1Institute of Biology, Free University of Berlin, 14195 Berlin, Germany, and 2School of Life Sciences, Arizona State University, Tempe, Arizona 85287
Materials and Methods
Key words: octopamine; Drosophila; tyramine; motor behavior; modulation; invertebrate
Materials and Methods
Neuromodulators play a major role in activating and modifying CPG activity (Marder and Bucher, 2001). The central release of specific neuromodulators or mixtures of different modulators can initiate distinct motor patterns (Nusbaum et al., 2001). Pioneering studies in locusts have demonstrated that microinjection of the biogenic amine octopamine (OA) into distinct neuropil regions elicits either walking or flight motor patterns in isolated ventral nerve cords (Sombati and Hoyle, 1984). This has led to the "orchestration hypothesis" (Hoyle, 1985) assuming that neuromodulator release into specific neuropils configures distinct neural assemblies to produce coordinated network activity. Monoamines have also been assigned to aggression, motivation, and mood in vertebrates and invertebrates (Baier et al., 2002; Kravitz and Huber, 2003; Stevenson et al., 2005; Popova, 2006). Furthermore, specific cognitive functions have been assigned to monoamine codes, such as that in flies OA mediates appetitive learning but dopamine mediates aversive learning (Schwaerzel et al., 2003; Riemensperger et al., 2005). In mammals, dysfunctions in monoamine neurotransmission are implicated in neurological disorders, including Parkinson's disease, schizophrenia, anxiety, and depression (Kobayashi, 2001; Taylor et al., 2005).
However, recent work from areas as diverse as Parkinson's disease (Scholtissen et al., 2006) and Drosophila larval motor behavior suggests that the chemical codes producing specific motor behavior outputs are bouquets of different amines rather than single ones (Saraswati et al., 2004; Fox et al., 2006). This study tests this hypothesis by genetic and pharmacological dissection of flight behavior in Drosophila. For >20 years, OA has been assigned as the sole modulator controlling insect flight. In contrast, we demonstrate that flight is controlled by the combined action of OA and tyramine (TA). OA and TA are decarboxylation products of the amino acid tyrosine, with TA as the biological precursor of OA. In insect flight systems, OA assumes a variety of physiological roles affecting central neuron excitability (Ramirez and Pearson, 1991), synaptic transmission (Evans and O'Shea, 1979; Leitch et al., 2003), sensory sensitivity (Matheson, 1997), hormone release (Orchard et al., 1993), and muscle metabolism (Mentel et al., 2003). Almost every organ is equipped with OA receptors (Roeder, 1999). TA receptors have been cloned recently in many insect species (Blenau and Baumann, 2003), and physiological functions for TA have been demonstrated (McClung and Hirsh, 1999; Nagaya et al., 2002). The multiple possible levels of OA and TA action on Drosophila flight behavior are discussed.
|Materials and Methods|
Materials and Methods
Gal4 driver lines. The Drosophila tyrosine decarboxylase 2 (dTdc2)–galactosidase-4 (Gal4) driver is expressed in clusters of neurons throughout brain and nerve cord. The gene encoding the neuronal enzyme tyrosine decarboxylase (TDC) was identified recently, and the coding section of the yeast GAL4 gene was inserted into it, immediately before the coding start (Cole et al., 2005). We made use of this genetic tool, driving the apoptosis-inducing construct upstream activating sequence (UAS)–reaper and the construct for the enhanced green fluorescent protein (UAS–2xeGFP).
Reporter strains. The cell death gene reaper (White et al., 1994) acts dominantly to kill cells in which it is expressed. Because it has been incorporated into a UAS vector (Zhou et al., 1997), cell-specific ablation can be accomplished efficiently and accurately. The F1 transheterozygote offspring of the dTdc2–Gal4 x UAS–reaper cross served as the experimental strain. Parent dTdc2–Gal4 and UAS–reaper strains were used as controls. The white-eyed w1118 strain was also chosen as control line, because it is the original nonrecombinant line from which the dTdc2–Gal4 and the UAS–reaper strains have been created. dTdc2–Gal4 and UAS–reaper were backcrossed with white, and the progeny was used as heterozygous control. For visualization of octopaminergic and tyraminergic cells, dTdc2–Gal4 virgins were crossed with UAS–2xeGFP (two times enhanced green fluorescent protein) (Halfon et al., 2002) males.
