mushroom body satellite area, the area between the vertical and the horizontal lobes, may provide the inputs for DA release in the mushroom body neuropil. Third, it is possible that the TH-GAL4 neurons are both pre- and postsynaptic to the mushroom body neurons, such that mushroom body activity would stimulate the release of DA from the nearby DA terminals and this release would stimulate DA receptors expressed by the mushroom body neurons (Mao, 2009).

The response of putative DA fibers to electric shock and odor were examined in four different subcompartments of the mushroom body lobes, the tips of α and α′ lobes, the upper stalk area, and the lower stalk/junction area, each shown to be innervated by a distinct subtype of PPL1 neuron. It was discovered that the DA fibers exhibit different responses to these stimuli. For instance, the α tip responded much more strongly to electric shock than to MCH, however, the α′ tip responded much more strongly to MCH than to electric shock. These observations suggest that the different PPL1 neurons have different functions, with some PPL1 neurons providing strong modulatory input to the mushroom body fibers in the α tip in response to electric shock and other PPL1 neurons providing strong modulatory input to the mushroom body fibers in the α′ lobe in response to odor stimulation. They also suggest that the different compartments of the mushroom body neuropil have different functions. It is also intriguing that the DA fibers respond to both electric shock and odor stimuli. These findings make clear that the DA fibers cannot be providing information about the US stimulus of electric shock only, as originally envisioned, but must provide a much richer input to the mushroom bodies presumably in support of their function in olfactory conditioning. It is not known which neurons convey information about electric shock and odors to the PPL1 neurons. It is possible that the different response properties of these neurons arise from different synaptic inputs onto their cell bodies or onto the neurite en route to the mushroom body neuropil (Mao, 2009).

The effect of pairing an odor with electric shock on subsequent response to the odor was examined. The responses observed were quite variable; the most consistent change was that the odor responses generally decreased after forward conditioning, backward conditioning, and the conditioned stimulus-only in the α tip, α′ tip, and the upper stalk areas. Since these treatments shared the element of a 1 min odor exposure, it is possible that this exposure caused the subsequent decrease. There is no current understanding of the mechanism for this change or it relevance, if any. There was no such decrease in response in the lower stalk/junction, indicating that DA processes that innervate different sub-areas of the mushroom bodies behave differently following exposure to certain combinations of odor and electric shock (Mao, 2009).

Previous work studied the activity using a FRET-based calcium reporter of DA terminals in a region that covers parts of the α lobe, β lobe and γ lobe. It was reported that this area responded weakly to odor but strongly to electric shock. It was also reported that pairing one of two odors with electric shock prolonged the subsequent calcium response to the paired odor 1 min after training. It was proposed that the prolonged calcium response represents the gain of relevance by a previously neutral stimulus, and that DA neurons thereby predict the reinforcement. The current study failed to observe any amplitude or time course change in response of the DA neurons 10 min after training that might be indicative of acquired predictive value or salience. However, the two studies are not directly comparable since the response properties were examined at different times after training and different calcium reporters were used (Mao, 2009).

The current functional imaging was performed in the vertical lobes and junction area, since TH-GAL4 expression in the horizontal lobes does not adequately reproduce the intensity and extent of tyrosine hydroxylase expression in these areas. In addition, the DA innervation in calyx is not dense or intense enough to be visible with the current drivers and reporter (Mao, 2009).

Dopaminergic modulation of arousal in Drosophila

Arousal levels in the brain set thresholds for behavior, from simple to complex. The mechanistic underpinnings of the various phenomena comprising arousal, however, are still poorly understood. Drosophila behaviors have been studied that span different levels of arousal, from sleep to visual perception to psychostimulant responses. This study investigated neurobiological mechanisms of arousal in the Drosophila brain by a combined behavioral, genetic, pharmacological, and electrophysiological approach. Administration of methamphetamine (METH) suppresses sleep and promotes active wakefulness, whereas an inhibitor of dopamine synthesis promotes sleep. METH affects courtship behavior by increasing sexual arousal while decreasing successful sexual performance. Electrophysiological recordings from the medial protocerebrum of wild-type flies showed that METH ingestion has rapid and detrimental effects on a brain response associated with perception of visual stimuli. Recordings in genetically manipulated animals show that dopaminergic transmission (interfered with by using the Tyrosine hydroxylase gene promoter to drive Shibire in Dopamine positive cells) is required for these responses and that visual-processing deficits caused by attenuated dopaminergic transmission can be rescued by METH. Therefore, changes in dopamine levels differentially affect arousal for behaviors of varying complexity. Complex behaviors, such as visual perception, degenerate when dopamine levels are either too high or too low, in accordance with the inverted-U hypothesis of dopamine action in the mammalian brain. Simpler behaviors, such as sleep and locomotion, show graded responses that follow changes in dopamine level (Andretic, 2005).

Behavioral performance is determined to a large degree by an animalís level of arousal. An optimal arousal level is required for proper cognitive and motor performance, and it is the result of an interaction between mechanisms controlling endogenous states and stimuli from the environment. An understanding of neural mechanisms determining the arousal level underlying behaviors is essential for understanding both normal and aberrant states (Andretic, 2005).

The extensive literature on the effects of psychostimulants such as cocaine, amphetamine, and methamphetamine on brain function and behavior universally point to the arousing properties of these drugs. The multiple behavioral consequences of psychostimulant administration have all been associated with changes in the extracellular concentration of the neurotransmitters dopamine, serotonin, and noradrenaline. Psychostimulants either block transporters for these neurotransmitters, thereby preventing their clearance from the synaptic cleft (cocaine), or in addition promote their release from the presynaptic neuron (amphetamines). The arousing impact of psychostimulants depends on the dose given and spans a range of cognitive and motor effects, from those that are beneficial at low doses to those that are detrimental for cognitive and behavioral functioning at higher doses. Low doses in humans improve selective attention, reaction time, and accuracy. In contrast, high doses induce hyperactive and stereotypical locomotor activity in rodents and lead to impulsive and distractive behavior in humans and rodents. Psychostimulants are also widely used in treatments for narcolepsy; their arousing effects suppress sleep and consolidate periods of wakefulness. Furthermore, psychostimulants counteract the negative effects of sleep deprivation by improving cognitive and motor performance in humans during periods of extended wakefulness. Whereas hyperactivity and the reinforcing effects of psychostimulants leading to addiction have been studied extensively, much less is known about the arousal-inducing effects at low doses (Andretic, 2005).

Attempts to understand the consequences for sleep and arousal of low psychostimulant doses have focused on the role of dopamine. Wake-promoting effects of METH in rodents have most often been associated with the enhancement of dopaminergic transmission, decreased activity of dopamine transporters, and stimulation of D1 and D2 receptors. Studies in rodents, in which the wake-promoting effects of amphetamine and/or methylphenidate were compared to those of the stimulant caffeine, indicated that psychostimulant effects depend on the enhancement of dopaminergic transmission whereas caffeine effects do not. Electrophysiological and microdialysis studies from mammalian brains argue for the activity of noradrenergic neurons from the locus coeruleus in maintaining wakefulness. However, there seems to be agreement that activation of dopaminergic transmission predominates as a mechanism through which psychostimulants maintain wakefulness (Andretic, 2005).

