Nicotine exposure alters the behavioral profile of Drosophila larvae.

A Legend and schematics for the set of observed locomotor and postural behaviors. B Fractional use of the set of behaviors following nicotine exposure. Between 5 and 7 wild type Canton-S (CS) larvae were used for each condition.

Nicotine affects multiple behaviors in larval locomotion.

(A) Larvae were rinsed (blue), exposed to a nicotine solution (red), rinsed again, and moved to an agar gel platform for behavioral recording (example track shown in green). (B) Extracted behavioral metrics. Example track comprised of an alternating sequence of runs (of duration Δti) and turns (yellow circles), which occur at rate γ. Properties of runs (efficiency d/𝓁, speed ⟨v⟩, peristalsis frequency f) and turns (size Δθ, acceptance probability P, angular velocity ω) are extracted from recorded video data. (C) Dose-response curve for moderate nicotine concentrations, with 1.125 mM producing the greatest speed increase. 2191 larva tracks were analyzed, between 88 and 394 for each NIC dose. (D) Nicotine changes specific behavioral components. Bar graphs compare six behaviors with and without NIC exposure. Error bars are s.e.m. *** indicates p < 0.001, ns indicates p > 0.05, Student’s t-test. 394 control (C) tracks of Canton-S larvae were analyzed, and 155 NIC larvae.

Speed and peristalsis frequency linked and modulated by nicotine.

(A) Histograms of the average speed (left) and peristalsis frequency (middle) from 4929 runs, and a scatter plot (right) of each run showing the correlation (blue line) between the behavioral features. (B) Same information as displayed in panel A, but for larae exposed to 1.125 mM nicotine solution. (C) One representative track each for control and nicotine conditions. Each track has a speed and peristalsis frequency extremely close to the corresponding distribution mean. Left: representative tracks plotted as starting from the same point. Middle: displacement of the larva over time for each tracks. Right: speed over time for each track, with vertical lines indicating adjacent peaks or troughs in the sinusoidal pattern. Canton-S larvae from 7 experiments for each condition (394 control tracks, 155 nicotine tracks) were analyzed, the same animals as Fig. 2D.

Dopamine is a key component of nicotine-driven hyperactivity.

(A) Confocal micrograph of an acutely dissected PLE-GAL4 > UAS-Kir2.1-eGFP brain, labeling dopamine neurons in the brain lobes. (B) Schematic of larval brain lobes and the ventral nerve cord (VNC) in gray with neural clusters of the dopaminergic system: dorsalateral 1, 2 (DL1,DL2), dorsomedial 1 (DM1), and primary protocerebral anterior medial (pPAM), and the mushroom body (MB), important in the dopaminergic system. (C-G) Left: Bar graphs showing the behavioral impact of four pharmacological treatments compared to control (C): nicotine (N), L-Dopa (L), 3IY (3) and nicotine and 3IY together (3+N). Treatments were administered as described in Methods and Materials. Right: Bar graphs showing the behavioral impact of genetic silencing of dopaminergic neurons on the response to nicotine exposure. Different strains are separated by dashed lines. Percent changes are indicated above lines (red for decreases, green for increases). H Schematic summary of the directional changes in behavioral components with and without silenced dopamine neurons. Between 150 and 400 larvae were used for each experimental condition (2381 total). Error bars are s.e.m. *** indicates p < 0.001, ** indicates p < 0.01, * indicates p < 0.05, ns indicates p > 0.05, Student’s t-test.

The mushroom body (MB) is an important brain region involved in nicotine-driven hyperactivity.

(A) Confocal micrographs of an acutely dissected OK107-GAL4 > UAS-Kir2.1-eGFP brain (top) and a 201Y-GAL4 > UAS-Kir2.1-eGFP brain (bottom), labeling the entire MB and the γ lobe, respectively. (B-F) Bar graphs for five behavioral features showing the impact of MB manipulation on the response to nicotine (N) treatment. Different strains separated by dashed lines. Percent changes are indicated above lines (red for decreases, green for increases). Between 150 and 400 larvae were used for each experimental condition (2372 total). Error bars are s.e.m. *** indicates p < 0.001, ** indicates p < 0.01, * indicates p < 0.05, ns indicates p > 0.05, Student’s t-test.

Recovery from single and repeated nicotine exposures.

Horizontal bars show exposure protocols, with red bar and text indicating nicotine, blue water control, and black the free crawling during recording. A Single nicotine exposure recovery. Crawling speed (circles) indicate the mean speed in 3-min time windows. Black curve is an exponential decay function fit (y(t) = y0 + Ae−t/τ). 88 larvae were recorded. B Repeated nicotine exposure recovery. Solid blue (control) and red (nicotine) circles indicate mean speeds in 3-min time windows, and larger black-outlined circles the mean of the whole 30 min segment between exposures. Black curves are exponential fits for nicotine-exposed larvae (control data is not exponential). 62 larvae used for control experiments and 54 larvae for nicotine experiments. Canton-S larvae were used in all experiments.

Larvae navigate towards nicotine after repeated exposure.

A Protocol for making nicotine gradient gels (blue dye solution is water and red the nicotine). B Photograph of the gradient gel, and a schematic graph of its linear horizontal nicotine gradient from 0 to 1.125 mM. C Protocol for the nicotine exposure prior to testing. Larvae are submerged in water (blue), or submerged in 1.125 mM nicotine (red), or freely moving on plain agar gel (black). D Net larval movement along the nicotine gradient summarized by the mean navigation index, each larva has an individual index NI = ⟨vx⟩/⟨v⟩. Positive NI indicates navigation towards nicotine. N indicates the number of larvae tested per condition. E Navigation indexes of individual larvae. Left: Distribution of NI for control (blue) or nicotine-exposed (red) larvae. Vertical dashed lines indicate the mean NI of the populations. Right: Crawling trajectories for all experiments and the average x component of velocity. * indicates p < 0.05, Student’s t-test. Canton-S larvae were used in these experiments.