Optogenetic control of serotonin and dopamine release in Drosophila larvae

Abstract

Optogenetic control of neurotransmitter release is an elegant method to investigate neurobiological mechanisms with millisecond precision and cell type-specific resolution. Channelrhodopsin-2 (ChR2) can be expressed in specific neurons, and blue light used to activate those neurons. Previously, in Drosophila, neurotransmitter release and uptake have been studied after continuous optical illumination. In this study, we investigated the effects of pulsed optical stimulation trains on serotonin or dopamine release in larval ventral nerve cords. In larvae with ChR2 expressed in serotonergic neurons, low-frequency stimulations produced a distinct, steady-state response while high-frequency patterns were peak shaped. Evoked serotonin release increased with increasing stimulation frequency and then plateaued. The steady-state response and the frequency dependence disappeared after administering the uptake inhibitor fluoxetine, indicating that uptake plays a significant role in regulating the extracellular serotonin concentration. Pulsed stimulations were also used to evoke dopamine release in flies expressing ChR2 in dopaminergic neurons and similar frequency dependence was observed. Release due to pulsed optical stimulations was modeled to determine the uptake kinetics. For serotonin, Vmax was 0.54 ± 0.07 μM/s and Km was 0.61 ± 0.04 μM; and for dopamine, Vmax was 0.12 ± 0.03 μM/s and Km was 0.45 ± 0.13 μM. The amount of serotonin released per stimulation pulse was 4.4 ± 1.0 nM, and the amount of dopamine was 1.6 ± 0.3 nM. Thus, pulsed optical stimulations can be used to mimic neuronal firing patterns and will allow Drosophila to be used as a model system for studying mechanisms underlying neurotransmission.

Figure 1

Representative serotonin release evoked by (A) 2 s continuous stimulation, (B) a pulsed stimulation train of 4 ms pulse width, 500 pulses at 20 Hz, and (C) a pulsed stimulation train of 4 ms pulse width, 500 pulses at 100 Hz in the same ventral nerve cord. The bottom panel shows false color plots with time on the x-axis, applied voltage on the y-axis and background-subtracted faradaic current in pseudocolor. The duration of the stimulation is marked as the black bar below the color plot. The concentration versus time profiles are plotted on top of the color plots by converting the current at the maximal oxidation potential for serotonin to concentration through in vitro calibration. The insets are background-subtracted cyclic voltammograms which confirm that serotonin is detected.

Figure 2

Frequency dependency of stimulated serotonin release for three different pulse widths: 4, 10, and 20 ms. The frequency dependency is tested in two patterns. (A–C) The total amount of light illumination is fixed at 2 s. (D–F) The total simulation duration is fixed at 2 s. It is 20, 40, 80, 120, 160, 200, and 240 pulses for 10, 20, 40, 60, 80, 100, and 120 Hz, respectively. Data are expressed as the ratio of serotonin release by pulsed stimulation to that released by 2 s continuous illumination, which normalizes for different release amounts in different samples. Each panel was evaluated with a one-way ANOVA: (A) F[6,41] = 11.27, p < 0.0001; (B) F[4,27] = 35.46, p < 0.0001; (C) F[2,15] = 16.14, p < 0.001; (D) F[6,50] = 24.89, p < 0.0001; (E) F[4,34] = 55.29, p < 0.0001; (F) F[2,21] = 44.35, p < 0.0001. Data are mean ± SEM, and n = 5–9.

Figure 3

Pulse width dependency of stimulated serotonin release with three different stimulation frequencies: (A) 10 Hz (one-way ANOVA, F[3,27] = 55.34, p < 0.0001), (B) 20 Hz (F[3,28] = 77.24, p < 0.0001), and (C) 40 Hz (F[2,21] = 24.49, p < 0.0001). The total stimulation duration is 2 s. Data are expressed as the ratio of serotonin release by pulsed stimulation to that of the 2 s continuous illumination. Data are mean ± SEM, and n = 7–8.

Figure 4

Effect of pulse number on stimulated serotonin release. The effect is tested at two different frequencies (A) 20 Hz and (B) 60 Hz with a 4 ms pulse width. Data are expressed as the ratio between serotonin release by pulsed stimulation and that of the 2 s continuous illumination. Data are mean ± SEM, (A) n = 4–7 and (B) n = 6. Insets are enlarged view of the first 120 pulses. (C) Data from one representative nerve cord at 4 ms, 20 Hz stimulation with two pulse numbers (20 pulses and 100 pulses). A steady-state is achieved with the higher pulse number while not with the lower one. (D) Similarly, data recorded from one nerve cord with 4 ms, 60 Hz stimulation using two pulse numbers (120 pulses and 480 pulses). The duration of the stimulation is marked below the concentration traces (black solid bar for the shorter stimulation and orange solid bar for the longer stimulation). The y scale in (D) is 10 times larger than the y scale in (C).

Figure 5

Kinetic modeling of pulsed optically stimulated serotonin release. Data from one representative nerve cord (black lines) with two different stimulation frequencies were fit to a Michaelis–Menten kinetic model to determine the parameters for serotonin release and uptake. Curves with steady-state (A) and non-steady-state (B) were selected for the kinetic modeling. Scale bar is the same for both panels. Simulation lines (orange) were calculated from best-fit parameters ([serotonin]_p = 4.3 nM, V_max = 0.48 μM/s and K_m = 0.61 μM). The duration of the stimulation is indicated by the black bar under the curves.

Figure 6

Effects of serotonin uptake inhibitor fluoxetine. (A) With 2 s continuous stimulation, the half decay time (t₅₀) significantly increased 15 min after 100 μM fluoxetine was applied (***p < 0.001, paired t test, n = 15). (B) Concentrations versus time profiles showing the effect of 100 μM fluoxetine on serotonin release by 4 ms pulse width stimulations at a low (20 Hz) and a high (100 Hz) frequency in the same nerve cord (black bar marks the stimulation duration). (C) Serotonin release is not frequency dependent after 100 μM fluoxetine (data mean ± SEM and n = 9–11). Data are expressed as the ratio of serotonin release by pulsed stimulation to that released by 2 s continuous illumination in the presence of fluoxetine. (D) Kinetic modeling of pulsed optically stimulated serotonin release in the presence of 100 μM fluoxetine. Simulation line (orange) to fit the representative data (black) is calculated with the parameters: [serotonin]_p = 4.4 nM, V_max = 0.54 μM/s, and K_m = 6.4 μM, with R² = 0.93.

Figure 7

Dopamine release evoked by pulsed optical stimulations. (A) Frequency dependence of stimulated dopamine release with 4 ms pulse width and the total stimulation duration is fixed at 2 s. Data are expressed as the ratio of dopamine release by pulsed stimulation to that of the 2 s continuous illumination. Data are mean ± SEM, n = 4. (B) Representative color plot of dopamine release evoked by a pulsed stimulation train of 4 ms, 40 Hz, 80 pulses. The green and blue areas show the oxidation and reduction peaks of dopamine, respectively. (C) The concentration versus time profile (black) is plotted, and kinetic modeling (orange) was calculated from the parameters: [dopamine]_p = 2.4 nM, V_max = 0.13 μM/s, and K_m = 0.45 μM, with R² = 0.91. (D) Background-subtracted cyclic voltammogram confirms that dopamine is detected.