Flight initiation and maintenance deficits in flies with genetically altered biogenic amine levels

Abstract

Insect flight is one of the fastest, most intense and most energy-demanding motor behaviors. It is modulated on multiple levels by the biogenic amine octopamine. Within the CNS, octopamine acts directly on the flight central pattern generator, and it affects motivational states. In the periphery, octopamine sensitizes sensory receptors, alters muscle contraction kinetics, and enhances flight muscle glycolysis. This study addresses the roles for octopamine and its precursor tyramine in flight behavior by genetic and pharmacological manipulation in Drosophila. Octopamine is not the natural signal for flight initiation because flies lacking octopamine [tyramine-beta-hydroxylase (TbetaH) null mutants] can fly. However, they show profound differences with respect to flight initiation and flight maintenance compared with wild-type controls. The morphology, kinematics, and development of the flight machinery are not impaired in TbetaH mutants because wing-beat frequencies and amplitudes, flight muscle structure, and overall dendritic structure of flight motoneurons are unaffected in TbetaH mutants. Accordingly, the flight behavior phenotypes can be rescued acutely in adult flies. Flight deficits are rescued by substituting octopamine but also by blocking the receptors for tyramine, which is enriched in TbetaH mutants. Conversely, ablating all neurons containing octopamine or tyramine phenocopies TbetaH mutants. Therefore, both octopamine and tyramine systems are simultaneously involved in regulating flight initiation and maintenance. Different sets of rescue experiments indicate different sites of action for both amines. These findings are consistent with a complex system of multiple amines orchestrating the control of motor behaviors on multiple levels rather than single amines eliciting single behaviors.

Figure 1.

Comparison of flight initiation and maintenance between TβH mutant and wild-type flies. For a–c, the black squares indicate the median, the boxes signify the 25 and the 75 percentiles, and the error bars range from the 15 to the 85 percentiles. a shows the flight duration until the first stop for wild-type (WT; light gray bar) and TβH null mutant (tbh; dark gray bar) flies. b indicates the duration of all flight bouts for wild-type and TβH flies. c shows the total flight duration for wild-type and TβH flies. d shows the mean number of stimuli to which wild-type and TβH mutant flies responded with flight bouts before they did not respond to three consecutive stimuli (error bars are SEMs). The number of animals per group is indicated in the bars. *p < 0.05, **p < 0.01, Mann–Whitney U test.

Figure 2.

The development of the flight system is not impaired in TβH mutant flies. a shows the mean wing-beat amplitudes for wild-type (WT; light gray bar) and TβH mutant (tbh; dark gray bar) flies. b shows the mean wing-beat frequencies for wild-type (WT; light gray bar) and TβH mutant (tbh; dark gray bar) flies. c shows representative fields of view of DLM flight muscle fibers with phalloidin-labeled actin bands for wild-type (WT), TβH mutant (tbh), and TβH mutant flies that were fed with octopamine (tbh OA). d shows the mean sarcomere lengths for the three groups shown in c. Numbers in bars indicate numbers of animals. Error bars are SEMs. n.s., Not significant.

Figure 3.

