Fig. 4

Developmental nicotine exposure does not alter the number of TH+ neurons in larval brains. 3rd instar larvae brains were dissected, immunostained, mounted and imaged on a confocal microscope. a-c are maximum projection images of a brain from a larva reared in control food and show representative images of larval brains used for analysis. a An anti-bruchpilot (BRP) antibody was used as background staining for the whole brain. b An anti-tyrosine hydroxylase (TH) antibody was used as marker for dopaminergic neurons. c Merged image of the BRP and TH channels. The scale bar is 50 μM. d-f show the average number of TH+ neurons counted in the DM, DL1 and DL2 dopaminergic clusters from larvae reared in control food (black bars) or nicotine food (red bars). The number of TH+ neurons was not affected by developmental nicotine exposure. Sample size was n = 19 brain hemispheres for control and n = 15 brain hemispheres for nicotine from n = 4 independent experiments. Student’s t-test was used to compare the control versus the nicotine condition