Opposite effects of 5-HT/AKH and octopamine on the crop contractions in adult Drosophila melanogaster: Evidence of a double brain-gut serotonergic circuitry

Abstract

This study showed that in adult Drosophila melanogaster, the type of sugar-either present within the crop lumen or in the bathing solution of the crop-had no effect on crop muscle contraction. What is important, however, is the volume within the crop lumen. Electrophysiological recordings demonstrated that exogenous applications of serotonin on crop muscles increases both the amplitude and the frequency of crop contraction rate, while adipokinetic hormone mainly enhances the crop contraction frequency. Conversely, octopamine virtually silenced the overall crop activity. The present study reports for the first time an analysis of serotonin effects along the gut-brain axis in adult D. melanogaster. Injection of serotonin into the brain between the interocellar area shows that brain applications of serotonin decrease the frequency of crop activity. Based on our results, we propose that there are two different, opposite pathways for crop motility control governed by serotonin: excitatory when added in the abdomen (i.e., directly bathing the crop) and inhibitory when supplied within the brain (i.e., by injection). Finally, our results point to a double brain-gut serotonergic circuitry suggesting that not only the brain can affect gut functions, but the gut can also affect the central nervous system. On the basis of our results, and data in the literature, a possible mechanism for these two discrete serotonergic functions is suggested.

Fig 1. Crop preparation and recording of muscle activity.

Recording layout of D. melanogaster crop activity (a) and schematic diagram of the typical dipteran foregut (b) showing various sphincters, pumps, crop nerve bundle and small segment of the muscles of the crop lobes. Shown in the circle area is a typical recording from a suction electrode from the crop P4. (c) Sample of a double simultaneous recording of crop muscle activity, showing the mAPs electrophysiologically recorded from P4 (upper trace), as compared to contractions of P5 (lower trace), that were determined by means of the Aviline software analysis [57]. Note that the two pumps can display either phase-locked or totally desynchronized (dashed rectangle) activities.

Fig 2. Effect of various sugars ingested and their influence on crop contraction rate in adult D. melanogaster.

Mean values ± SE (vertical bars) from 20 insects (yw-1118 strain) for each sugar tested. No significant differences were detected amongst the sugars ingested (P > 0.05; Tukey-Kramer test subsequent to one-way ANOVA).

Fig 3. Effect of crop volume on contraction rate in adult D. melanogaster.

Mean values ± SE (vertical bars) from 12 insects (yw-1118 strain) for each volume tested. Bars followed by different letters are significantly different (P < 0.05; Tukey-Kramer test subsequent to one-way ANOVA).

Fig 4. Effect of various sugars bathing the crop and their influence on crop contraction rate in adult D. melanogaster.

Frequency of mPAs electrophysiologically recorded from P4 (a) and of contractions visually recorded from P5 (b) of Drosophila Canton-S strain, determined over a 1-min interval, following replacement of hemolymph with 0.1 M trehalose, glucose or fructose with respect to Drosophila saline (basal activity). Mean values ± SE (vertical bars) from 8 (trehalose and glucose) or 7 (fructose) replicates in (a) and from 18 (trehalose and 20 (glucose and fructose) replicates in (b). No significant differences were detected among sugars for both P4 and P5 (P > 0.05; Tukey test subsequent to one-way ANOVA).

Fig 5. Dose-response curves for various neuromodulators on P5 crop contraction rate.

Frequency of contractions visually recorded from P5 of Drosophila Canton-S strain, determined over a 1-min interval, following replacement of hemolymph with 0.01–10 mM 5-HT and octopamine (OA) and 0.001–1 mM AKH, as compared to the activity in Drosophila saline (basal). Mean values ± SE (vertical bars) from 10 replicates for each compound.

Fig 6. Effect of various neuromodulators on crop contraction rate in adult D. melanogaster.

Samples of electromyograms (a) and frequency of mPAs (b) recorded from P4 in Drosophila Canton-S strain, determined over a 1-min interval, following replacement of hemolymph with 1 mM 5-HT, octopamine (OA) and ketanserin (ket) and 0.1 mM AKH, as compared to the activity in Drosophila saline (basal). Mean values ± SE (vertical bars); number of replicates for each compound is indicated in brackets. Bars followed by different letters are significantly different (P < 0.05; Tukey test subsequent to one-way ANOVA).

Fig 7. Effect of various neuromodulators on crop contraction rate in adult D. melanogaster.

(a) Frequency of contractions visually recorded from P5 in Drosophila Canton-S strain, determined over a 1-min interval, following replacement of hemolymph with 1 mM 5-HT, octopamine (OA) and ketanserin (ket) and 0.1 mM AKH, as compared to the activity in Drosophila saline (basal). Mean values ± SE (vertical bars); number of replicates for each compound is indicated in brackets. Bars followed by different letters are significantly different (P < 0.05; Tukey test subsequent to one-way ANOVA). (b) Frequency of P5 contractions in the same Canton-S strain, classified on the basis of the percentage of maximal contraction amplitude elicited, as assessed by the Aviline software analysis [57]. Mean values ± SE (vertical bars); number of replicates for each compound is indicated in brackets. Filled symbols represent significant differences with respect to the basal level (P < 0.05; Tukey test subsequent to one-way ANOVA).

Fig 8. Opposite effects of 5-HT injected into the brain vs. perfusion application on the crop.

Frequency of mPAs, electrophysiologically recorded from P4 and of contractions visually recorded from P5 in Drosophila Canton-S strain, determined over a 1-min interval, following injection of 5-HT into the brain as compared to the activity in Drosophila saline (basal) (a) or to the activity previously enhanced by perfusion application of 5-HT into the abdomen. Mean values ± SE (vertical bars) from 15 replicates in (a) and 16 replicates in (b). The asterisk indicates a significant difference (P < 0.05; Tukey test subsequent to repeated-measures ANOVA) with respect to the basal level.