As in mammals, Drosophila exhibits behavioral states spanning the full continuum of arousal, from general anesthesia and sleep to visual discrimination. Inactive states that predominate during the night, and which are associated with increased arousal thresholds and decreased brain activity, are analogous to sleep in mammals. On the other extreme of this continuum, volatilized cocaine induces hyperactive and stereotypical behaviors, and intermittent exposure to the same drug concentration will lead to behavioral sensitization. Recent advances in recording of brain activity from flies responding to sensory stimuli have made it possible to correlate behavioral performance with changes in local field potentials (LFPs) in the animalís brain (van Swinderen, 2003). These electrophysiological studies in Drosophila showed not only that distinct arousal states in the fly can be determined by looking at locomotor output (the only method available in the past) but also that they can be inferred from analyzing changes in brain activity (Andretic, 2005).

The results of this study show that changes in dopaminergic transmission modulate levels of arousal in Drosophila for behaviors of varying complexity. Sleep and locomotion show graded responses that follow changes in dopamine level, and drug concentrations that promote wakefulness were detrimental to courtship success. Neural correlates of visual perception, on the other hand, degenerate when dopamine levels are either too high or too low (Andretic, 2005).

The most obvious effect of feeding METH to Drosophila is a general arousal increase manifested as a decrease in average sleep time (even in flies that have significantly increased sleep need) and an increase in average activity when awake. The following similar behavioral effects have been reported in mammals: a decrease in sleep amount, consolidation of periods of wakefulness, and improved vigilance during extended sleep deprivation. These findings complement previously published work that studied the behavioral effects of volatilized-cocaine exposure in Drosophila and addressed issues of acute behavioral sensitization, whereas the feeding protocol investigates chronic changes in arousal. It is important to note that METH feeding to flies has in no case induced the same kind of stereotypical, hyperkinetic, or uncoordinated behaviors seen with volatilized cocaine (Andretic, 2005).

The opposing effects on average sleep time of METH versus 3IY, drugs that have been shown to have opposing effects on the concentration of dopamine, agree well with those in mammalian studies in which administration of low concentrations of D1 and D2 dopamine-receptor agonists promote active wakefulness and in which blockade of those receptors leads to sedation (Isaac, 2003). Decreasing dopamine concentration with 3IY has a selective effect on sleep, whereas increasing it with l affects both average sleep and activity, suggesting that sleep time is more sensitive than locomotor activity to perturbations in the neurotransmitter concentration. Similarly, in rodents, the METH-induced decrease in sleep is inseparable from its motor-activating effects, whereas another wake-promoting substance, modafinil, whose activity appears to be mediated by dopamine, does not lead to increased locomotor activity. Thus, drugs, such as 3IY or modafinil, that selectively influence the dopaminergic system produce a more selective effect on sleep. In Drosophila, as in mammals, locomotor-activating effects of METH at low doses are likely to be mediated by the combined action of the drug on multiple transmitter systems (Andretic, 2005).

It has been proposed that arousal levels in the fly are a function of the degree of coupling among various parts of the nervous system. This was seen physiologically during sleep in the uncoupling of peripheral responses to visual stimuli from the CNS (van Swinderen, 2003) and in the uncoupling of movement from brain local field potentials (LFPs) during a putative intermediate stage of sleep (van Swinderen, 2003). At the high end of the arousal scale, it is seen in the increased coherence between central brain sites during a visual-discrimination task (Andretic, 2005).

In light of these findings, it may seem paradoxical that METH reduces the correlation between brain LFPs and movement while at the same time producing an increase in wakefulness and locomotor activity. This apparent paradox may be explained, however, by reference to another previous finding: Presentation of a visual stimulus to a fly also reduces the correlation between brain LFPs and movement. Both of these results suggest that the LFP-movement correlation decreases when the fly is 'distracted' by something: the visual stimulus in one case and METH in the other. For the visual stimulus, it is likely that the LFP-movement coupling is being replaced by a specific coupling, such as the coherence increase seen during visual discrimination, among other brain regions. METH, in contrast, is likely to be inducing nonspecific brain activity, uncoupled from the flyís sensory input (Andretic, 2005).

A further possible consequence of a nonspecific, METH-induced uncoupling relates to the restorative functions of sleep. If one considers that brain LFPs are generally uncoupled from movement and from sensory input in the intermediate state preceding quiescent sleep, then perhaps some of the restorative functions of sleep are being carried out during that time. If so, then the dramatic reduction in quiescent sleep in METH-fed flies and the suppression of a homeostatic response to sleep deprivation in these flies may result from the partial fulfillment of some sleep functions during their prolonged periods in this state of LFP uncoupling from sensory stimuli and movement (Andretic, 2005).

The finding that central visual perception is impaired by manipulation of dopamine, whether by increasing its action (METH) or by suppressing its release using ectopic shibire, agrees well with the hypothesis of an inverted-U functional-response curve corresponding to increasing dopamine signaling in prefrontal cortex. When human subjects are given low doses of amphetamine, their cognitive performance will depend on the level of dopaminergic signaling in the prefrontal cortex. The same concentration of amphetamine enhancedperformance for subjects with low prefrontal dopamine and caused deterioration in subjects with high prefrontal dopamine (Andretic, 2005).

The effects of METH on courtship may resemble those on visual perception with respect to the requirement for an optimal setting of arousal level. The METH-induced increase in sexual arousal is defined by the latency to initiate courtship; however, this high level of arousal appears to be detrimental for the completion of the entire complex behavioral sequence. Males may persist in particular courtship steps longer because of their inability adequately to interpret and respond to female behavior, consistent with the finding that central visual processing is impaired after METH administration. Dopaminergic effects on courtship have been shown previously, where inhibition of dopamine synthesis during development in males increased the latency to initiate courtship and to copulate (Neckameyer, 2001). The possibility that the effects reported in this study on visual perception and courtship might be due merely to primary visual defects is unlikely for several reasons. First, dopamine-depleted flies (Neckameyer, 2001) are normal for phototaxis. Second, although vision is not essential for courtship, the lack of it produces an increase in courtship latency but no impairment to copulation. Thus, the effects observed in the current study are likely to be central rather than peripheral and more involved in the modulation of overall arousal than in the primary sensory response (Andretic, 2005).

Arousal has been defined operationally as a state in which 'an animal is more responsive to a wide variety of external stimuli spanning sensory modalities and is more motorically active'. The current results suggest that the situation is more complex and nuanced. Not all behaviors show a graded arousal change correlating with changes in dopaminergic activity. METH concentrations that lead to a gradual increase in locomotor activity (without hyperactivity or loss of coordination) and a decrease in average sleep time produce maladaptive arousal in the context of more complex behaviors. Performance of complex behaviors degenerates when dopamine levels are either too high or too low, as seen also in mammalian brain. Although the idea that the observed effects of dopamine in Drosophila are acting primarily through its effect on arousal, the possibility is recognized of alternative explanations involving more restricted actions ,yet to be identified, of central dopaminergic circuits on particular aspects of behavior (Andretic, 2005).

