A gustatory second-order neuron that connects sucrose-sensitive primary neurons and a distinct region of the gnathal ganglion in the Drosophila brain

Abstract

Although the gustatory system provides animals with sensory cues important for food choice and other critical behaviors, little is known about neural circuitry immediately following gustatory sensory neurons (GSNs). Here, we identify and characterize a bilateral pair of gustatory second-order neurons (G2Ns) in Drosophila. Previous studies identified GSNs that relay taste information to distinct subregions of the primary gustatory center (PGC) in the gnathal ganglia (GNG). To identify candidate G2Ns, we screened ∼5,000 GAL4 driver strains for lines that label neural fibers innervating the PGC. We then combined GRASP (GFP reconstitution across synaptic partners) with presynaptic labeling to visualize potential synaptic contacts between the dendrites of the candidate G2Ns and the axonal terminals of Gr5a-expressing GSNs, which are known to respond to sucrose. Results of the GRASP analysis, followed by a single-cell analysis by FLP-out recombination, revealed a pair of neurons that contact Gr5a axon terminals in both brain hemispheres and send axonal arborizations to a distinct region outside the PGC but within the GNG. To characterize the input and output branches, respectively, we expressed fluorescence-tagged acetylcholine receptor subunit (Dα7) and active-zone marker (Brp) in the G2Ns. We found that G2N input sites overlaid GRASP-labeled synaptic contacts to Gr5a neurons, while presynaptic sites were broadly distributed throughout the neurons’ arborizations. GRASP analysis and further tests with the Syb–GRASP method suggested that the identified G2Ns receive synaptic inputs from Gr5a-expressing GSNs, but not Gr66a-expressing GSNs, which respond to caffeine. The identified G2Ns relay information from Gr5a-expressing GSNs to distinct regions in the GNG, and are distinct from other, recently identified gustatory projection neurons, which relay information about sugars to a brain region called the antennal mechanosensory and motor center (AMMC). Our findings suggest unexpected complexity for taste information processing in the first relay of the gustatory system.

Keywords:

• Higher-order circuits
• interneuron
• taste
• subesophageal zone

Acknowledgements

We thank Moyi Li and Peter Nguyen for helping to maintain fly stocks, Mihaela Serpe for sharing fly resources, and Chun-yuan Ting, Yan Li, Kazumichi Shimizu, and members of the Stopfer Lab for helpful discussion and technical advice. We thank Kristin Scott (UC Berkeley), Marco Gallio (Northwestern University), Craig Montell (UC Santa Barbara), and Bloomington Drosophila Stock Center (Indiana University) for providing fly strains. This work was supported by the Intramural Research Programs of the National Institutes of Health (NIH), Eunice Kennedy Shriver National Institute of Child Health and Human Development (grant 1ZIAHD008760 to M.S.; grant Z01-HD008776 to C.-H.L). T. M. received a Japan Society for Promotion of Science Research Fellowship for Japanese Biomedical and Behavioral Researchers at NIH (Mar., 2011–Feb., 2013).

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Supplementary material available online

Supplementary Figure 1 available online at http://informahealthcare.com/abs/doi/10.3109/01677063.2015.1054993.

Supplementary Movie 1. Separation of signals from R12C04-labeled neurons. Confocal serial sections of the brain sample used in Figure 2D are shown from anterior to posterior, dorsal to the top, and medial to the left. The top-right and bottom-left show distributions of separated signals from the R12C04-labeled fibers extending from the ventrolateral neurons and the dorsal neurons, respectively. The bottom-right shows the distribution of axons of Gr5a-expressing GSNs. The top-left shows overlay of these signals. The separate signals from the ventrolateral neurons and the dorsal neurons are shown in green and purple, respectively. The axons of Gr5a-expressing neurons are shown in orange. In addition, R12C04-labeled signals outside the GNG are shown in blue. The green and purple signals are separated in 3D space (available online at http://informahealthcare.com/abs/doi/10.3109/01677063.2015.1054993).