Evoked peak serotonin concentration varies with blue light stimulus duration. (a) Representative traces showing the effect of different stimulation lengths (2, 5, 10, and 30s) on peak height in the same sample. (b) Pooled data (mean +/− SEM, n = 6 samples) shows an increase in peak height with increasing duration of blue light exposure. Peak height appears to reach a plateau after 10s; peak height at 30s is not significantly different from that at 10s (p = 0.78, student t-test, 2 tailed).

Effect of PCPA on serotonin release. When VNCs are dissected in the serotonin synthesis inhibitor PCPA, serotonin release decreases with repeated stimulation. All data is from 10 s long stimulations performed 10 min apart. (a) Representative traces from a VNC that has been incubated in PCPA, superimposed to show progressive decrease in peak height. (b) Pooled data (mean +/− SEM, n = 6) showing that peak height in PCPA incubated VNCs (triangles) decreases to 50 +/− 3% of initial value by the 4th stimulation. Asterisks indicate data are significantly different (** p < 0.01, *** p < 0.001, student t-test, 2 tailed) than samples incubated in buffer (circles).

Kinetic parameters governing serotonin clearance. (a) Parameters can be calculated from the elicited serotonin peaks. The decay portion of the signal is fit with a one phase exponential decay curve. From this equation, k, the exponential constant is used to calculate an initial velocity (V). These parameters are shown (b, c) plotted against peak height (mean +/− SEM of 3–12 signals per point) (b) The initial velocity of serotonin clearance data were fit to a 4 parameter logistics equation (line). (c) The derived clearance decay rate (k) decreases as larger serotonin release is elicited.

Characterization of evoked serotonin and dopamine signals. Blue light stimulation of isolated larval Drosophila VNCs elicits depolarization in neurons expressing ChR2. Left column: Color plots with dashed white line denoting oxidation potential utilized for signal trace. Duration of blue light exposure (10 s) indicated by blue bars. Color plots are scaled so the maximum oxidative current corresponds to 750 nM monoamine. Middle: The CVs verify the compound being detected. The CV from the fly (black line) is compared to that obtained during electrode calibration with 1 μM monoamine (blue line). The asterisks identify the oxidation and reduction peaks, which correspond to the green and blue areas in the color plots, respectively. Right: Signal traces show neurotransmitter concentration changes over time. Currents were converted to concentration based on electrode calibration values. (a–c) Serotonin release from a VNC expressing ChR2 under the control of Tph-GAL4. The color plot (a) and CV (b) exhibit serotonin-specific peaks as evident by the signal CVs match to the calibration CV. The signal trace (c) shows the serotonin peak is time-locked to the stimulus. Data from a control VNC lacking ChR2 expression shows minor fluctuations throughout the color plot (d) upon blue light exposure but the CV (e, black line) does not show any neurotransmitter specific peaks. (f) Trace shows a small error fluctuation in current.(g–i) Dopamine release from a VNC expressing ChR2 under the control of TH-GAL4. Note a different voltage waveform was used than for serotonin. The color plot (g) and CV (h) verify that dopamine was detected. Dopamine release is also time-locked to the stimulation (i). Data from a control VNC lacking ChR2 expression shows changes similar to those observed with the other waveform. Minor fluctuations are present at all voltages during the stimulation (j) but the CV (k) does not show any neurotransmitter specific peaks. (l) Trace shows a small fluctuation in current.

Stability of serotonin signal. (a) Diagram of measured parameters. Peak height, which is the maximal concentration change from baseline, and time to half maximal signal decay (t50), which is the time from the end of the blue light stimulation until the signal decays to half its maximal value, were calculated. (b) Representative traces from same VNC showing repeated stimulations. All data are 10s long stimulations performed 10 min apart. (c,d) Pooled data (mean +/− SEM, n = 4) from VNCs dissected in buffer showing that t50 (c) and peak height (d) remain relatively stable over 8 stimulations repeated 10 min apart.

Pharmacological characterization of serotonin release and clearance. VNCs were incubated in drugs for 30 minutes prior to the initiation of stimulation. (a) Representative trace from a VNC incubated in the VMAT inhibitor reserpine, superimposed on a trace from a control VNC that lacks ChR2 expression showing that they are similar in size and shape. (b) Representative traces from VNCs incubated in buffer, cocaine, or fluoxetine, superimposed to highlight differences in time course. (c, d) Pooled data (mean +/− SEM, n as shown). (c) Peak height is not affected by incubation in cocaine or fluoxetine (signal not different from buffer control group). Reserpine leads to a significant reduction in released serotonin (p<0.0001, student t-test, 2 tailed), with the signal size falling below the noise threshold (signal not significantly different from control VNC). (d) Cocaine and fluoxetine-incubated VNCs exhibit significantly longer time to half maximal signal decay (p < 0.0001, student t-test, two-tailed). Clearance measures were not performed for control or reserpine-incubated VNCs because serotonin was not detected.