The findings suggest that courtship and visual perception in Drosophila display a complex response to changes in dopaminergic activity, whereas sleep and locomotor activity give a more linear response. Similar observations have been reported on the actions of drugs, such as volatile general anesthetics, that decrease general arousal, where complex behaviors are more susceptible to the sedating effects of these agents. This commonality suggests that neural mechanisms governing behaviors of varying degrees of complexity have evolved corresponding degrees of sensitivity to changes in the neuromodulatory milieu of an organism, with more primitive or basic behaviors showing greater robustness. On a more practical note, this finding indicates that locomotion alone is too crude an indicator of changes in the arousal of a fly, especially for more complex behaviors (Andretic, 2005).

These explanations fit well with the role of dopamine as a key component of neuromodulatory 'value' systems in the brain. Such systems have been shown to play an important role in conferring salience on particular stimuli, either intrinsically as part of the animalís heredity or adaptively when paired with specific sensory inputs. In vertebrates, these functions have been attributed to diffusely ascending systems, employing biogenic amines as neurotransmitters. In the fly brain, the dopaminergic and octopaminergic systems have been shown to play such a role in aversive and appetitive conditioning, respectively. These systems are generally nonspecific, both anatomically, in the sense that their projections are diffuse, and physiologically, in the sense that they provide general reinforcement (positive or negative) to more restrictively stimulated sensory or motor systems. The interaction between relatively specific sensory and motor systems, on the one hand, and relatively nonspecific value systems, on the other, thus underlies much of the brainís combinatorial versatility (Andretic, 2005).

In this formulation, too much dopaminergic transmission would be as dysfunctional as too little, disrupting the balance between specific input and value-system modulation. Thus, nonspecific arousal producing sleep loss, increased activity, and overly stereotypical, unsuccessful courtship would have a common etiology with the failure of the visual response: a failure of regulation of the animalís value system (Andretic, 2005).

Roles of dopamine in circadian rhythmicity and extreme light sensitivity of circadian entrainment

Light has profound behavioral effects on almost all animals, and nocturnal animals show sensitivity to extremely low light levels. Crepuscular, i.e., dawn/dusk-active animals such as Drosophila melanogaster are thought to show far less sensitivity to light. This study reports that Drosophila respond to extremely low levels of monochromatic blue light. Light levels three to four orders of magnitude lower than previously believed impact circadian entrainment and the light-induced stimulation of locomotion known as positive behavioral masking. GAL4;UAS-mediated rescue of tyrosine hydroxylase (DTH) mutant (ple) flies was used to study the roles of dopamine in these processes. Evidence is presented for two roles of dopamine in circadian behaviors. First, rescue with either a wild-type DTH or a DTH mutant lacking neural expression leads to weak circadian rhythmicity, indicating a role for strictly regulated DTH and dopamine in robust circadian rhythmicity. Second, the DTH rescue strain deficient in neural dopamine selectively shows a defect in circadian entrainment to low light, whereas another response to light, positive masking, has normal light sensitivity. These findings imply separable pathways from light input to the behavioral outputs of masking versus circadian entrainment, with only the latter dependent on dopamine (Hirsch, 2010).

Sensitivity to extremely low levels of light is most commonly found in nocturnal animals. These animals, such as nocturnal geckos or insects such as nocturnal hawkmoths, can not only sense extremely low levels of light but can also discern colors at light intensities well below those to which diurnal animals are sensitive. Humans and diurnal vertebrates lose color vision at light intensities comparable to dim moonlight at irradiances of 3-10 nW/cm2. In contrast, nocturnal hawkmoths and geckos can discern colors even at intensities of ~0.01-0.3 nW/cm2 and normally function in starlight, ~0.001 nW/cm2. Extreme light sensitivity in nocturnal insects commonly involves adaptations to their compound eyes to allow summation of photons from many individual ommatidia. These visual system adaptations are not seen in diurnal insects such as the fruit fly Drosophila melanogaster. Accordingly, current data accord Drosophila with rather modest light sensitivity. For light-dependent entrainment of circadian rhythmicity, ~40 nW/cm2 blue light was thought to be required, although subsequent studies show entrainment by 1-5 nW/cm2 white light. Wild-type flies are now thought to entrain at ~0.04 nW/cm2 blue light (C. Helfrich-Forster, personal communication to Hirsch, 2010). An intensity of ~0.5 nW/cm2 white light is reported to cause positive behavioral masking, the largely circadian clock-independent stimulation of locomotion. For comparison, this study found that a dark-adapted human observer loses the ability to perceive the diffuse planar blue light sources used in the present study at intensities of ~0.01-0.03 nW/cm2. This intensity is difficult to compare to published human perception studies, which commonly use short duration flashes of focal light (Hirsch, 2010).

This study found unexpectedly strong light sensitivity for Drosophila melanogaster, with behavioral masking and circadian entrainment at intensities as low as 0.001 nW/cm2 and at least two roles for dopamine in circadian rhythmicity. First, DTH rescue flies showed poor behavioral rhythmicity in constant dark conditions, independent of whether dopamine levels were rescued in the nervous system. Second, it was found that neuronal DTH rescue flies lacking neuronal dopamine showed reduced light sensitivity for circadian entrainment, whereas light sensitivity of behavioral masking was unaffected. Dopamine has several roles in Drosophila neural function, from modulation of locomotor behaviors and arousal states to learning and memory, but a role for dopamine in insect light-dependent circadian behavioral entrainment is novel (Hirsch, 2010).

The two circadian phenotypes likely represent separate roles for dopamine, presumably in different regions of the nervous system, because reduced amplitude of rhythmicity, as seen in DTH rescue lines, is normally associated with higher rather than lower efficacy of reentrainment. The dopaminergic system in Drosophila is highly rhythmic, as evidenced by rhythmicity in responsiveness to dopamine agonists and by the rhythmic transcription of the tyrosine hydroxylase gene ple, which encodes the rate-limiting enzyme in dopamine biosynthesis. The rhythmicity of the ple transcript may explain the poor rhythmicity in ple rescue animals. These animals have near-normal levels of brain dopamine in an apparently normal cellular pattern, but the inclusion of the GAL4 transcription factor into the regulatory cascade will almost certainly interfere with normal temporal cycling of the DTH transcript. Note that significant diurnal variation in levels of brain dopamine in brain extracts have not been detected, but this does not preclude diurnal variation in dopamine neuron subsets (Hirsch, 2010).

Low-light circadian entrainment is disrupted in the brain dopamine-deficient DTHgFS±;ple flies. The simplest mechanism for the disruption of low-light circadian entrainment would be due to alterations in the photoreceptive pathway, which could be via cryptochrome (CRY) or visual photoreceptors. There is some support for dopaminergic involvement in the CRY pathway, because Sathyanarayanan (2008) identified ple in a screen for genes that, when targeted by RNA interference, have a strong inhibitory effect on light-dependent degradation of CRY and timeless (TIM) in cultured cells. This could indicate a positive role for dopamine in light-dependent degradation of these molecules, providing a potential mechanism for the reduced light sensitivity for circadian entrainment that was observed in the absence of dopamine (Hirsch, 2010).

Alternatively, it is known that visual photoreceptors are involved in dim-light entrainment because genetic loss of all photoreceptive visual organs results in at least a three-order-of-magnitude reduction in blue light sensitivity for circadian entrainment. Analogous studies in mice show an ~60-fold reduction in dim-light sensitivity for entrainment in animals lacking both rods and cones (Hirsch, 2010).

