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).

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).

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).

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).

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).

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 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).

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).