Treatments for behavioral rescue experiments
Octopamine. Flies were raised on OA-containing medium. To obtain an OA (O0250; Sigma, St. Louis, MO) concentration of 10 mg/ml, each vial containing 15 ml of freshly prepared standard food was supplemented with 150 mg of octopamine diluted in 900 µl of distilled water. The OA solution was added while the food was still liquid but at a temperature below 50°C. Distilled water without OA (also 900 µl) was added to control vials. Four-day-old flies were transferred to the vials for oviposition and removed after 24 h. The progeny was raised on the OA-supplemented food and used for experiments later.
Yohimbine. To feed yohimbine (YH) (Y3125; Sigma), a 5% sucrose (S1888; Sigma) solution with or without yohimbine added (10 mg/ml) was pipetted onto five pieces of filter paper in cylindrical vials before transferring 10–20 mutants into the vials. After 1–2 h, the animals were singled out and prepared for testing.
Heat shock. Flies (TßHnM18 hsp–TßH) were kept at 37°C for 45 min twice with a 6 h interval and were then allowed to recover for 12 h before experiments.
Three- to 5-d-old male flies were briefly immobilized by cold anesthesia and glued [clear glass adhesive (Duro; Pacer Technology, Rancho Cucamonga, CA)] with head and thorax to a triangle-shaped copper hook (0.02 mm diameter). Adhesion was achieved by exposure to UV light for 10 s. The animals are then kept individually in small chambers containing a few grains of sucrose until testing (1–5 h).
The fly, glued to the hook as described above, was attached to the experimental setup via a clamp to accomplish stationary flight. For observation, the fly was illuminated from behind and above (150 W, 15 V; Schott, Elmsford, NY) and fixed in front of a polystyrene panel. Additionally, it was shielded by another polystyrene panel from the experimenter. Tarsal contact with a bead of polystyrene prevented flight initiation before the experiment started. A digital high-speed camera (1000 pictures per second; Motion Scope; Redlake Imaging, Morgan Hill, CA) was positioned behind the test animal. To initiate flight, the polystyrene bead was removed, and the fly was gently aspirated. The time until the fly ceased flying was recorded (initial flight). The fly was aspirated as a stimulation to fly, each time it stopped flying. When no flight reaction was shown after three consecutive stimulations, the experiment was completed and the total flight time was recorded (extended flight). Every stimulus after the first one, to which the fly showed a response, was recorded. Each fly was filmed during the first few seconds of flight, and the recordings were saved on a personal computer for later analysis. The person scoring the flight time was unaware of the treatment group of the animal. All animals were included in the study, including those that did not show any flight behavior.
Immunocytochemistry. For immunohistochemical stainings of Drosophila CNS with GFP antibody (Ab), fly CNS was removed in saline. After fixation for 1 h in 4% paraformaldehyde (PFA) (10 ml of PBS plus 0.4 g of PFA, pH 7.4), the CNS was treated with a mixture of enzymes (collagenase/dispase, 1 mg/ml each) for 1 min to ensure better penetration of antibodies (Abs) into the tissue and then washed in PBS (0.1 M) overnight at 4°C. Preparations were then washed six times for 30 min in 0.5% Triton X-100 in PBS (PBSTx), again to increase the penetration of Ab into the tissue. Subsequently, the CNS was placed for 2 d in a 1:200 dilution of the anti-GFP primary Ab mouse serum in 0.3% PBSTx at 4°C. They were then rinsed eight times for 15 min in PBS and then incubated at 4°C overnight in a 1:500 dilution of the secondary Ab serum that was coupled to a fluorescent dye [anti-mouse cyanine 2 (Cy2)] in PBS. After rinsing the preparations eight times for 15 min in PBS, they were dehydrated in an ascending ethanol series (50, 70, 90, and 100%, 10 min each) and then transferred to a microscope slide and cleared in methylsalicylate. For immunohistochemical stainings of Drosophila CNS for presynaptic active zones with bruchpilot antibody (Wagh et al., 2006) (gift from E. Buchner, University of Wuerzburg, Wuerzburg, Germany), the same protocol was followed with the exception that the primary Ab was diluted 1:100 in 0.3% PBSTx.