A role for dopamine in fly visual function has some support in that cyclic AMP (cAMP) can slow the response to light in a preparation of isolated Drosophila photoreceptors (Chyb, 1999), and this effect can be mimicked by application of octopamine or dopamine, an effect interpreted as enhanced adaptation to dark. Dopamine signaling, via cAMP second-messenger pathways, is not currently considered part of the main insect visual transduction pathway. However, dopamine involvement could have been missed if it has an exclusive role in a neural pathway selectively required for circadian entrainment by dim light (Hirsch, 2010).

There is strong support of a role for dopamine functioning in the vertebrate retina, which makes visual involvement of dopamine in the fly all the more likely. The vertebrate retina contains autonomous circadian oscillators that are thought to allow the retina to prepare for the large difference in light intensity between day and night. Central to this rhythmicity are opposing and rhythmic roles for melatonin and dopamine, with release of each modulator inhibiting synthesis and/or release of the other. The best defined role for dopamine in the vertebrate circadian oscillator is in entraining fetal rodents prior to light exposure, a capacity lost in adults. This role of dopamine could be related to the roles that have been uncovered in adult Drosophila (Hirsch, 2010).

The selective effect of neural dopamine on low-light entrainment versus low-light masking behavior implies separable pathways involved in modulating these behaviors, a novel finding because previous studies have only identified circadian components with parallel effects on masking (Mazzoni, 2005). The best defined synaptic connections from eye to circadian neurons are the projections from the Drosophila eyelet, a remnant of the larval photoreceptive Bolwig's organ. This photoreceptive organ makes projections that terminate in close apposition to neurites from the small and large ventral lateral neurons, neurons key to circadian rhythmicity. Connections from the main visual photoreceptors to these circadian neurons must be indirect because the rod-like outer photoreceptor ommatidia terminate in the optic lamina, and the cone-like central ommatidia terminate in the optic medulla. Nonetheless, dopamine could be acting as a neuromodulator in any of these pathways to increase sensitivity to a light-dependent signal. The genetic tools available in Drosophila should prove useful to precisely identify these pathways (Hirsch, 2010).

Dopamine acts through Cryptochrome to promote acute arousal in Drosophila

The fruit fly, Drosophila melanogaster, is generally diurnal, but a few mutant strains, such as the circadian clock mutant ClkJrk, have been described as nocturnal. This study reports that increased nighttime activity of Clk mutants is mediated by high levels of the circadian photoreceptor Cryptochrome (Cry) in large ventral lateral neurons (l-LNvs). Cry expression is also required for nighttime activity in mutants that have high dopamine signaling. In fact, dopamine signaling is elevated in ClkJrk mutants and acts through Cry to promote the nocturnal activity of this mutant. Notably, dopamine and Cry are required for acute arousal upon sensory stimulation. Because dopamine signaling and Cry levels are typically high at night, this may explain why a chronic increase in levels of these molecules produces sustained nighttime activity. It is proposed that Cry has a distinct role in acute responses to sensory stimuli: (1) circadian responses to light, as previously reported, and (2) noncircadian effects on arousal, as shown in this study (Kumar, 2012).

Both dopamine and Cry are required for acute arousal at night. An arousal-promoting role for dopamine is supported by earlier studies. Dopamine transporter mutants were shown to exhibit a decreased arousal threshold, whereas the pale mutants exhibit an increased arousal threshold. This effect on arousal reflects a novel role for dopamine in sensory responses at night. Cry has not been implicated in arousal, although it promotes neural activity in a light-dependent manner. As in the case of the neural activity assay, this study found that arousal in response to sensory stimuli is reduced but not eliminated by the cryb mutant, indicating that the mechanism is distinct from the circadian response that is eliminated by cryb. Both neural activity and behavioral arousal responses are eliminated by the cry0 mutant, suggesting that the neural response underlies the behavioral effect. It is proposed that Cry is required at multiple levels for acute responses to sensory stimuli. In the case of circadian photoreception, it is absolutely required for phase-shifting in response to pulses of light, although not for entrainment to LD cycles. In the case of responses to sensory stimuli, again it is required for the startle response. Any effects of Cry on light-induced activity (physiological or behavioral) are likely to be acute, since Cry gets degraded with increased light treatment. Interestingly, in two different species of Bactrocera, cry mRNA levels are positively correlated with the timing of mating, which is also indicative of a regulated response required for a specific purpose. A chronic effect is seen only in the case of Drosophila Clk mutants, where levels of Cry are considerably higher than normal, and dopamine signaling is also elevated. It is hypothesized that Cry only promotes nocturnal activity in flies with chronically elevated dopamine signaling because dopamine acts as a trigger to activate Cry. However, this activation may be different from activation in a circadian context, given that different mechanisms appear to underlie the circadian and arousal-promoting roles of Cry. Dopamine- and Cry-mediated locomotor activity is restricted largely to the night because of light-induced Cry degradation and light-induced inhibition of dopamine signaling (Kumar, 2012).

At night, animals sleep, and the arousal threshold is increased. However, they still need to be able to respond in case of sudden events. It is speculated that dopamine and Cry are essential for this. In the case of Cry, it may arouse the animal and also reset the clock. For instance, the immediate response of an animal to a pulse of light at night is to wake up, which may be driven by the arousal-promoting role of Cry. In addition, the circadian clock must be reset, which requires the circadian function of Cry. Whether or not these roles of Cry are conserved, it is speculated that dopamine functions similarly in mammals. Interestingly, melanopsin, which is the circadian photoreceptor in mammals (analogous to Cry in flies), is regulated by dopamine in intrinsically photosensitive retinal ganglion cells (ipRGCs). Like Cry, melanopsin is also required for acute behavioral responses to light, specifically for sleep induction in nocturnal animals during the day. These ipRGCs have been proposed as functionally similar to l-LNvs, so a conserved function for the relevant molecules is intriguing. Finally, it is noted that elevated dopamine has been linked to increased nighttime activity in humans, which are, of course, diurnal like Drosophila. People with Sundown syndrome or nocturnal delirium show increased agitation and sleep disturbances in the early evening, which can be treated with anti-psychotic medications that target dopamine signaling (Kumar, 2012).

A developmental role for catecholamines in Drosophila behavior

Tyrosine hydroxylase (TH), the enzyme which catalyzes the conversion of tyrosine to L-DOPA and is the rate limiting step in catecholamine biosynthesis, is genetically expressed during development in Drosophila. Null mutant alleles of the single copy gene which codes for this enzyme are developmentally lethal as is a conditional TH mutant at its restrictive temperature. In adult flies, inhibition of TH by alpha-methyl-p-tyrosine (alphaMT) decreases locomotor activity in a dose-dependent manner. This behavioral effect is accompanied by reductions in brain levels of dopamine, the primary CNS catecholamine in Drosophila, and can be prevented by the coadministration of L-DOPA. Similar effects are found with reserpine and at the restrictive temperature in flies with a temperature conditional mutation for TH. In agreement with published studies in mammals, inhibition of TH by alphaMT during Drosophila development results in enhanced expression of this enzyme in the progeny of surviving adults. This biochemical outcome is accompanied behaviorally by increased sensitivity to the locomotor effects of both alphaMT and reserpine, drugs which act via depletion of brain catecholamines. Since TH is the rate limiting enzyme responsible for the conversion of tyrosine to L-DOPA and L-DOPA is converted to dopamine by aromatic amino acid decarboxylase (AAAD), the results indicate that depletion of catecholamine levels in the fly embryo results in increased dopamine biosynthesis in the next generation accompanied by alterations in behavior (Pendleton, 2005).

