Functional analysis of an olfactory receptor in Drosophila melanogaster

Abstract

Fifty nine candidate olfactory receptor (Or) genes have recently been identified in Drosophila melanogaster, one of which is Or43a. In wild-type flies, Or43a is expressed at the distal edge of the third antennal segment in about 15 Or neurons. To identify ligands for the receptor we used the Gal4/UAS system to misexpress Or43a in the third antennal segment. Or43a mRNA expression in the antenna of transformed and wild-type flies was visualized by in situ hybridization with a digoxigenin-labeled probe. Electroantennogram recordings from transformed and wild-type flies were used to identify cyclohexanol, cyclohexanone, benzaldehyde, and benzyl alcohol as ligands for the Or43a. This in vivo analysis reveals functional properties of one member of the recently isolated Or family in Drosophila and will provide further insight into our understanding of olfactory coding.

Figure 1

Or43a is expressed in neuronal cells. mRNA expression of Or43a in a 10-μm thick cryosection through the third antennal segment of (A) wild type and (B) line 87-3c as visualized by in situ hybridization with a digoxigenin-labeled cDNA probe. Although few cells are stained in wild type, the number of Or43a-expressing cells is increased in the antenna of 87-3c. Immunostaining of 10-μm horizontal cryosections of the antenna. (C) Interphase contrast picture of the third antennal segment of the Gal4/UAS-LacZ line. (D) The same section with anti Elav-antibody (green) as neuronal marker and with (E) anti LacZ-antibody (red). (F) Fluorescence labeling was visualized with a Zeiss filter Nr.24 (emission at 460 nm, 530 nm, and 610 nm). Colocalization of Elav and LacZ can be identified by yellow color indicating neuronal expression of both proteins. Few neuronal cells do not have LacZ expression (arrows). (Bar = 5 μm.)

Figure 2

Increased EAG response of lines 87-1c and 87-3c to Bz. (A) Average of 10 EAG-recordings each from 1) transformed line 87-3c misexpressing Or43a and 2) wild-type flies. (Upper) Black bar indicates the given odorant pulse (1.0 sec) with a concentration of 10⁻² in paraffin oil. (B) EAG response of wild type, Or43a misexpressing lines 87-1c and 87-3c, and Or22b misexpressing line 67-3c to Bz diluted 10⁻² in paraffin oil (n = 20) for each genotype and (C) to acetone (Ac), butanol (But), Ea, and propionaldehyde (Pa), all diluted 10⁻² in paraffin oil (oil). n = 20 for each genotype tested with each odorant.

Figure 3

Increased EAG response to Bz is concentration dependent. EAG response to different concentrations of (A) Bz and (B) Ea dissolved in paraffin oil. In most cases error bars are too small to be shown on the graphs. GH230 and UAS-Or43a-3c are parental lines whereas 87-3c misexpresses Or43a. n = 25 tests for each genotype.

Figure 4

EAG response to different concentrations for (A) Cyl and (B) Cyc dissolved in paraffin oil. In most cases error bars are too small to be shown on the graphs. n = 25 tests for each genotype.