Phalloidin stainings. Flies were opened via a dorsal longitudinal cut in saline and then fixed in 4% PFA. After 1 h, they were transferred into PBS, and flight muscles were removed and washed three times for 1 h in 0.5% PBSTx. After treatment with 2 µl/ml Oregon Green phalloidin, 0.3% PBSTx for 36 h, the muscles were washed six times for 15 min in PBS and finally embedded in glycerin on a microscope slide.
Confocal microscopy. The preparations were viewed under a Leica (Bensheim, Germany) SP2 confocal laser-scanning microscope with 40x oil immersion objective. Stacks of optical sections (0.5 µm) were acquired. Both Cy2 and Oregon Green phalloidin were excited with an argon laser at 488 nm, and emitted light was detected between 500 and 530 nm.
Wing-beat amplitude. For wing-beat amplitude measurements, Redlake Imaging MotionScope software (DEL Imaging Systems, Cheshire, CT) was used to capture the first 100 frames. After image inversion, the image stacks were imported into AMIRA software (TGS, San Diego, CA) for overlaying of all frames (projection view) and then measuring wing angles using the angle-measuring tool.
Wing-beat frequency. To measure the wing-beat frequency, the number of frames per 10 wing beats was counted, starting from frame 1, 100 and 300 in each sequence, and subsequently the mean was calculated.
Sarcomere length. For sarcomere-length survey, the images of phalloidin-stained muscles were imported into AMIRA software, and sarcomeres were measured with the line-measuring tool. For each animal, the lengths of 31–41 sarcomeres were measured.
Flight time per stimulation. To calculate flight time per stimulation, the total flight time was divided by the number of stimulations, including the initial one.
Statistics. The flight data approximately conformed to a Poisson distribution, and hence nonparametric tests were used. For comparison of more than two groups, a Kruskal–Wallis ANOVA was used to test the hypothesis that the samples were drawn from the same population. When differences between the samples occurred, Mann–Whitney U tests were performed for planned comparisons of two samples. Two groups were always compared with a Mann–Whitney U test. To display the measurements, box-and-whisker plots were chosen, and medians were used as central values. Boxes included the medial 25–75%, and, because the data show many extreme scores, the whiskers included 15–85% of the data values. Outliers were not shown. Significant differences were accepted at p < 0.05.
A full rescue is scored when the rescue group differs significantly from the mutant but not from the wild-type control. For a partial rescue, the rescue line must either differ significantly from both mutant and wild type or not differ from both. No rescue is achieved when no significant difference is obtained between the mutant flies and the rescue line and a significant difference remains for the wild-type controls.
Materials and Methods
Average flight duration per stimulation is at least partially rescued in all experimental groups (Fig. 3b, see inset for comparison of medians). A full rescue is obtained only by feeding YH alone (median of 4; p < 0.001 compared with TßH flies, p = 0.114 compared with wild-type flies). Partial rescues can be achieved with HS + YH (median of 7; p < 0.001 compared with TßH flies, p = 0.047 compared with wild-type flies), with HS (median of 2; p = 0.025 compared with TßH flies, p = 0.032 compared with wild-type flies), and by feeding OA (median of 3; p = 0.025 compared with TßH flies, p = 0.015 compared with wild-type flies). In summary, a full rescue of the average flight duration in multiple subsequent flight bouts is achieved only by blocking TA receptors but not by replacing OA either genetically or pharmacologically.