Tissue-specific developmental requirements of Drosophila tyrosine hydroxylase isoforms

Drosophila tyrosine hydroxylase (DTH) is a key enzyme in dopamine (DA) biosynthesis, which is expressed in neural and hypodermal DA-synthesizing cells. Two DTH isoforms are produced in flies through tissue-specific alternative splicing that show distinct regulatory properties. In this study each DTH isoform was selectively expressed in vivo in a pale (ple, i.e., DTH-deficient) mutant background. The embryonic lethality of ple can be rescued by expression of the hypodermal, but not the neural, DTH isoform in all DA cells, indicating that the hypoderm isoform is absolutely required for cuticle biosynthesis and survival in Drosophila. In addition, new observations are reported on the consequences of DTH overexpression in the CNS and hypoderm. The results provide evidence that tissue-specific alternative splicing of the DTH gene is a vital process in Drosophila development (Friggi-Grelin, 2003).

Neuroarchitecture of aminergic systems in the larval ventral ganglion of Drosophila melanogaster

Biogenic amines are important signaling molecules in the central nervous system of both vertebrates and invertebrates. In the fruit fly Drosophila melanogaster, biogenic amines take part in the regulation of various vital physiological processes such as feeding, learning/memory, locomotion, sexual behavior, and sleep/arousal. Consequently, several morphological studies have analyzed the distribution of aminergic neurons in the CNS. Previous descriptions, however, did not determine the exact spatial location of aminergic neurite arborizations within the neuropil. The release sites and pre-/postsynaptic compartments of aminergic neurons also remained largely unidentified. This study used gal4-driven marker gene expression and immunocytochemistry to map presumed serotonergic (5-HT), dopaminergic, and tyraminergic/octopaminergic neurons in the thoracic and abdominal neuromeres of the Drosophila larval ventral ganglion relying on Fasciclin2-immunoreactive tracts as three-dimensional landmarks. With tyrosine hydroxylase- (TH) or tyrosine decarboxylase 2 (TDC2)-specific gal4-drivers, the distribution of ectopically expressed neuronal compartment markers was examined in presumptive dopaminergic TH and tyraminergic/octopaminergic TDC2 neurons, respectively. The results suggest that thoracic and abdominal 5-HT and TH neurons are exclusively interneurons whereas most TDC2 neurons are efferent. 5-HT and TH neurons are ideally positioned to integrate sensory information and to modulate neuronal transmission within the ventral ganglion, while most TDC2 neurons appear to act peripherally. In contrast to 5-HT neurons, TH and TDC2 neurons each comprise morphologically different neuron subsets with separated in- and output compartments in specific neuropil regions. The three-dimensional mapping of aminergic neurons now facilitates the identification of neuronal network contacts and co-localized signaling molecules, as exemplified for DOPA decarboxylase-synthesizing neurons that co-express crustacean cardioactive peptide and myoinhibiting peptides (Vömel, 2008).

This study used gal4-driven marker gene expression and immunocytochemistry to three-dimensionally map presumed serotonergic, dopaminergic and tyraminergic/octopaminergic neurons within the Fas2 landmark system of the larval VG. Furthermore, several ectopically expressed pre- and postsynaptic markers were employed to reveal the in- and output compartments of presumptive dopaminergic TH and tyraminergic/octopaminergic TDC2 neurons. The results allow comparison of the segmental distribution patterns of aminergic neurons and to trace aminergic projections to defined neuropil areas within the VG. In the following, the morphology of aminergic neurons are related to known biogenic amine (BA) functions and describes putative neuronal network interactions with other VG neurons. This work also exemplifies how Fas2-based mapping can simplify the identification of co-localized signaling molecules, and allocate all neurons within the complex Ddc-gal4 expression pattern to distinct neuron subsets (Vömel, 2008).

Throughout the insects, similar neuron groups synthesize BAs. These groups typically comprise only few neurons with large branching patterns. In agreement with previous studies, 5-HT neurons in t1-a8 of the Drosophila larval VG represent interneurons with intrasegmental neurites. The 5-HT neurons of a8, however, appear to supply only the neuropil of a7, but not that of a8 and the adjacent 'terminal plexus'. Like 5-HT neurons, the presumptive dopaminergic TH neurons lack peripheral projections and appear to exclusively represent interneurons. In contrast, presumptive tyraminergic/octopaminergic TDC2 neurons mostly represent efferent vumTDC2 neurons. The vumTDC2 neurons obviously project to larval body wall muscles including M1 and M2 since these muscles showed TA- and OA-immunoreactive type II boutons. In a8, dorsally located dmTDC2 neurons send axons through the associated segmental nerves, and hence are efferent neurons as well. These dmTDC2 neurons probably innervate the reproductive tract in the adult female fly. Besides the dmTDC2 neurons of a8, typically two additional dmTDC2 neurons reside in the dorsal cortex between the last subesophageal neuromere and t1. These dmTDC2 neurons were not described in previous morphological studies on TA- and OA-/TβH-immunoreactive neurons. Nevertheless, all dmTDC2 neurons in the VG consistently showed strong Tdc2-gal4-driven mCD8GFP expression as well as TßH immunoreactivity. Thus, they likely synthesize both TA and OA. Although their neurites could not be traced, the dmTDC2 neurons resemble a pair of anterior medial neurons in locusts and crickets that localize to t1 and innervate the anterior connectives. Alternatively, dmTDC2 neurons may correspond to a single dorsal unpaired median neuron which resides in t1 of the locust and supplies the subesophageal nerves. Like dmTDC2 neurons, pmTDC2 neurons are probably interneurons as well. The soma position of pmTDC2 neurons highly resembles that of descending OA-immunoreactive interneurons detected in the subesophageal and thoracic neuromeres of bees, crickets, cockroaches, locusts, and moths (Vömel, 2008).

Within the larval VG of Drosophila, aminergic neurons typically show a segmentally reiterated distribution. The number of aminergic modules, however, often varies between different neuromeres. 5-HT neurons, for instance, typically occur as two bilateral pairs per neuromere. Yet, t1 comprises three 5-HT neuron pairs and a8 only one pair. The presumptive dopaminergic TH neurons also lack a strict serial homology since three ventral median TH (vmTH) neurons are present in t1, but only one in t2-a7. Furthermore, dlTH neurons locate to a1-7, but appear to be missing in t1-3. The neuromere a8 lacks TH neurons. The number of presumptive tyraminergic/octopaminergic TDC2 neurons differs between various neuromeres as well. Whereas t1 comprises one or two dmTDC2 neurons, comparable neurons are absent in t2-a7. Putative descending pmTDC2 interneurons localize to t1-a1, but appear to be missing in the remaining abdominal neuromeres. Taken together, the number of aminergic modules in t1 and a8 often deviated from that of t2-a7. This difference may-at least partially-reflect unique neuronal circuits in t1 and a8. While t1 specific physiological functions in larvae are unknown, a8 and the adjacent 'terminal plexus' are associated with the tail region, and hence contain a specific set of sensory neurons and motoneurons. The terminal neuromeres also supply several unique structures such as the spiracles or the anal pads (Vömel, 2008).

