The serotonin 5-HT7Dro receptor is expressed in the brain of Drosophila, and is essential for normal courtship and mating

Abstract

The 5-HT(7) receptor remains one of the less well characterized serotonin receptors. Although it has been demonstrated to be involved in the regulation of mood, sleep, and circadian rhythms, as well as relaxation of vascular smooth muscles in mammals, the precise mechanisms underlying these functions remain largely unknown. The fruit fly, Drosophila melanogaster, is an attractive model organism to study neuropharmacological, molecular, and behavioral processes that are largely conserved with mammals. Drosophila express a homolog of the mammalian 5-HT(7) receptor, as well as homologs for the mammalian 5-HT(1A), and 5-HT(2), receptors. Each fly receptor couples to the same effector pathway as their mammalian counterpart and have been demonstrated to mediate similar behavioral responses. Here, we report on the expression and function of the 5-HT(7)Dro receptor in Drosophila. In the larval central nervous system, expression is detected postsynaptically in discreet cells and neuronal circuits. In the adult brain there is strong expression in all large-field R neurons that innervate the ellipsoid body, as well as in a small group of cells that cluster with the PDF-positive LNvs neurons that mediate circadian activity. Following both pharmacological and genetic approaches, we have found that 5-HT(7)Dro activity is essential for normal courtship and mating behaviors in the fly, where it appears to mediate levels of interest in both males and females. This is the first reported evidence of direct involvement of a particular serotonin receptor subtype in courtship and mating in the fly.

Figure 1. Map of 5-HT₇Dro region.

The 5-HT₇Dro locus on the third chromosome is approximately 40 kb with a large first intron that contains two additional short annotated transcripts. The 5 kb region of genomic DNA immediately upstream of the mRNA transcript start site used to generate the 5-HT₇Dro GAL4 strain is shown (white box). Grey boxes indicate untranslated regions of exons, darker boxes represent translated regions. Arrows indicate direction of transcription. Scale bar is shown.

Figure 2. 5-HT₇Dro-GAL4 expression in the brain of third instar larva.

5-HT₇Dro-GAL4 driven expression of GFP (green) is detected in distinct circuits within the brain of wandering third instar larva as well as in the ventral ganglia. Antisera to 5-HT detected with secondary antibodies conjugated to alexafluor 568 (magenta) highlights the presynaptic serotonergic circuitry. No overlap indicates that 5-HT₇Dro-GAL4 expression is postsynaptic.

Figure 3. 5-HT₇Dro-GAL4 expression in the adult brain.

A) Expression of the 5-HT₇Dro-GAL4 driver is highly localized to large field R-neurons of the ellipsoid body. There are additional groups of cells that cluster with, but do not express peptide dispersal factor (PDF, magenta), between the central brain and the optic lobes (inset). B) High resolution cross-eyed stereo view of a typical cluster of large field R-neurons expressing 5-HT₇Dro-GAL4. This particular cluster consists of 48 large-field R neurons that express the driver. (EB ellipsoid body; LTR  =  lateral triangle, CB  =  cell bodies; ATL  =  antennal lobe).

Figure 4. Distribution of 5-HT₇Dro-GAL4 expression and serotonin in adult Drosophila brain.

Ai) Within the ellipsoid body (EB) of the central complex, the innermost and outermost rings display 5-HT₇Dro-GAL4 expression (green). The lateral triangles (LTR) of the central complex also display GFP. Expression is also seen more ventrally in antennal lobe neurons. Aii) Serotonin-immunoreactive neuron processes highlight the presence of 5-HT in the innermost and outermost rings of the EB as well as the lateral triangles and antennal lobes (magenta). Bi) Close up view of 5-HT₇Dro-GAL4 expression in the central EB. Bii) The same field as in Bi showing serotonin-immunoreactive neuron processes (magenta). Biii) Merge of Bi and Bii. Note the prominent superposition of receptor and serotonin distribution in the two rings of the EB and in the LTR. Ci-Ciii) Distribution of 5-HT₇Dro-GAL4 expression and serotonin-immunoreactivity in antennal lobes (AL). The receptor is seen in select glomeruli of the lobes (Ci), whereas serotonin is distributed in varicose processes throughout the lobes (Cii). The arrow indicates the cell body of the left serotonergic antennal lobe interneuron. The merged channels are seen in Ciii.

Figure 5. Distribution of 5-HT₇Dro-GAL4 expression and serotonin in adult Drosophila ventral nerve cord.

