A pair of dopamine neurons target the D1-like dopamine receptor DopR in the central complex to promote ethanol-stimulated locomotion in Drosophila

Abstract

Dopamine is a mediator of the stimulant properties of drugs of abuse, including ethanol, in mammals and in the fruit fly Drosophila. The neural substrates for the stimulant actions of ethanol in flies are not known. We show that a subset of dopamine neurons and their targets, through the action of the D1-like dopamine receptor DopR, promote locomotor activation in response to acute ethanol exposure. A bilateral pair of dopaminergic neurons in the fly brain mediates the enhanced locomotor activity induced by ethanol exposure, and promotes locomotion when directly activated. These neurons project to the central complex ellipsoid body, a structure implicated in regulating motor behaviors. Ellipsoid body neurons are required for ethanol-induced locomotor activity and they express DopR. Elimination of DopR blunts the locomotor activating effects of ethanol, and this behavior can be restored by selective expression of DopR in the ellipsoid body. These data tie the activity of defined dopamine neurons to D1-like DopR-expressing neurons to form a neural circuit that governs acute responding to ethanol.

Figure 1. Blockade of evoked release from dopaminergic neurons reduced ethanol-induced hyperactivity.

A. Expression of active tetanus toxin (TeTx) or an inactive form (IMP) as a control in tyrosine hydroxylase (TH) neurons (full genotype: TH-GAL4/+;UAS-TeTx/+). Open box indicates time of ethanol vapor exposure (47% ethanol vapor concentration). ‘A’ indicates locomotor speed of acclimated flies in humidified air just prior to ethanol exposure. B. Higher resolution analysis of the olfactory startle response. Peak speed achieved between 0–1 min ethanol exposure did not differ between genotypes (P = 0.2722, 1 way ANOVA, n = 10). C. Distance traveled for hyperactivity onset. TH/TeTx was different from indicated control genotypes (**P<0.01, 1-way ANOVA, Dunnett's multiple comparison test, n = 10). Expression of TeTx with Ddc(HL7) but not Ddc(HL5) resulted in reduced ethanol-induced hyperactivity (Ddc(HL7): **P = 0.0006, Ddc(HL5): P = 0.139, 2 sample t-test, n = 9–10). All transgenes were heterozygous in animals tested for behavior. D. Dopaminergic neuron cell body positions in one hemisphere of the adult brain. Drawing was adapted from Friggi-Grelin et al. . DA neuron nomenclature describes the location of the cell bodies in the adult brain (for example the PPM3s are one of three clusters of protocerebral posterior medial DA cells) . E–G. The average number of TH-positive cells that expressed the indicated GAL4 transgenes (red). Only PAM cell number is underrepresented. Number of hemispheres counted is indicated below the diagrams. The TH-GAL4 expression pattern has been reported previously , .

Figure 2. TH-positive PPM3 neurons that project to the ellipsoid body promote ethanol-induced hyperactivity.

A. c346 (detected by UAS-GFP, green) was expressed in two TH-positive (magenta) PPM3 neurons per hemisphere. The c346 PPM3 processes projected anteriorly (arrowheads) towards the central complex. c346 was also detected in the mushroom body Kenyon cells (KC) and in neurons that projected to the lobula (lo). B. c346 (green) processes project to the lateral triangles (LTR) and ring of the ellipsoid body (EB), counterstained with FasII (magenta). C. c346 expression in TH-positive cells is limited to the PPM3s. D,E. Labeling of individual TH-GAL4 cells using the flp-out technique. Two GFP-labeled TH cells in the PPM3 cluster projected anteriorly to the LTR region (entry point is marked with an arrow in E). The process branched to innervate the EB ring and LTR, and the ventral body region. F,G. Distance traveled was reduced in c346/TeTx (*P<0.05, **P<0.01, 1-way ANOVA, Tukey's multiple comparison test) whereas the startle response was not (P = 0.0925, 1-way ANOVA, n≥6). H. Transient activation of TH neurons increased locomotor activity. Expression of the heat-activated TrpA1 ion channel in TH neurons at 15°C (off) and 30°C (on). I. Distance traveled from 20–30 min at 30°C for TH/TrpA1 (**P<0.01, 1-way ANOVA, Dunnett's multiple comparison test, n≥5). c346/TrpA1 also increased locomotor activity. Flies of the genotype c346/Y;TH-GAL80/+;UAS-TrpA1/+, where GAL4 activity was blocked by GAL80 solely in c346 PPM3 neurons, showed no increase in locomotor activity (*P<0.05, **P<0.01, 1-way ANOVA, Dunnett's multiple comparison test to c346/Y; UAS-TrpA1/+, n≥6). Activation of TrpA1 in the Ddc(HL5) pattern had no effect (P = 0.6243, 1-way ANOVA, n≥5). J,K. Overexpression of DAT in TH-GAL4 or c346 neurons increased ethanol-induced hyperactivity. (*P = 0.0123, **P = 0.0005, 2 sample t-test, n≥10).

Figure 3. Functional mapping of ellipsoid body neurons in ethanol-induced hyperactivity.

