Drosophila Dopamine2-like receptors function as autoreceptors

Abstract

Dopaminergic signaling pathways are conserved between mammals and Drosophila and D2 receptors have been identified in Drosophila. However, it has not been demonstrated whether Drosophila D2 receptors function as autoreceptors and regulate the release of dopamine. The goal of this study was to determine if Drosophila D2 receptors act as autoreceptors by probing the extent to which D2 agonists and antagonists affect evoked dopamine release. Fast-scan cyclic voltammetry was used to measure stimulated dopamine release at a carbon-fiber microelectrode implanted in an intact, larval Drosophila nervous system. Dopamine release was evoked using 5-second blue light stimulations that open Channelrhodopsin-2, a blue light activated cation channel that was specifically expressed in dopaminergic neurons. In mammals, administration of a D2 agonist decreases evoked dopamine release by increasing autoreceptor feedback. Similarly, we found that the D2 agonists bromocriptine and quinpirole decreased stimulated dopamine release in Drosophila. D2 antagonists were expected to increase dopamine release and the D2 antagonists flupenthixol, butaclamol, and haloperidol did increase stimulated release. Agonists did not significantly modulate dopamine uptake although the modulatory effects of D2 drugs on release were affected by prior administration of the uptake inhibitor nisoxetine. These results demonstrate that the D2 receptor functions as an autoreceptor in Drosophila. The similarities in dopamine regulation validate Drosophila as a model system for studying the basic neurobiology of dopaminergic signaling.

Figure 1

Effect of D2 agonist bromocriptine on evoked dopamine. (A) Control data show that when buffer is added instead of drug, stimulations evoked 15 and 30 min after the initial stimulation are stable (n = 5). (B) Background-subtracted cyclic voltammogram from a single nerve cord comparing evoked dopamine release before the addition of drug and 15 and 30 min after application of 50 μM bromocriptine. (C) Concentration vs time profile showing the effect of bromocriptine on stimulated dopamine release in a single nerve cord. The bar underneath marks the duration of the blue-light stimulation. (D) Pooled data (n = 7) show 50 μM bromocriptine decreased evoked dopamine release. Data are normalized to the initial stimulation in each animal. Statistics were determined via comparison of evoked release before and after addition of drug using paired t tests. **p < 0.01; ***p < 0.001. (E) Preincubation with 50 μM bromocriptine also decreased evoked dopamine release (n = 6). Significance is determined using an unpaired t test. *p < 0.05. (F) Dopamine clearance (t₅₀) is not significantly different in the presence of bromocriptine (paired t test; n = 6).

Figure 2

Effect of D2 agonist quinpirole. (A) Concentration–time traces show 50 μM quinpriole decreased evoked dopamine release. (B) Averaged data demonstrate (n = 7) evoked release is significantly decreased 15 and 30 min after addition of quinpirole (paired t test comparing evoked release before and after addition of drug). (C) Dopamine clearance (t₅₀) is not significantly reduced in the presence of quinpirole (paired t test; n = 6). *p < 0.05. **p < 0.01.

Figure 3

Effect of dopamine D2 antagonists. (A) An example concentration–time profile shows 5 μM flupenthixol increased dopamine release. Release is higher 15 min than 30 min after addition of drug. (B) Averaged data for 5 μM flupenthixol show that release was significantly increased 15 and 30 min after addition of drug and that the time for dopamine clearance significantly increases after addition of 5 μM flupenthixol (n = 6). (C) Averaged data for 5 μM butaclamol show that evoked release is significantly increased 15 min after addition of drug but not 30 min, and t₅₀is not significantly different (n = 5). (D) Averaged data for 5 μM haloperidol show a significant increase in release after 15 min and no change in t₅₀ (n = 6). All statistics are from paired t tests of release or clearance before and after addition of drug. *p < 0.05. **p < 0.01. ***p < 0.001.

Figure 4

Effects of the DAT inhibitor with D2R agonists and antagonists. (A) Concentration–time profile for a VNC incubated in 50 μM nisoxetine followed by addition of 50 μM bromocriptine. (B) Clearance of dopamine is significantly increased in the presence of both nisoxetine and bromocriptine (n = 6). The effect of 50 μM bromocriptine is suppressed in the presence of 50 μM nisoxetine (n = 6). (C) Concentration–time profile for a VNC incubated in 5 μM nisoxetine followed by addition of 5 μM flupenthixol. (D) Clearance is not significantly changed in VNCs incubated in nisoxetine followed by flupenthixol. The effect of flupenthixol is suppressed in the presence of nisoxetine. All statistics are from paired t tests of release or clearance before and after addition of drug. *p < 0.05. **p < 0.01. ***p < 0.001.