Anterior is up in all panels and scale bar applies to all. GFP-expression is shown in green and serotonin-immunolabeling in magenta. A) Overview of ventral nerve cord with segmental distribution of 5-HT₇Dro-GAL4-expressing neurons in pro-, meso- and metathoracic and abdominal (Abd) neuromeres. Note the large lateral cell bodies in meso- and metathoracic neuromeres. B) Different optical section plane of pro- and anterior mesothoracic neuromeres. Note tracts of GFP-labeled neuronal processes. C) Very dense distribution of serotonin-immunoreactive processes in abdominal neuromeres. Di-iii) A sagital view of the metathoracic and abdominal neuromeres with receptor and serotonin distribution. Note that markers are not colocalized in any neuronal structures, suggesting postsynaptic distribution of the 5-HT₇Dro. However, processes of the two types of neurons superimpose in neuropil regions. At arrows: ant, anterior and d, dorsal. Ei-iii) Horizontal views of the same neuromeres. No colocalized markers can be detected, but overlap between fibers. At arrows: a, anterior and lat, lateral.

Figure 6. 5-HT₇Dro modulates courtship and mating.

Mating pairs fed increasing amounts of the antagonist SB285719 (black bars) were observed for ten minutes to assay the latency of orient, wing vibration, licking, and curling (A, B, C, D, F), the number of copulation attempts (E), the duration of copulation (G), and the frequency at which each behavior occurs (H-L). The latencies of SB-treated flies performing a specific behavior did not differ significantly from untreated controls (gray bars) with increasing drug doses (A, B, C, D, F) with the exception of curling and licking at 3 mM. The number or copulation attempts (E) were significantly decreased at doses above 0.7 mM, while the frequency of attempted copulations decreased with doses above 0.05 mM. Whereas flies fed doses of SB greater than 0.07 mM did not successfully copulate (F, L), the duration of successful copulations at lower doses did not differ from controls (G). (n = 10 pairs observed for each behavior; * p<0.01, ANOVA with Dunnett's post hoc test for multiple comparison; φ = No successful copulation). M) Wild-type OR male flies were loaded into the DAMS monitoring system and maintained on 10% sucrose, 1% agarose (gray bar) or the same medium supplemented with 3 mM SB (Black bars). Locomotion was measured by counting the total number of beam breaks in a 24 hour period. Number of beam breaks per hour were counted and averaged over a three-day period. SB-treated flies exhibited only a slight increase in levels of activity compared to control flies.

Figure 7. 5-HT₇Dro influences mating behaviors in both male and female Drosophila.

Courtship rituals were observed in pairs where either the female (SB-F, white bars) or male (SB-M, black bars) were fed 3mM of the antagonist SB285719 and paired with an untreated partner. Pairs consisting of two untreated flies were used as controls (Gray bars). The time it took for the pairs to perform the courtship behaviors and copulation, the number of copulation attempts and the duration of copulations were measured. In pairs with SB-treated females and untreated males, orient, wing vibration and lick latency (A, B, C) were increased while curl latency (D) was decreased. Pairs with an SB-treated male and untreated male differed significantly only in curl latency (D). The number of copulation attempts was significantly decreased for both experimental sets and neither exhibited any successful copulation (F). Frequencies of courtship behaviors and copulations are listed in Table 1. (n = 10 pairs observed for each behavior; *p<0.01, ANOVA with Bonferroni post hoc test for multiple comparison; φ = No successful copulation)

Figure 8. dsRNAi construct effectively reduces 5-HT₇Dro transcript levels.

RNA from the heads of male flies carrying either the sym-p5-HT₇RNAi (white box), the 5-HT₇Dro-GAL4 (gray box), or both (F1, black box) transgenes was used in quantitative real-time PCR to examine 5-HT₇Dro gene expression. Flies carrying both transcripts show an approximately 80% decrease in 5-HT₇Dro transcript levels. Reactions were performed in quadruplicate for each gene. RpL32 expression was used as the reference control to normalize expression between treatment groups (Error bars indicate SEM).

Figure 9. 5-HT₇Dro knock-down results are consistent with pharmacological studies.

A) The average latencies of courtship behaviors for flies that performed those behaviors are shown. Transgenic F1 lines expressing 5-HT₇Dro double stranded RNA under the control of the 5-HT₇ Dro-GAL4 promoter (black bars) exhibit increased licking and curling latencies, and did not copulate (φ = no successful copulation; * p<0.01; **p<0.001; two-way ANOVA with Bonferroni post hoc analysis). B) The average number of copulation attempts per mating pair are significantly decreased in the F1 knockdowns compared to the parental strains (n = 10 pairs observed per behavior; * p<0.01; one-way ANOVA with Tukey post hoc analysis). C) The activity of male flies carrying either the 5-HT₇Dro-GAL4 element (GAL4, white bar), the UAS-sym-p5-HT₇RNAi element (UAS-RNAi, black bar), or both (F1, gray bar) was measured using the DAMS system for five days. The average daily count of beam breaks per 24 hours is slightly increased in the F1 flies with respect to the GAL4 driver parental strain (*), but there is no significant increase in activity when compared with the UAS-RNAi parental. (n = 16 flies; * p<0.05; one-way ANOVA with Tukey post hoc analysis).