Blockade of evoked release in patterns that included R2/R4 neurons of the ellipsoid body (B–D, GAL4 transgenic lines 5.30, 11.148, and c819) resulted in reduced ethanol-induced hyperactivity (A). Ethanol-induced hyperactivity was not reduced by TeTx expression in patterns that included R1 (c561), R3 (189Y, c232), or R4d (c232) neurons of the ellipsoid body (E–G). *P<0.05, **P<0.01, 1-way ANOVA for a given GAL4 transgene, Tukey's multiple comparison test, n≥5. For c819/TeTx vs. c819/IMP, **P = 0.0041, 2 sample t-test, n = 8. B–G. Expression of UAS-GFP (green) by the indicated GAL4 transgene, counterstained with FasII antibodies (magenta). Arrowheads indicate innervation of the ellipsoid body ring. PI: pars intercerebralis, MB: mushroom bodies.

Figure 4. The D1-like DA receptor DopR expression pattern in the adult brain includes the R neurons of the central complex ellipsoid body.

A–B. Presynaptic regions of DopR-expressing neurons. GAL4 enhancer trap PL00420 in the DopR locus driving expression of tdTomato-GFP tagged with presynaptic protein HIP14. A. Expression in the mushroom body lobes (mb), ellipsoid body neurons (eb), and central complex noduli (n). B. Expression in the central complex fan shaped body (fsb) and noduli, and the mushroom body peduncles. C–E. Genetic background control brains stained with DopR (green) and FasII (magenta) antisera. Each panel is a 4.3 µm confocal section of a whole mount brain. Ellipsoid body (arrowhead), mushroom body peduncles, and cellular cortex (ctx) labeling with DopR antisera. F–H. Ellipsoid body lateral triangles (arrowhead) and ring showed elevated DopR levels that were coincident with the 5.30 GAL4 enhancer trap expression pattern in 10 µm thin sections. I. Mushroom bodies and cellular cortex staining in controls. J. Fan-shaped body and noduli DopR staining in controls. K. DopR^f02676 mutant brain (DopR^−/−) stained with DopR (green) and FasII (magenta) antisera. 100 µm confocal projection shows nearly absent DopR staining throughout the brain. L–N. Colabeling of c346 presynaptic sites (using n-syb-tagged GFP) and DopR in the ring and lateral triangles.

Figure 5. Molecular and behavioral characterization of DopR mutants.

A. DopR gene structure. Positions of the transposons f02676 and PL00420 are indicated in the DopR locus. Arrows indicate orientation of UAS sites for f02676 and GAL4 for PL00420. Black rectangles indicate the single predicted open reading frame. B. Relative to genetic background controls, DopR transcript levels were nearly eliminated in DopR^f02676 homozygotes (−/−), whereas transcript levels of the DA receptors DopR2 and D2R were unchanged, as determined by quantitative RT-PCR (*P<0.05, **P<0.01, 1 way ANOVA, Dunnet's multiple comparison test to control, n = 3 replicates). DopR PCR probeset location is indicated in A. C, D. Ethanol-induced locomotor activity was reduced in DopR^f02676 homozygotes (**P<0.01, 1 way ANOVA, Tukey's multiple comparison test, n≥11). E. Precise excision of f02676 reverted the ethanol-induced hyperactivity phenotype (**P<0.01, 1 way ANOVA, Dunnett's multiple comparison test, n = 12). F. Reduced ethanol-induced hyperactivity in DopR^PL00420 homozygotes (*P = 0.0116, 2 sample t-test, n = 11). G. Non-complementation for ethanol-induced hyperactivity by DfED5634, a deficiency that deletes the entire DopR locus, for the DopR f02676 and the dumb¹ inversion alleles (**P<0.01, 1 way ANOVA, Dunnett's multiple comparison test, n≥6). H–K. DopR mutants develop normal ethanol tolerance. H. Ethanol exposure scheme to induce and measure rapid ethanol tolerance. Flies were exposed twice for 26 min each to ethanol vapor (60%), separated by a 3.5 hr rest period. Locomotor activity was quantified for both exposures, and the number of flies sedated was counted immediately after each exposure. I. Locomotor activity profiles for exposure 1 and 2 for genetic background control and DopR^f02676 flies. J. No change in distance traveled was detected from 2–25 min, Exposure 2 minus Exposure 1 (ΔDist) (P = 0.6691, paired t-test, n = 15). K. Sedation tolerance, the fraction awake exposure 2 minus exposure 1, was unaffected (P = 0.27, 2 sample t-test, n = 15). L. Ethanol sensitivity as measured in the inebriometer was unaffected (P = 0.16, 2 sample t-test, n = 5). M. Rapid tolerance, as measured in the inebriometer, was unaffected (P = 0.09, 2 sample t-test, n = 5).

Figure 6. Genetic rescue of DopR mutant ethanol-induced hyperactivity by restricted expression of DopR in the ellipsoid body.

A. Distance traveled for animals homozygous for f02676 (DopR/DopR), and heterozygous for GAL4 transgenes to drive expression of DopR in patterns that include the ellipsoid body R2/R4 neurons (11.148, 5.30), R3/R4d neurons (c232, 189Y), or the mushroom bodies (17d, 201Y). Restored expression of DopR in the R2/R4 neuron patterns but not others resulted in increased ethanol-induced hyperactivity as compared to DopR/DopR (**P<0.01, 1 way ANOVA, Dunnett's multiple comparison test, n≥10). B. Rescuing strategy results in selectively localized DopR protein expression. Confocal section of an animal homozygous for f02676 and carrying the GAL4 enhancer trap 5.30, stained for DopR protein (green) and FasII (magenta). 5.30-GAL4 drives expression of DopR protein in the ring of the ellipsoid body (eb) and the lateral triangles (ltr), but not in the mushroom bodies (mb). DopR expression in the R2 neurons is indicated by the arrowhead.