The duration of total flight (Fig. 3c) can be fully rescued by HS + YH (median of 72; p < 0.001 compared with TßH flies, p = 0.259 compared with wild-type flies), by only feeding YH (median of 40; p < 0.001 compared with TßH flies, p = 0.441 compared with wild-type flies), and by HS (median of 30; p = 0.002 compared with TßH flies, p = 0.076 compared with wild-type flies) but not by supplementing OA alone (median of 11; p = 0.163 compared with TßH flies, p = 0.005 compared with wild-type flies). Total flight duration is the product of the number of flight initiations times the average time of the flight bouts. The average time of the flight bouts is partially rescued by feeding OA (Fig. 3b), but the number of responses (flight initiations) is not rescued by feeding OA to TßH flies (Fig. 3d).
The responsiveness to stimulation (Fig. 3d) was fully rescued by feeding YH (median of 10; p < 0.001 compared with TßH flies, p = 0.083 compared with wild-type flies) and by HS (median of 8,1; p = 0.021 compared with TßH flies, p = 0.599 compared with wild-type flies). Feeding OA only did not rescue this phenotype (p = 0.994 over TßH flies, p = 0.053 over wild-type flies) but even caused a slight but nonsignificant decrease in the responsiveness to stimulation. HS + YH-treated animals responded to stimulation even more often than wild-type flies (median of 9.3; p < 0.001 compared with TßH flies, p = 0.028 compared with wild-type flies).
This complex set of full and partial rescues depending on OA and TA manipulation demonstrates that flight behavior depends on OA and on TA. One possibility is that OA and TA each act on different aspects of the flight machinery, such as sensory sensitivity, muscle metabolism, or CPG activation. Alternatively, OA and TA might act antagonistically on similar aspects of motor behavior, and thus, the absolute levels of one modulator are not important, but the relative levels of both modulators influence flight behavior. In a first test of the latter hypothesis, we ablated all neurons synthesizing TA from tyrosine by expressing the apoptosis-inducing gene reaper under control of the dTdc2 promotor (for details, see Materials and Methods). The dTdc2 gene codes for the neural version of two TDC enzymes converting tyrosine to TA.
Because TA is the precursor of OA, dTdc2 expresses in all neurons containing TA or OA, as can be visualized by expressing eGFP under the control of dTdc2 and enhancing the eGFP signal by anti-GFP immunocytochemistry (Fig. 4a). Cell bodies of dTdc2 neurons are located in the midlines of each thoracic and each abdominal neuromere, bilateral symmetric processes of efferent unpaired median neurons can clearly be seen, and a large number of finer aminergic processes with numerous varicosity-like structures can be visualized within the CNS (Fig. 4a).
We compared initial flight (Fig. 6a), mean flight bout duration (Fig. 6b), total flight duration (Fig. 6c), and the number of stimulations causing flight (Fig. 6d) in wild-type flies that were fed with yohimbine and wild-type controls that were fed with sucrose solution only. Feeding yohimbine yields the most effective rescues of flight initiation and maintenance in TßHnM18 mutants (Fig. 3). However, none of these flight parameters is different among sucrose-fed and yohimbine-fed wild-type flies (Fig. 6). Consequently, flight initiation and maintenance do not depend strictly on the relative levels of OA and TA but are affected by some concerted interaction of both amines. Depleting OA and increasing TA impairs flight motor behavior, as does ablation of all OA- and TA-containing neurons. In OA-depleted flies with increased TA, flight initiation and maintenance can be rescued either by restoring OA levels or blocking TA action. In contrast, blocking TA action in flies with normal OA and TA levels does not affect any of the flight motor behavior parameters measured in this study.
