The mushroom body D1 dopamine receptor controls innate courtship drive

Abstract

Mating is critical for species survival and is profoundly regulated by neuromodulators and neurohormones to accommodate internal states and external factors. To identify the underlying neuromodulatory mechanisms, we investigated the roles of dopamine receptors in various aspects of courtship behavior in Drosophila. Here, we report that the D1 dopamine receptor dDA1 regulates courtship drive in naïve males. The wild-type naïve males actively courted females regardless their appearance or mating status. On the contrary, the dDA1 mutant (dumb) males exhibited substantially reduced courtship toward less appealing females including decapitated, leg-less and mated females. The dumb male's reduced courtship activity was due to delay in courtship initiation and prolonged intervals between courtship bouts. The dampened courtship drive of dumb males was rescued by reinstated dDA1 expression in the mushroom body α/β and γ neurons but not α/β or γ neurons alone, which is distinct from the previously characterized dDA1 functions in experience-dependent courtship or other learning and memory processes. We also found that the dopamine receptors dDA1, DAMB and dD2R are dispensable for associative memory formation and short-term memory of conditioned courtship, thus courtship motivation and associative courtship learning and memory are regulated by distinct neuromodulatory mechanisms. Taken together, our study narrows the gap in the knowledge of the mechanism that dopamine regulates male courtship behavior.

Keywords: Conditioning; D1 receptor; Drosophila; courtship; dopamine; learning; mating decision; memory; motivation; mushroom body.

Figure 1

Dopamine receptors are dispensable for acquisition and short‐term memory in associative courtship conditioning. The wild‐type CS and dopamine receptor mutants were trained with a mated female or mock‐exposed and then tested with a decapitated virgin female right after or 1 h training. (a) The CI of the mock‐exposed (Mo) and trained (Tr) dopamine receptor mutants right after training. Mann–Whitney U test: ***P < 0.0001; **P < 0.01; n = 32–48. (b) PI right after training. Kruskal–Wallis test, P = 0.0193; the letters on the bars denote significant difference from the control CS by Dunn method for Joint Ranking; P = 0.049 for b. (c) PI at 1 h after training. Kruskal–Wallis test, P > 0.05; b, P = 0.0189, Dunn for Joint Ranking with CS; n = 22–73.

Figure 2

dDA1 is needed to court less appealing mates. (a) Naïve CS and dumb ² males were paired with either an intact or decapitated virgin female, and the percent time that a male courting a female (CI) was measured. With an intact female: Kruskal–Wallis, P < 0.0001; b, P = 0.0130 by Dunn for Joint Ranking with the control CS; ns, not significant; n = 20–28. With a decapitated female: Kruskal–Wallis, P < 0.0001; ***P < 0.0005 by Dunn for Joint Ranking with the control CS; n = 27–28. (b) The percent reduction of CI with a decapitated virgin female calculated from the mean CI with an intact virgin female. Kruskal–Wallis, P < 0.0001; ***P < 0.0005 by Dunn for Joint Ranking with the control CS; n = 27–28. (c) Transposon locations in dumb alleles (top) and dDA1 immunoreactivity (bottom). Boxes indicate exons and triangles denote the transposons piggyBac{WH} containing UAS and Mi{MIC} containing splice acceptor (SA) in dumb ² and dumb ⁴, respectively. The orange‐colored boxes represent the open reading frame downstream of the UAS in dumb ², which corresponds to the previously characterized dDA1 (Sugamori et al. 1995). The whole mount CS and dumb ⁴ brains were stained with anti‐dDA1 antibody and the Alexa 488‐labeled secondary antibody. The stacked optical sections of the MB lobe areas in CS and dumb ⁴ are shown (scale bar, 50 µm). Yellow arrowheads and arrows demarcate the α, α′, β, and γ lobes in CS and the MB lobe areas in dumb ⁴, respectively. (d) CI of the naïve dumb ⁴ males paired with either an intact or decapitated virgin CS female (left two columns) and the percent reduction of CI with a decapitated virgin female calculated from the mean CI with an intact virgin female (right column). ***P < 0.0001 by Mann–Whitney U test, n = 32–34.

Figure 3

dDA1 is required to court suboptimal females. (a, b) CI of CS and dumb ² males with a mated female (a) or a leg‐less virgin female (b). *P < 0.05 by Mann–Whitney U test, n = 20–67. (c) CS, dumb ¹ and dumb ² males exhibit comparable courtship activity with a male of the same genotype. The boxplot delineates minimum, the first quartile, median, third quartile and maximum as long with outliers in each genotype. ns, P > 0.05 by Kruskal–Wallis test, n = 49–75.

Figure 4

dDA1 is important for courtship drive but not for courtship rigor. (a) Courtship latency. The time that CS or dumb ² males began courting an intact or decapitated virgin female was measured. Mann–Whitney U test: ns, P > 0.05; **P < 0.005; n = 30–42. (b) Interval between courtship bouts. Mann–Whitney U test: **P < 0.005; n = 30–42. (c) Courtship rigor. The percentage of CI ended at each courtship step such as singling, licking or copulation attempt with the decapitated virgin female was calculated from the total CI to represent percent courtship advancement. Mann–Whitney U test: ns, P > 0.05, n = 30–42. (d) Locomotor activity. The number of times that a male crosses a midline drawn across the courtship chamber per min is shown. ns, P > 0.05 by two‐tailed Student's t‐test, n = 32–42.

Figure 5

Restored dDA1 expression in the MB. dDA1 immunoreactivity was visualized by the Alexa 488‐labeled secondary antibody. Shown are the stacked optical sections of the MB lobe areas in CS, dumb ², dumb ² with reinstated dDA1 expression driven by MB247‐GAL4 in the α/β and γ lobes, NP1131‐GAL4;NP3061‐GAL4 in the α/β and γ lobes, c739‐GAL4 in the α/β lobe, and NP1131‐GAL4 in the γ lobe. dDA1 expression is visible in all MB lobes in CS but undetectable in dumb ². Yellow arrowheads demarcate the α, α′, β, and γ lobes. Scale bar, 25 µm.

Figure 6

dDA1 in the MB α/β and γ lobes regulates courtship drive. The percent reduction of the CI with the decapitated female calculated from the mean CI with the intact female was measured in CS and dumb ² males along with the dumb ² males with restored dDA1 expression in the α/β and γ lobes (MB247‐GAL4 and NP1131‐GAL4;NP3061‐GAL4), γ lobe (NP1131‐GAL4), α/β lobe (c739‐GAL4), or PDF neurons (PDF‐GAL4). Kruskal–Wallis test, P < 0.0001; ns, P > 0.05; *P < 0.05 by Dunn for Joint Ranking with the control CS; n = 31–42.