Figure 2.

The s-LNvs, but not the l-LNvs, respond to HB eyelet excitation with increases in calcium and cAMP. A, P2X2-mediated excitation of HB eyelets. Top two traces, Average GCaMP3.0 fluorescence plots (±SEM) for HB eyelet nerves coexpressing P2X2 and GCAMP in Rh6-GAL4/UAS-GCaMP3.0;UAS-P2X2/+ brains in response to 30 s perfusion of 1 mm ATP (top, N = 24) or vehicle (bottom, N = 24). Histogram represents comparison of average maximum GCaMP responses (±SEM) for the same data. ATP caused significant GCaMP fluorescence increases compared with vehicle control. B, Effect of eyelet excitation on l-LNv Ca²⁺ in Pdf-lexA,LexA-GCaMP3.0/Rh6-GAL4; UAS-P2X2/+ brains in which P2X2 is expressed in the HB eyelet and GCaMP in the LNvs. Data arranged as in A. N = 14 for ATP and N = 15 for vehicle. The l-LNvs did not display significant GCaMP fluorescence increases in response to eyelet excitation. C, Effect of eyelet excitation on s-LNv Ca²⁺ in Pdf-lexA,LexAop-GCaMP3.0/Rh6-GAL4;UAS-P2X2/+ brains. Data arranged as in A. N = 20 for ATP, N = 13 for vehicle. Excitation of the eyelet resulted in significant GCaMP fluorescence increases relative to vehicle controls. D, In brains lacking a driver for UAS-P2X2 expression in the eyelets (Pdf-lexA,LexAop-GCaMP3.0/+;UAS-P2X2/+). ATP failed to result in an increase in s-LNv GCaMP fluorescence relative to vehicle controls. Data arranged as for A. N = 13 for ATP, N = 13 for vehicle. Calibration: A–D, 1 min (x-axis) and a 50% change in GCaMP3.0 fluorescence over baseline (y-axis). E, Effect of eyelet excitation on l-LNv cAMP in Pdf-lexA,LexAop-Epac1-camps/Rh6-GAL4;UAS-P2X2 brains. The two traces represent average inverse Epac1-camps FRET (CFP/YFP) plots for l-LNvs in response to 1 mm ATP (top, N = 17) and vehicle (bottom, N = 17). Histogram represents comparison of average maximum Epac1camps responses (±SEM) for the same data. ATP perfusion failed to produce significant inverse FRET increases in l-LNvs relative to vehicle controls. F, Excitation of the eyelet causes cAMP increases in the s-LNvs of Pdf-lexA,LexAop-Epac1-camps/Rh6-GAL4;UAS-P2X2. Data arranged as for E. ATP produced significant inverse FRET increases in the s-LNvs relative to vehicle controls. N = 15 for ATP, N = 14 for vehicle. G, Average inverse Epac1-camps FRET (CFP/YFP) plots for l-LNvs in Pdf-lexA,LexAop-Epac1-camps/+;UAS-P2X2/+ brains that express Epac1-camps in the LNvs but fail to drive P2X2 in the eyelet. The l-LNvs did not display inverse FRET increases in response to ATP (middle, N = 14) relative to vehicle controls (bottom, N = 14). The l-LNvs did display inverse FRET increases to 10⁻⁴ m nicotine (top plot, N = 12). Histogram represents comparison of maximum Epac1-camps responses for the same data. ATP perfusion failed to produce significant increases in CYP/YPF ratio in the l-LNvs relative to vehicle controls, whereas nicotine (10⁻⁴ m) produced significant cAMP increases. H, Average inverse Epac1-camps FRET (CFP/YFP) plots for s-LNvs in Pdf-lexA,LexAop-Epac1-camps/+;UAS-P2X2/+ brains. Data organized as for G. ATP perfusion (N = 19) caused small but significant increases in CYP/YPF ratio relative to vehicle controls (N = 19) in the s-LNvs, whereas nicotine (10⁻⁴ m, N = 15) produced large and significant inverse FRET increases. Calibration: E–H, 1 min (x-axis) and a 25% changes in CFP/YFP ratio (y-axis). Bars under averaged plots represent 30 s of ATP perfusion switched from a constant saline flow. n.s., Not significant (p ≥ 0.05). *p < 0.05. **p < 0.01. ***p < 0.001. I, Response of a representative l-LNv to local pressure injection of 100 mm histamine (HA) into the ipsilateral Me. Black bars represent stimulus length (0.5 s, 1 s, 10 s). Duration-dependent inhibition of ipsilateral spiking activity (large spikes) was apparent, although contralateral activity (small spikes) was not affected. J, Enlargements of the boxed regions in I showing the inhibition after drug application. Spiking stops abruptly but comes back gradually before returning to previous levels. mp, Membrane potential.