Materials and Methods
Concerted action of OA and TA on flight behavior
A novel finding is that flies lacking OA and with TARs blocked show wild-type-like flight behavior. It is important to note that the TßH phenotype comprises OA knock-out plus eightfold increased TA levels. Pharmacological blockade of TARs yields the most efficient rescue of the TßH mutants, even outscoring replacement of OA by heat-shock plus TAR blockade. However, blocking TARs in wild-type flies does not increase flight initiation or maintenance. This indicates that TA inhibits flight behavior only at abnormally high TA levels. Furthermore, with regard to flight maintenance, the inhibitory effects of TA take place only at low OA levels, because OA replacement without affecting the TA system also yields rescues of the initial and the average flight bout durations. In contrast, the responsiveness to stimulation is rescued best by blocking TA. Therefore, flight initiation is most likely inhibited by high TA levels, regardless of the OA levels. Accordingly, feeding TßH mutants OA does not rescue flight initiation but restoring tyramine-ß-hydroxylase activity by heat shock does, because only the latter manipulation decreases the levels of TA by conversion of TA into OA. Therefore, the most parsimonious interpretation is that OA is necessary for flight maintenance, and TA acts most likely as an inhibitor, especially for flight initiation at high concentrations.
This interpretation is further supported by ablating all OA/TA neurons by expressing the apoptosis factor reaper in these cells. Flies without OA/TA neurons show the same massive changes in flight behavior as TßH mutants. Therefore, genetic ablation of all TA/OA-containing neurons does not phenocopy genetic ablation of the OA-producing enzyme paired with pharmacological block of TA action. How can these seemingly contradictory results be explained? Clearly, the pharmacological treatment with yohimbine is effective; it fully rescues the mutant phenotype. The ablation of the OA/TA neurons is equally effective, ruling out methodological flaws. However, yohimbine does most likely not block all TA action, whereas genetic ablation of all TA-containing neurons does. Thus, the action of TA presumably follows a bell-shaped curve, with its presence necessary for normal flight but hindering flight initiation and maintenance at high concentration. OA is required most likely for flight maintenance because feeding it to TßH mutants fully rescues normal flight maintenance. However, OA supplementation in the food might also exert rescuing effects in TßH mutants by downregulating TA via feedback inhibition. In summary, the most compelling explanation for the data are that OA is boosting flight maintenance, low levels of TA are required for flight maintenance and initiation, and inhibitory TA actions fall in place at high TA and low OA levels.
TA as neurotransmitter/modulator
Our finding that OA and TA are involved in regulating flight emphasizes the role of TA as an independent neurotransmitter in invertebrates. Further supporting this role, tyramine-like immunoreactivity has been demonstrated in non-octopaminergic cells of Caenorhabditis elegans and locusts (Stevenson and Spoerhase-Eichmann, 1995; Donini and Lange, 2004; Alkema et al., 2005). Moreover, at least one Drosophila amine receptor is specific for TA and does not cross-react with OA (Cazzamali et al., 2005). Furthermore, OA and TA receptor distributions in the insect CNS differ considerably from each other [J. Erber (Technical University Berlin, Berlin, Germany), personal communication]. Functionally, exogenous TA increases chloride conductances in Drosophila malphigian tubules (Blumenthal, 2003), alters body wall muscle excitatory junction potentials (Kutsukake et al., 2000), and can rescue cocaine sensitization in Drosophila (McClung and Hirsh, 1999). In mammals, the physiological roles for trace amines such as TA and OA are mostly unknown, but they have been implicated in a variety of neurological disorders (Branchek and Blackburn, 2003), and receptors specific for TA have been identified (Borowsky et al., 2001). In invertebrates, a role of endogenous TA as an important transmitter/modulator has been shown for Drosophila locomotor (Saraswati et al., 2004; this study) and olfactory avoidance (Kutsukake et al., 2000) behavior, as well as for C. elegans motor behavior (Alkema et al., 2005).
Sites of OA and TA action
Previous studies suggested that OA acts as a potent, direct stimulator of flight muscle metabolism (Wegener, 1996; Mentel et al., 2003). Accordingly, we expected that especially prolonged flight would be affected in TßH mutants, attributable to insufficient fuel supply. In contrast, all flight parameters are similarly affected in TßH mutants. The initial flight bout duration is decreased 40 times, and the total flight duration is decreased 30 times in TßH mutants. Moreover, flight behavior changes in TßH mutants are rescued by blocking TA action alone, leaving OA levels unaltered. This is hard to reconcile with direct effects of OA on flight metabolism and would require independent effects of OA and TA on flight metabolism. These considerations render metabolism unlikely as the site of action for OA. Therefore, amine effects on Drosophila flight initiation and maintenance are more likely to be mediated by effects on the nervous system.