Besides the segmental differences in neuron number, the density of aminergic innervation and the amount of immunolabeling/marker gene expression varies between neuromeres as well. In particular, presumptive dopaminergic TH neurons show a striking neuromere-specific labeling pattern. Whereas a1-5 contain only few labeled TH projections, t1-3 and a6-7 comprise a comparably dense network of TH neurites. Similar to TH neurons, 5-HT neurons most densely innervate the neuropil of a7. Since a high extracellular concentration of 5-HT decreases the density of 5-HT-immunoreactive arborizations within the neuropil, a7 may represent a minor 5-HT release site. In contrast to a7, the neuropil of a8 and the adjacent 'terminal plexus' (which receive prominent peptidergic innervation) typically lack aminergic neurite arborizations. Consequently, larval aminergic neurons may play a subordinate role in tail-related physiological processes (Vömel, 2008).

To reveal putative synaptic in- and output zones of aminergic neurons, the neuronal compartment markers neuronal synaptobrevin-GFP, synaptotagmin 1-GFP, and Drosophila Down syndrome adhesion molecule [17.1]-GFP were employed. Neuronal synaptobrevin is a vesicle associated membrane protein that plays a role in the SNARE complex during vesicle transport and fusion with the plasma membrane. In accordance with this function, ectopically expressed neuronal synaptobrevin-GFP (SybGFP) accumulates at nerve terminals. SybGFP therefore served to define the presynaptic compartments of several Drosophila neurons, e.g. in the visual system. However, neuronal synaptobrevin is not restricted to small synaptic vesicles, but also locates to the membrane of large dense core vesicles, which contain BAs or neuropeptides. Consequently, in a7, SybGFP localized to putative release sites of presumptive serotonergic DDC neurons. SybGFP was also used to identify non-synaptic release sites in several peptidergic neurons. In aminergic neurons, the distribution of gal4-driven SybGFP highly resembled the corresponding mCD8GFP expression pattern. SybGFP localized in dotted patterns to aminergic neuron somata and associated neurites. It is therefore suggested that SybGFP does not exclusively label the presynaptic compartments of aminergic neurons. This fits to the assumption that ectopically expressed synaptic proteins can either localize to transport vesicles or non-synaptic compartments in peptidergic neurons. On the other hand, the ubiquitous distribution of SybGFP in aminergic neurites may suggest a widespread BA release/recycling from non-synaptic active sites. In mammals, BA release/recycling is not restricted to synapses. Vesicular monoamine transporters, which transport BAs into secretory vesicles, reside within neuron somata, axons, and dendrites. In Drosophila, the vesicular monoamine transporter DVMAT-A localizes to somata as well as neurites of several aminergic neurons both in the larval. Thus, the widespread distribution of SybGFP and DVMAT-A in aminergic neurons suggests that a considerable amount of aminergic vesicles resides at non-synaptic sites. Non-synaptic BA release/recycling might therefore play a major role for aminergic neuronal network signaling (Vömel, 2008).

Like neuronal synaptobrevin, synaptotagmins also represent integral membrane proteins of both small synaptic and large dense core vesicles. In Drosophila, the products of seven synaptotagmin genes localize to distinct neuronal compartments including the postsynaptic site. At the presynaptic site, synaptotagmin 1 does not participate in the SNARE complex, but acts as a Ca2+-sensor for synaptic vesicle fusion. Furthermore, synaptotagmin 1 appears to be the only crucial isoform for synaptic vesicle release. Consequently, a synaptotagmin 1-GFP fusion construct (SytGFP) was developed as a synaptic vesicle marker that specifically labels presynaptic sites. In aminergic neurons, the distribution pattern of SytGFP strikingly differed from the observed mCD8GFP and SybGFP labeling. Primary neurites of aminergic neurons always completely lacked SytGFP. Varicose neurite structures which were less evident in the mCD8GFP and SybGFP expression patterns showed strong SytGFP labeling. In agreement with the SytGFP distribution in other Drosophila neuron types, SytGFP hence appears to exclusively accumulate at the presynaptic sites of aminergic neurons. Thus, SytGFP represents a valuable marker to separate synapses from other neuronal compartments in aminergic neurons. However, since BA release is not restricted to synapses, SytGFP may not label all BA release sites of aminergic neurons. The sparse co-localization of SytGFP and SybGFP in aminergic neurites in fact suggests that aminergic vesicles-which are located distal to presynaptic sites-generally lack SytGFP. Consequently, non-synaptic BA release appears to be independent of synaptotagmin 1, but may depend on other synaptotagmin isoforms such as synaptotagmin α or β. The differing distribution of SytGFP and SybGFP also suggests that aminergic neurons contain several types of aminergic vesicles which are either associated with presynaptic or non-synaptic BA release. Alternatively, aminergic neurons may synthesize additional non-aminergic neurotransmitters like acetylcholine, GABA, or glutamate. Presumed octopaminergic efferent neurons, for instance, appear to release glutamate from type II terminals at the neuromuscular junction. In such neurons, SytGFP likely labels presynaptically located transmitter vesicles and may not reveal BA release sites (Vömel, 2008).

In contrast to SybGFP and SytGFP, ectopically expressed Drosophila Down syndrome adhesion molecule [17.1]-GFP (DscamGFP) localized to postsynaptic compartments and not to axons or presynaptic sites. Consequently, DscamGFP has served as dendrite marker in mushroom body lobe neurons. Aminergic neurons showed only weak DscamGFP labeling. DscamGFP primarily localized to neurites that lacked SytGFP labeling. Since SytGFP accumulates at presynaptic sites, DscamGFP appears to represent a valuable marker to define dendritic compartments in aminergic neurons (Vömel, 2008).

In 5-HT neurons, the distribution of ectopically expressed neuronal compartment markers was not examined since specific gal4 drivers are not available. The Ddc-gal4 driver induces marker gene expression not only in presumed serotonergic, but also in dopaminergic and additional peptidergic neurons. Consequently, neurites of different DDC neuron subsets overlap in specific neuropil areas. Presumptive serotonergic as well as dopaminergic DDC neurites, for instance, localize to the VG neuropil above the CI tracts. These conditions prevent an accurate description and interpretation of the compartment marker distribution in presumptive serotonergic DDC neurons. Thus, appropriate gal4 drivers (e.g. Dtph-gal4) are needed to further analyze 5-HT neuron morphology (Vömel, 2008).

5-HT neurons bifurcate strongly in the whole neuropil of t1-a7, and hence may influence various VG neurons including sensory, inter- as well as motoneurons. However, putative neuronal network contacts of 5-HT neurons were not examined since previous morphological studies on Drosophila 5-HT receptors did not describe the exact spatial location of the respective receptors in the larval VG (Vömel, 2008).