Different types of rescues of the TβH^nM18 caused flight behavior phenotypes. For a–c, the black squares indicate the median, the boxes signify the 25 and the 75 percentiles, and the error bars range from the 15 to the 85 percentiles. To allow for a better between-group comparison, insets in a to c depict bar graphs of the respective medians at a higher y-axis resolution. a shows the duration of the initial flight bout for each experimental group, b shows the average duration of a flight bout for each group, c shows the total flight duration, and d shows the number of stimuli to which the flies responded with flight before they did not respond to three consecutive stimuli. fr, Full rescue; pr, partial rescue; nr, no rescue (for definition, see Materials and Methods). The experimental groups were wild-type flies (WT), a genetic rescue in which TβH expression in TβH mutant flies was induced in all cells via a heat-shock inducible TβH transgene in the TβH null mutant genetic background (tbh, hsp–tbh HS), a combined genetic and pharmacological rescue in which TβH expression was induced via a heat shock and in which the flies were also fed the tyramine receptor blocker yohimbine (tbh, hsp–tbh HS + YH), a pharmacological rescue in which TβH mutant flies containing the inducible TβH transgene received no heat shock but were fed yohimbine (tbh, hsp–tbh YH), a pharmacological rescue in which TβH mutant flies were fed octopamine (tbh OA), and TβH mutant flies (tbh). e shows the biosynthesis pathway of tyramine and octopamine from tyrosine. Genetic and pharmacological blocks are depicted in light gray. TA synthesis is blocked by killing all cells containing tyrosine decarboxylase by expressing reaper. OA synthesis is blocked in tyramine hydroxylase null mutants (TβH^nM18). TARs are blocked by yohimbine. Rescues are depicted in dark gray. Octopamine levels were increased by either expressing tyramine hydroxylase under the control of a heat shock promoter or by feeding OA.

Figure 4.

Genetic ablation of all tyraminergic and octopaminergic neurons. a, Visualization of all tyraminergic and octopaminergic neurons in the thoracic and abdominal ventral nerve cord by expressing 2xeGFP under the control of Tdc2 and enhancing the signal by anti-GFP immunocytochemistry. To test the effectiveness of neuron ablation by targeted ectopic expression of the cell death gene reaper, animals expressing either only GFP or GFP together with reaper were subjected to standard immunohistochemistry. Animals expressing only GFP reveal the expression pattern typical of Tdc2 neurons. b1 shows double labels of the ventral nerve cord for Tdc2 neurons labeled by targeted expression of eGFP (green) and all synapses labeled with bruchpilot antibody Nc82 (Kittel et al., 2006) (red) to visualize presynaptic active zones in the neuropil regions. b2 and b3 show the Tdc2 and the Nc82 signal separately as grayscale images. b1–b3 show a ventral nerve cord from heterozygous progeny of dTdc2–Gal4 crossed with y w P{w^+mC=UAS–2xEGFP}. c, Gal4-driven apoptosis was induced by crossing dTdc2–Gal4 with w;; P{UAS-rpr}/TM3 Sb, and eGFP was from y w P{w^+mC=UAS–2xEGFP}. No GFP expression can be detected in animals with targeted expression of both GFP and reaper to these OA/TA cells (c1), but Nc82 immunostaining appears unaffected in these animals (c3), demonstrating effective and specific ablation.

Figure 5.

Genetic ablation of all tyraminergic and octopaminergic neurons decreases flight initiation and maintenance. For a–c, the black squares indicate the median, the boxes signify the 25 and the 75 percentiles, and the error bars range from the 15 to the 85 percentiles. a shows the flight duration until the first stop in control flies (gen. controls; light gray bar) and for flies expressing reaper under the control of TDC2 (TDC2rpr; dark gray bar). b indicates the mean duration of all flight bouts for control and TDC2rpr flies. c shows the total flight duration for control and TDC2rpr flies. d shows the mean number of stimuli to which control and TDC2rpr responded with flight bouts before they did not respond to three consecutive stimuli (error bars are SEMs). **p < 0.01, Mann–Whitney U test.

Figure 6.

Blocking TA receptors in wild-type flies does not affect flight behavior. For a–c, the black squares indicate the median, the boxes signify the 25 and the 75 percentiles, and the error bars range from the 15 to the 85 percentiles. a shows the flight duration until the first stop in control wild-type flies fed with sucrose (WT; light gray bar) and for wild-type flies fed with the TA receptor blocker yohimbine (WT + YH; dark gray bar). b indicates the average duration of flight bouts for WT control and WT + YH flies. c shows the total flight duration for WT control and WT + YH flies. d shows the mean number of stimuli to which WT control and WT + YH responded with flight bouts before they did not respond to three consecutive stimuli (error bars are SEMs). n.s. indicates that no significant differences were found, Mann–Whitney U test.