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A single pair of interneurons commands the Drosophila feeding motor program

Abstract

Many feeding behaviours are the result of stereotyped, organized sequences of motor patterns. These patterns have been the subject of neuroethological studies1,2, such as electrophysiological characterization of neurons governing prey capture in toads1,3. However, technical limitations have prevented detailed study of the functional role of these neurons, a common problem for vertebrate organisms. Complexities involved in studies of whole-animal behaviour can be resolved in Drosophila, in which remote activation of brain cells by genetic means4 enables us to examine the nervous system in freely moving animals to identify neurons that govern a specific behaviour, and then to repeatedly target and manipulate these neurons to characterize their function. Here we show neurons that generate the feeding motor program in Drosophila. We carried out an unbiased screen using remote neuronal activation and identified a critical pair of brain cells that induces the entire feeding sequence when activated. These ‘feeding neurons’ (here abbreviated to Fdg neurons for brevity) are also essential for normal feeding as their suppression or ablation eliminates sugar-induced feeding behaviour. Activation of a single Fdg neuron induces asymmetric feeding behaviour and ablation of a single Fdg neuron distorts the sugar-induced feeding behaviour to become asymmetric, indicating the direct role of these neurons in shaping motor-program execution. Furthermore, recording neuronal activity and calcium imaging simultaneously during feeding behaviour5 reveals that the Fdg neurons respond to food presentation, but only in starved flies. Our results demonstrate that Fdg neurons operate firmly within the sensorimotor watershed, downstream of sensory and metabolic cues and at the top of the feeding motor hierarchy, to execute the decision to feed.

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Figure 1: Thermogenetic activation reproduced coordinated natural feeding behaviour.
Figure 2: Thermogenetically induced food ingestion through the pharyngeal pump.
Figure 3: Identification of the Fdg neuron.
Figure 4: Functional analyses of Fdg neuron.

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Acknowledgements

We thank S. Waddell for discussions, fly stocks and reading of the manuscript; T. Lee for discussions and fly stocks; A. Sakurai for reading of the manuscript; S. Reppert for support; M. Alkema and T. Ip for discussions; members of the NP consortium for NP lines; T. Awasaki., C. Kao, V. Budnik, P. Garrity, M. Freeman, M. Rosbash, Y.-N. Jan, L. Luo, S. Sigrist, K. Scott, T. Tanimura, L. Looger, M. Ramaswami and K. Gajewski for fly stocks; K. Ikeda, T. Tanimura and H. Ishimoto for technical advices; A.Taylor and R. Seeham for technical help; and N. Yoshihara for material information. This work was supported by National Institute of Mental Health Grant MH85958, and the Worcester Foundation (to M.Y.), the National Institute of Mental Health Intramural Research Program (B.W.), the summer program of the Japan Society for the Promotion of Science/National Science Foundation (to T.F.F.), and a Japan Science and Technology Agency CREST grant (to K.I.).

Author information

Authors and Affiliations

Authors

Contributions

M.Y., S.I., T.F.F. and M.G. designed the research. T.F.F. screened GAL4 lines under the supervision of K.I and M.Y. T.F.F., S.I and M.Y. performed behavioural analyses. S.I. performed analyses of ingestion and pump movement while M.Y. visualized the pump movement. M.G., S.I., K.I. and M.Y. performed neuroanatomy. S.I. and M.Y. did the fly genetics. M.Y. performed calcium imaging. S.I. and M.Y. performed experiments of laser activation and laser ablation with the technical assistance of M.G. B.W. contributed TRPM8 and essential advice. M.Y., M.G. and T.F.F. wrote the paper with assistance from S.I., B.W. and K.I.

Corresponding author

Correspondence to Motojiro Yoshihara.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-17, Supplementary Tables 1-2, Supplementary Note 1, Supplementary Methods and additional references. (PDF 10764 kb)

Natural Drosophila feeding behaviour

WT flies were starved for 24 hours. See Figure 1 and text. (MOV 9114 kb)

Genetically-induced feeding behaviour

NP883>TrpA1 satiated flies were activated by restrictive temperature. All aspects of natural feeding behaviour were reproduced. (MOV 16186 kb)

Movement of pharyngeal pump and food ingestion by the pump

Movement of the pharyngeal pump was visualized by Myosin heavy chain (Mhc)-GFP and blue dye. Induced feeding of a NP883>TrpA1 fly at 29˚C was compared with natural feeding of a starved WT fly on 100 mM sucrose at 29˚C. At this high temperature, pump movement in natural feeding is two times faster (6-8 Hz) than that at 21˚C (3-4 Hz). Pattern of muscle contraction in the induced feeding at 29˚C was quite similar to that in the natural feeding at 29˚C. See Fig. 2, Supplementary Figure 7 and text. (MOV 26601 kb)

Feeding behaviour by activation of Fdg-neuron in flip-out flies

The first fly with TrpA1 expression in an Fdg-neuron at the fly’s left almost exclusively was activated by raising temperature (fly is same as in Figure 3d and Supplementary Figures 10c, d, e). All aspects of natural feeding behaviour were reproduced in a substantially coordinated manner (see text). See Supplementary Table 2 for information of all flies from the flip-out Gal80 screening in this video. See Figure 3, Supplementary Figure 10 and text. (MOV 14650 kb)

3D-structure of Fdg-neuron

See Figure 3, Supplementary Figure 12 and text. (MOV 9156 kb)

Suppression of feeding behaviour by Kir

Comparison of PER and feeding on food in free running condition between Kir and WT flies, See Supplementary Figure 14. In free running condition, a starved Kir fly rarely extends their proboscis to the food, and when it does, it does not continue. (MOV 4279 kb)

PER simultaneous with Ca2+ imaging at the cell body of Fdg-neuron

Illumination is provided by the confocal microscope laser used for GCaMP Ca2+ imaging. The proboscis of a NP883>GCaMP3.0 fly was immobilized by glue, and 400 mM sucrose solution on Washi wick was used for stimulation, with its head capsule open to expose the SEG for Ca2+ imaging. Simultaneous Ca2+ imaging is shown with the labella opening. See Figure 4 and text. (MOV 3094 kb)

Laser activation of Fdg-neuron

Laser-activation of an Fdg-neuron induced proboscis extension and pump movement in NP883>TrpA1; mCD8-GFP flies. Infrared illumination is detected by the CCD camera as a red-tint flash. See Figure 4 and text. (MOV 25944 kb)

Effect of laser-ablating Fdg-neurons on sucrose-induced proboscis extension

Two examples are shown. The first fly; Proboscis extension before (to the front) and after laser-ablation of the fly’s left Fdg-neuron (to the right) and after ablation of both Fdg-neurons (no response) in a NP883>mCD8-GFP fly. Ablation at the opposite side for the second fly. See Figure 4 and text. (MOV 27934 kb)

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Flood, T., Iguchi, S., Gorczyca, M. et al. A single pair of interneurons commands the Drosophila feeding motor program. Nature 499, 83–87 (2013). https://doi.org/10.1038/nature12208

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