Figure 1.
Repeated subthreshold stimulation sensitizes odorant receptors.
A, Recordings of neuronal activity from ab3 sensilla (large action potentials, ab3A neuron expressing Or22a; small action potentials, ab3B neuron expressing Or85b) upon before and after 20 s repeated ethyl butyrate (Etb) stimulation (−10 v/v; 0.5 s, shaded area). The first stimulation fails to elicit a response while the second does so. B, Dependence of normalized ab3A neuron spike frequency (fnorm) upon 1st and 2nd subthreshold Etb stimulation (−10 v/v; 0.5 s) on the interval between stimulations (n = 12). C, Time course of fnorm for 1st and 2nd stimulation (interval 20 s, n = 12). D–F, Mean fnorm for ab3A (D), ac3B (E) and ab2A (F) neuron to repetitive subthreshold Etb (D), ethyl acetate (Eta, E) and methyl acetate (Mea, F) stimulations (interval 20 s, n = 12). *P<0.05, **P<0.01, ***P<0.001; Paired Wilcoxon Signed Ranks test.
Figure 2.
Repeated subthreshold stimulation does not sensitize ionotropic receptors (IRs).
A, Recordings of neuronal activity from ac3 sensilla (large action potentials, Ir75abc neuron; small action potentials, Or35a neuron) upon before and after 20 s repeated butyric acid (Ba) stimulation (−7 v/v; 0.5 s, shaded area). Both stimulations fail to elicit a response. B, Dependence of normalized Ir75abc neuron spike frequency (fnorm) during 1st and 2nd subthreshold Etb stimulation (−7 v/v; 0.5 s) on the interval between stimulations (n = 12). C, Time course of fnorm for 1st and 2nd stimulation (interval 20 s, n = 12). D–F, Mean fnorm for Ir75abc (D), Ir41a (E) and Ir84a (F) neuron to repetitive subthreshold Ba (D), Dab (E) and Paa (F) stimulations (interval 20 s, n = 12). N.s.; Paired Wilcoxon Signed Ranks test.
Figure 3.
Manipulation of cAMP signalling in Drosophila ab3 sensilla affects the odorant response.
A, Recordings of neuronal activity (large action potentials, Or22a neuron; small action potentials, Or85b neuron) before and after Etb stimulation (−5 v/v; 0.5 s, shaded area) in the presence of indicated compounds. While 8-br-cAMP enhances the Etb response, inhibition of adenylyl cyclase with SQ22536 attenuates it. B, Normalized spike frequency (fnorm) of ab3A upon Etb stimulation (0 to 0.5 s, shaded area) at indicated dilution after injection of saline solution (Control; n = 11), of 8-bromo-cAMP (n = 11; P<0.05, Mann-Whitney U test) and of the adenylyl cyclase inhibitor SQ22536 (n = 17; P<0.01, U test). C, Concentration dependence of the maximum frequency fmax of fnorm to Etb stimulation after saline, forskolin and SQ22536 injection (**P<0.01, ***P<0.001, ANOVA). D, fnorm as described in (B) after injection of saline solution (Control; n = 11), U73122 plus 8-br-cAMP (n = 10; P = 0.18, U test), and Gö6976 plus 8-br-cAMP (n = 17; P = 0.16, U test). In the presence of the PLC or PKC inhibitors 8-br-cAMP fails to enhance the odor response. E, Comparison of treatment effects on Etb response before and after microinjection. fnorm on Etb stimulation (0.5 s) as determined from area under the curve measurements of the total response (1.35 s). Responses to Etb were measured 20 s after commencement of recording (before injection) and 200 s after injection (after injection) of the control (n = 11), SQ22536 (n = 17), 8-br-cAMP (n = 11), forskolin (n = 9; data from Olsson et al., 2011), cholera toxin (CTX; n = 12), 8-br-cAMP plus U73122 (n = 10), and 8-br-cAMP plus Gö6976 (n = 17). Error bars represent s.e.m. Asterisks indicate significant differences (P<0.05, Paired Wilcoxon Signed-Rank Test).
Figure 4.
Regulation of OR response by cAMP signaling is intrinsic.
A, Orco (left), Orco mut (middle) and Or22a (right) proteins visualized in adult antennal sections with specific antibodies (red). The proteins show expression in cell bodies (arrowhead) and dendrites (arrow). Or22a-expressing cells are housed in few sensilla opposite to arista (a). Scale bar 50 mm. B, Normalized ab3A neuron spike frequency (fnorm) upon Etb stimulation wild type flies (Orco, n = 12), for Orco null mutants (no Orco, n = 15), and mutants rescued with Orco mut (“Orco mut flies”; n = 14; P = 0.016 vs. Control, Mann-Whitney U test). C, fnorm as in B upon Etb stimulation in Orco mut flies (n = 17) before (Control) and after forskolin injection.
Figure 5.
OR sensitization is mimicked by Orco activation and disrupted by Orco inhibition.
A, B, fnorm for ab3A neurons expressing Or22a upon initial subthreshold Etb stimulations (log [Etb] −10) after injection of saline (Control;, forskolin (A, n = 8; P<0.05, Mann-Whitney U test), and the protein kinase C activators PMA (B, n = 7; P<0.001, U test) and OAG (B, n = 7; P = 0.001, U test). C, Time course of fnorm upon 1st and 2nd subthreshold Etb stimulation (log [Etb] −10, interval 20 s) after injection of SQ22536 (n = 13). D, Mean fnorm for Or22a neurons to repetitive subthreshold Etb stimulations (interval 20 s) after injection of SQ22536 (n = 13). E, Time course of fnorm for neurons expressing Orco mut upon 1st and 2nd subthreshold Etb stimulation (log [Etb] −10, interval 20 s, n = 12). F – H, Mean fnorm for ab3A (F), ac3B (G) and ab2A (H) neurons expressing Orco mut to repetitive subthreshold Etb (F), Eta (G) and Mea (H) stimulations (interval 20 s, n = 8–14). N.s.; Paired Wilcoxon Signed Ranks test.