In TH neurons, the distribution of ectopically expressed mCD8GFP, SybGFP, SytGFP and DscamGFP differed only slightly. This might relate to the fact that the VG contains two different TH neuron groups, the vmTH and dlTH neurons, whose neurites contact each other at longitudinal projections. Consequently, pre- and postsynaptic compartments of both TH neuron groups appeared to overlap, e.g. at longitudinal projections next to the VL tracts. Since additional TH neurons located in the brain or subesophageal ganglia also innervate the VG, it was not possible to clarify which TH neuron group attributes to a particular neuronal projection. Several morphological findings, however, suggest that TH neurons possess distinct in- and output sites: Most strikingly, a1-5 contained less TH neurites labeled with mCD8GFP, SybGFP and DscamGFP, as compared to t1-3 and a6-7. In t1-a7, high amounts of SybGFP and SytGFP located to lateral longitudinal projections next to the VL tracts. These longitudinal TH neurites also contained a comparably high amount of DscamGFP, and hence likely represent synaptic in- as well as output compartments of different TH neuron groups. Besides lateral longitudinal TH projections, SybGFP and SytGFP also co-localized to the median neuropil between the DM/VM tracts. At least in a1-5, this neuropil area lacked DscamGFP, and hence probably represents a presynaptic output site of TH neurons. In a6-7, a comparably strong SybGFP and SytGFP labeling was observed in arborizations around transversal TH neurites. Whereas SybGFP mainly located to the dorsal branches of the transversal TH neurite loops, SytGFP and DscamGFP primarily labeled the ventral branches. Thus, the dorsal branches of the transversal TH neurite loops may represent non-synaptic DA release sites, while the ventral branches seem to comprise overlapping synaptic in- and output compartments of different TH neuron groups (Vömel, 2008).

Both vmTH and dorso-lateral TH (dlTH) neurons innervate distinct neuropil areas within the VG. The vmTH neurons send their primary neurites dorsally and then project through the dorsal part of the neuropil above Transversal projection (TP) 3. Since the dorsal neuropil comprises the dendritic compartments of most motoneurons, vmTH neurites are ideally located to modulate locomotor activity. This fits to the finding that DA application onto intact larval CNS-segmental preparations rapidly decreased the rhythmicity of CNS motor activity and synaptic vesicle release at the neuromuscular junction. Unlike vmTH neurons, dlTH neurons exclusively innervate the ventral part of the VG neuropil beneath TP 3. There, putative dendritic compartments of TH neurons mainly localize to lateral longitudinal and to transversal projections adjacent to the main output site of several afferent sensory neurons, e.g. tactile and proprioreceptive neurons. Thus, some TH neurons may receive synaptic input from specific sensory neurons. In contrast, TH neurons also seem to have output sites in the ventral part of the neuropil, and hence may influence the signal transmission between sensory neurons and interneurons. This fits to the finding that peptidergic apterous neurons, which appear to transmit sensory input from the VG to the brain, express DA receptors. Concomitantly, dendritic compartments of apterous neurons seem to reside adjacent to the putative DA release sites of TH neurons at the CI tracts. Besides the overlap between transversal TH neurites and sensory/interneuron projections in the ventral neuropil, TH neurons may influence several neuron groups at other locations within the VG. For instance, the putative synaptic output sites of TH neurons in the median neuropil between the DM/VM tracts overlap with presumptive input compartments of both interneurons and efferent neurons expressing peptides such as CCAP, corazonin, FMRFa, or MIP. Furthermore, the putative output sites at longitudinal TH projections next to the VL tracts lay adjacent to presumptive input compartments of e.g. efferent leucokininergic neurons (Vömel, 2008).

In the VG, most TDC2 neurons are efferent vumTDC2 neurons and showed a differential distribution of ectopically expressed SybGFP, SytGFP, and DscamGFP. The primary neurites and transversal projections of vumTDC2 neurons were labeled with DscamGFP, but lacked SytGFP. Therefore, these neurites likely represent dendritic input sites. This fits to the finding that vumTDC2 neurons possess output sites at larval body wall muscles. However, vumTDC2 neurites within the VG also contained high amounts of SybGFP, and hence may release TA/OA from non-synaptic sites. Besides vumTDC2 neurites, SybGFP strongly labeled longitudinal TDC2 neurites and associated arborizations in the dorso-lateral neuropil between TP 1 and 3. These TDC2 projections showed prominent SytGFP labeling and TßH immunoreactivity, but largely lacked DscamGFP. Thus, the dorsal part of the VG neuropil likely contains output compartments of TDC2 neurons. Since the larval brain seems to contain only tyramine- and no octopamine-immunoreactive neurons, these output sites likely derive from descending interneurons located in the subesophageal ganglia, dmTDC2 or pmTDC2 neurons. Noteworthy, the strong SybGFP and SytGFP labeling in TDC2 neurites projecting through the dorso-lateral neuropil of the VG overlapped with DscamGFP in transverse vumTDC2 neurites. Thus, descending TDC2 neurons may interact with vumTDC2 neurons (Vömel, 2008).

The VG comprises efferent vumTDC2 neurons as well as several putative TDC2 interneuron groups. Since all vumTDC2 neurons appear to have synapses at peripheral targets and dendrites in the dorsal neuropil, they show the typical motoneuron morphology. This corresponds to the finding that OA inhibited synaptic transmission at the neuromuscular junction by affecting both pre- and postsynaptic mechanisms. In addition, T?H mutant larvae, with altered levels of TA and OA, showed severe locomotion defects, which seemed to be linked to an imbalance between TA and OA signaling. Hence, vumTDC2 neurons likely regulate peripheral processes such as body wall muscle activity, whereas TDC2 interneurons centrally modulate the neuronal activity of motoneurons and interneurons involved in locomotor control. Interestingly, presumptive presynaptic compartments of descending TDC2 interneurons reside adjacent to transversal vumTDC2 dendrites. Thus, both TDC2 neuron groups may interact to modulate larval locomotor activity. Besides their function for locomotion, descending TDC2 neurons may also influence other neurons which project into the dorsal neuropil between TP 1 and 3. The putative output sites of TDC2 interneurons, for instance, lay adjacent to several peptidergic projections showing allatostatin-A, FMRFa, MIP or tachykinin immunoreactivity. However, nothing is known about TA/OA receptor distribution in the larval VG (Vömel, 2008).

During the morphological analysis of DDC neurons in the L3 larval VG, two DDC neuron groups were identified that obviously synthesize neither 5-HT nor DA. This corresponds to the previous finding that Ddc-gal4-driven marker gene expression is not restricted to presumptive serotonergic 5-HT and dopaminergic TH neurons. However, it cannot be excluded that the putative non-aminergic DDC neurons transiently synthesize BAs during other developmental stages. Ddc-gal4-driven mCD8GFP expression never revealed the dlTH neurons. This may relate to the fact that the onset of Ddc expression varies between different DDC neuron groups, and high DDC and TH levels do not temporally coincide. Taken together, these results suggest that-at least in the L3 larval VG-the Ddc-gal4 expression pattern 1) contains additional non-aminergic neurons, and 2) typically comprises most, but not all 5-HT and TH neurons. These particular characteristics of the Ddc-gal4 driver line should be carefully considered for the interpretation of studies that employed Ddc-gal4-driven expression to genetically manipulate serotonergic or dopaminergic neurons. Nevertheless, since all Ddc-gal4 expressing neurons within the VG showed at least faint DDC immunoreactivity, the Ddc-gal4 driver appears to restrict ectopical gene expression to DDC neurons. Noteworthy, the DDC neurons which lacked 5-HT and TH immunoreactivity showed corazonin and CCAP/MIP immunoreactivity respectively. In the moth Manduca sexta, these peptides play vital roles during ecdysis. At least the CCAP/MIP neurons are also necessary for the proper timing and execution of ecdysis behavior in Drosophila. Since dopaminergic DDC neurons regulate the titers of the molting hormones 20-hydroxyecdyson and juvenile hormone, both aminergic and peptidergic DDC neurons may interact to control ecdysis-related events. Recent findings indeed suggest that CCAP/MIP neurons modulate TH activity after eclosion to control the precise onset of tanning (Vömel, 2008).