Two main OA/TA effects on flight behavior can be observed: maintenance of flight and the probability of initiating flight. In principle, both could be controlled by aminergic action on the CPG and/or on the fly's sensory system. It is well established that OA acts on the CPG in a number of insect species (Sombati and Hoyle, 1984; Claassen and Kammer, 1986; Stevenson and Kutsch, 1987), but central actions of TA are not known. OA has also been reported to increase the responsiveness of flight-associated sensory cells in insects (Ramirez and Orchard, 1990), and TA could conceivably reduce excitability of sensory neurons as Drosophila TARs activate chloride currents (Cazzamali et al., 2005).
Motor behavior specificity of combined amine effects
OA and TA have been implicated as agonist and antagonist, respectively, controlling locomotor behavior in Drosophila larvae (Saraswati et al., 2004; Fox et al., 2006) and in C. elegans (Alkema et al., 2005). This raises the possibility of a general, opponent OA/TA control of locomotor behavior in invertebrates. Our results make it unlikely that OA and TA simply act antagonistically on the same targets because, with regard to flight initiation and maintenance, OA and TA probably have different sites of action and TA effects are important only at high TA and low OA levels. Nevertheless, in some preliminary experiments, we tested whether TßHnM18 mutant adults show also walking behavior deficits. Neither the overall motor activity per unit time nor the number of walking bouts differed between wild-type and TßHnM18 mutant flies. However, we found a slight but statistically significant reduction in walking speed in TßHnM18 mutants (data not shown). These findings indicate that aminergic modulation by OA and TA does not act generally on locomotor performance but specifically affects different aspects of motor behaviors.
In summary, the emerging picture is that, for some motor behaviors, the concerted interaction of specific biogenic amines is more important than the concentration of single amines (Scheiner et al., 2002; Schwaerzel et al., 2003; Saraswati et al., 2004; Alkema et al., 2005; Fox et al., 2006; Fussnecker et al., 2006). The current study is the first to suggest that the antagonistic actions of OA and TA are not a general feature of all invertebrate locomotor behaviors but specifically affect distinct aspects of different motor behaviors. It provides evidence that OA and TA do not simply act antagonistically on the same targets but most likely mediate their effects on motor performance by affecting different targets in a dose-dependent manner. The next steps toward understanding amine function for motor behavior is to determine their sites of action during behavior. One possibility addressing this question is to combine pharmacological and genetic rescues and test immunocytochemically where the OA and TA levels are restored in which rescue procedure, how behavior is affected in these different manipulations, and where the various subtypes of TA and OA receptors are localized. Ultimately, a complete understanding of the mechanism by which various modulators interact on different parts of the brain and other tissues to control motor behavior will require a large number of targeted manipulations of each individual circuit component separately.
*B.B. and F.C. contributed equally to this work.
B.B. helped with experiments and contributed to the design of the study and the writing of the manuscript. F.C. conducted most experiments and contributed to the design of the study. H.J.P. contributed funding and intellectual exchange. C.D. participated in some experiments and contributed to the design of the study and the writing of this manuscript.
This work was supported by the German Science Foundation (C.D., H.J.P.). We are grateful for the help of Fernando Vonhoff with our behavioral experiments. We thank Drs. R. B. Levine (University of Arizona, Tucson, AZ) and J. A. Mustard (Arizona State University, Tempe, AZ) for many critical comments on this manuscript and Marinus de Bruyne and Wernher Fouquet for discussions concerning fly genetics.
Correspondence should be addressed to Carsten Duch, School of Life Sciences, Arizona State University, Tempe, AZ 85287. Email: firstname.lastname@example.org
Copyright © 2007 Society for Neuroscience 0270-6474/07/2711122-10$15.00/0
Materials and Methods
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