Functional feedback from mushroom bodies to antennal lobes in the Drosophila olfactory pathway

Abstract

Feedback plays important roles in sensory processing. Mushroom bodies are believed to be involved in olfactory learning/memory and multisensory integration in insects. Previous cobalt-labeling studies have suggested the existence of feedback from the mushroom bodies to the antennal lobes in the honey bee. In this study, the existence of functional feedback from Drosophila mushroom bodies to the antennal lobes was investigated through ectopic expression of the ATP receptor P2X(2) in the Kenyon cells of mushroom bodies. Activation of Kenyon cells induced depolarization in projection neurons and local interneurons in the antennal lobes in a nicotinic receptor-dependent manner. Activation of Kenyon cell axons in the betagamma-lobes in the mushroom body induced more potent responses in the antennal lobe neurons than activation of Kenyon cell somata. Our results indicate that functional feedback from Kenyon cells to projection neurons and local interneurons is present in Drosophila and is likely mediated by the betagamma-lobes. The presence of this functional feedback from the mushroom bodies to the antennal lobes suggests top-down modulation of olfactory information processing in Drosophila.

Fig. 1.

Depolarization responses in PNs and LNs resulted from activation of mushroom bodies. (A) KC cell bodies or βγ-lobes of mushroom bodies were activated by local puffing of ATP at positions marked 1 and 2, respectively. PN or LN responses were monitored by whole-cell recording. (B–E) Representative traces showed the responses in PNs and LNs resulted from activation of KC cell bodies (position 1) or βγ-lobes (position 2) by 10 mM ATP (B, C) or 1 mM ATP (D, E). Inset depicts the response evoked by ATP application at position 2 with higher time resolution. Graphs on the right show the peak amplitudes of depolarization responses evoked by paired ATP applications at positions 1 and 2 (Materials and Methods). Points with error bars depict the average ± SEM. Data sets marked *** are significantly different with P < 0.001 (paired t test). (F) Average amplitudes of depolarization responses in PNs and LNs evoked by local puffing of 10 mM ATP and 1 mM ATP at position 2. (G) The average input resistance of PNs (n = 10) and LNs (n = 6). Data sets marked *** are significantly different with P < 0.001 (t test).

Fig. 2.

Mushroom body activation-induced PN/LN responses depend on specific expression of P2X₂ in KCs. (A and B) (Left) Representative responses from PNs/LNs of 247-Gal4, UAS-P2X₂ and 247-Gal4:UAS-P2X₂ flies caused by local puffing of 10 mM ATP at βγ-lobes. (Right) Average peak amplitudes ± SEM (**, P < 0.01; ***, P < 0.001 by t test). (C and D) Average peak amplitudes (Left) and latencies (Right) of PNs/LNs responses evoked by puffing of ATP on the βγ-lobes of mushroom bodies or directly to antennal lobes (position 3 in Fig. 1A). Results are shown as the average ± SEM (*, P < 0.05; **, P < 0.01; and ***, P < 0.001 by paired t test). (E and F) (Left) Representative responses of PNs/LNs recorded from fly brains after removal of the calyx (calyx¯) or mushroom body (MB¯), with ATP puffing at position 2 shown in Fig. 1A. (Right) Average peak amplitudes ± SEM (*, P < 0.05; **, P < 0.01; and ***, P < 0.001 by t test). Number in line/bar graph indicates sample size.

Fig. 3.

Functional feedback is mediated by cholinergic transmission. (A–D) (Left) Responses from PNs/LNs evoked by ATP puffing at position 2, before and after application of MCA (A and B) or α-BTX (C and D), and after MCA washout (A and B). (Right) Average peak amplitudes ± SEM (**, P < 0.01; ***, P < 0.001 by paired t test). Number in column indicates sample size.

Fig. 4.

Identification of candidate neurons underlying functional feedback from mushroom bodies to antennal lobes. Neurons recorded using the whole-cell method were loaded with biocytin, and the morphology of the neuron was examined by post hoc staining combined with confocal microscopy. (A) Example of a neuron with the cell body located near the mushroom body calyx in a 247-Gal4:UAS-GFP fly. Biocytin-stained projections from the neuron are shown in red, GFP-labeled mushroom bodies in green. The soma (arrowhead) is located in or next to the mushroom body calyx, the dendrites are primarily distributed in the calyx, and the axon projects (arrows) to the ipsi-lateral antennal lobe, proto-cerebrum, and the contralateral mushroom body (For additional data, see Movie S1). (Scale bar, 50 μm.) (B) An example of a neuron found in the antennal lobes of the 247-Gal4:UAS-GFP fly. The soma of the neuron (arrowhead) is located in the dorso-lateral antennal lobe, and the processes (red, with white arrows to show the shaft of the process) project to the mushroom body lobes (green) and spread over the majority of the antennal lobes (For additional data, see Movie S2). (Scale bar, 50 μm.)