Dopamine and sensory tissue development in Drosophila melanogaster

Dopamine is an important signaling molecule in the nervous system; it also plays a vital role in the development of diverse non-neuronal tissues in Drosophila. The current study demonstrates that males depleted of dopamine as third instar larvae (via inhibition of the biosynthetic enzyme tyrosine hydroxylase) demonstrated abnormalities in courtship behavior as adults. These defects were suggestive of abnormalities in sensory perception and/or processing. Electroretinograms (ERGs) of eyes from adults depleted of dopamine for 1 day as third instar larvae revealed diminished or absent on- and off-transients. These sensory defects were rescued by the addition of L-DOPA in conjunction with tyrosine hydroxylase inhibition during the larval stage. Depletion of dopamine in the first or second larval instar is lethal, but this is not due to a general inhibition of proliferative cells. To establish that dopamine is synthesized in tissues destined to become part of the adult sensory apparatus, transgenic lines were generated containing 1 or 4 kb of 5' upstream sequences from the Drosophila tyrosine hydroxylase gene (DTH) fused to the E. coli beta-galactosidase reporter. The DTH promoters directed expression of the reporter gene in discrete and consistent patterns within the imaginal discs, in addition to the expected expression in gonadal, brain, and cuticular tissues. The beta-galactosidase expression colocalized with tyrosine hydroxylase protein. These results are consistent with a developmental requirement for dopamine in the normal physiology of adult sensory tissues (Neckameyer, 2001).

Functional interactions between GTP cyclohydrolase I and tyrosine hydroxylase in Drosophila

Tyrosine hydroxylase requires the regulatory cofactor, tetrahydrobiopterin, for catecholamine biosynthesis. Because guanosine triphosphate cyclohydrolase I is the rate limiting enzyme for the synthesis of this cofactor, it has a key role in catecholamine production. GTP cyclohydrolase and tyrosine hydroxylase (TH) are co-localized in the Drosophila central nervous system. Mutations in the Punch locus, which encodes GTP cyclohydrolase, reduce TH activity; addition of cofactor to crude extracts could not fully rescue this activity in all mutant strains. The decrease in TH activity and the inability to increase it with added cofactor is not due to loss or decreased production of TH protein. TH co-immunoprecipitates with GTP cyclohydrolase when wild type head extracts are incubated with anti-GTP cyclohydrolase antibody. It is suggested that regulation of TH by its cofactor may require its association with GTP cyclohydrolase, and that the ability of GTP cyclohydrolase to associate with TH and its role in tetrahydrobiopterin synthesis may be separable functions of this enzyme. These results have important implications for understanding catecholamine-related neural diseases and designing strategies for gene therapy (Krishnakumar, 2000).

Differential regulation of Drosophila tyrosine hydroxylase isoforms by dopamine binding and cAMP-dependent phosphorylation

Tyrosine hydroxylase (TH) catalyzes the first step in dopamine biosynthesis in Drosophila as in vertebrates. Tissue-specific alternative splicing of the TH primary transcript generates two distinct TH isoforms in Drosophila, DTH I and DTH II. Expression of DTH I is restricted to the central nervous system, whereas DTH II is expressed in non-nervous tissues like the epidermis. The two enzymes present a single structural difference; DTH II specifically contains a very acidic segment of 71 amino acids inserted in the regulatory domain. The enzymatic and regulatory properties of vertebrate TH are generally conserved in insect TH, and the isoform DTH II presents unique characteristics. The two DTH isoforms were expressed as apoenzymes in Escherichia coli and purified by fast protein liquid chromatography. The recombinant DTH isoforms are enzymatically active in the presence of ferrous iron and a tetrahydropteridine co-substrate. However, the two enzymes differ in many of their properties. DTH II has a lower Km value for the co-substrate (6R)-tetrahydrobiopterin and requires a lower level of ferrous ion than DTH I to be activated. The two isoforms also have a different pH profile. As for mammalian TH, enzymatic activity of the Drosophila enzymes is decreased by dopamine binding, and this effect is dependent on ferrous iron levels. However, DTH II appears comparatively less sensitive than DTH I to dopamine inhibition. The central nervous system isoform DTH I is activated through phosphorylation by cAMP-dependent protein kinase (PKA) in the absence of dopamine. In contrast, activation of DTH II by PKA is manifest only in the presence of dopamine. Site-directed mutagenesis of Ser32, a serine residue occurring in a PKA site conserved in all known TH proteins, abolishes phosphorylation of both isoforms and activation by PKA. It is proposed that tissue-specific alternative splicing of TH has a functional role for differential regulation of dopamine biosynthesis in the nervous and non-nervous tissues of insects (Vié, 1999; full text of article).

A novel and major isoform of tyrosine hydroxylase in Drosophila is generated by alternative RNA processing

Two isoforms of Drosophila tyrosine hydroxylase protein are encoded via alternatively spliced exons. The major isoform (Type II) contains a novel acidic extension of 71 amino acids in the amino-terminal regulatory domain, which is likely to alter the regulatory properties of the tyrosine hydroxylase protein. The minor isoform (Type I) corresponds to the cDNA sequence reported previously. The structure of the Drosophila tyrosine hydroxylase (DTH) gene is reported and the diversity and tissue localization of its transcripts. At least three types of DTH mRNA are generated from a single primary transcript through alternative splicing and polyadenylation. Type II mRNA is the most abundant tyrosine hydroxylase transcript in Drosophila and is found predominantly in the hypoderm throughout all stages of development. Type I mRNA is present only in the CNS, where it is the primary form. The DTH transcripts detected in the CNS contain a longer 3'-untranslated region than the transcript expressed in the hypoderm, due to differential polyadenylation. In contrast, the same start site is used for DTH gene transcription in both tissues. These results show unexpected diversity in the DTH transcripts and point out possible mechanisms for differential regulation of tyrosine hydroxylase activity in the CNS and in the hypoderm (Birman, 1994).

Drosophila tyrosine hydroxylase is encoded by the pale locus

An 8 kb genomic fragment from the Drosophila tyrosine hydroxylase (DTH) locus was reintroduced into the genome of mutant pale flies. ple was first recovered as a recessive embryonic lethal by Jurgens (1984) and maps to the same chromosomal region as DTH (65A-E). Mutant ple alleles affect pigmentation of the cuticle (L-DOPA, the product of the reaction catalyzed by TH, is an intermediate in the cuticular sclerotization and pigmentation pathways) and catecholamine biosynthesis. This report demonstrates that ple does encode the structural gene for TH, since the reintroduced sequences rescue ple flies from lethality to viable adults. Morphological, immunocytochemical, and behavioral characterization of three transformant lines suggests that the reintroduced sequences contain the necessary elements for correct temporal and spatial expression of the gene, but may not contain all the sequences essential for quantitative expression (Neckameyer, 1993).


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