Conceived and designed the experiments: JB OJ DRN CDN. Performed the experiments: JB OJ JL CDN. Analyzed the data: JB OJ DRN CDN. Contributed reagents/materials/analysis tool: DRN CDN. Wrote the paper: JB DRN CDN.
The authors have declared that no competing interests exist.
The 5-HT7 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,
Serotonin (5-HT) is a monoamine neurotransmitter that regulates a variety of
behaviors and physiological processes including circadian rhythms, sleep, appetite,
aggression, locomotion, perception and sexual behavior in mammals
In mammals, 5-HT7 mRNA has been observed in both the CNS and peripheral
tissues including the suprachiasmatic nucleus of the hypothalamus, thalamus,
hippocampus and cortex, as well as coronary artery, gastrointestinal tract, kidney,
and spleen
General laboratory chemicals and reagents were obtained from Sigma (St. Louis, MO). The 5-HT7 receptor antagonist SB258719 was obtained from TOCRIS, (Ellisville, MO).
Fly strains obtained from other sources were Canton-S (CS), and UAS-mCD8::GFP (Bloomington Stock Center, Bloomington, IN). For routine maintenance, flies were reared on standard cornmeal-molasses food at 25°C under 12 hour light/dark conditions.
For the mating assays, bottles of wild type CS flies were cleared and newly
eclosed, virgin females and males were collected and matured for 5-6 days prior
to testing in 15 mL conical tubes containing ∼300 µL of food
(10% sucrose, 1% agarose and the appropriate drug) and plugged
with cotton at the open end. In the courtship and mating assays, flies were
maintained on food + drug for 5 days to ensure that steady-state levels
were reached. To determine if the presence of SB258719 affected the feeding
behaviors of the flies, a CAFÉ assay was performed following established
protocols
Between five and six virgin females were housed together during this process,
while sexually naïve males were individually housed. During the maturation
period, all flies were maintained at 25°C under a 12 hour light/dark cycle
until testing. Following the maturation period, one male and one female were
transferred to a single chamber of a mating wheel. The mating wheel is a
circular piece of 1.0 cm thick plexiglass 10.0 cm in diameter with ten circular
chambers are drilled into the wheel at the outer edge, approximately 1.0 cm in
diameter and 5.0 mm deep. A second circular piece of 2.0 mm plexiglass that is
able to rotate freely is attached to the lower plexiglass wheel and serves as a
cover for the mating chambers. A single 3.0 mm hole in the top is used to insert
flies into the chambers. Our mating chambers are slightly larger than those used
by Ejima and Griffith
Between 100 and 150 1–3 day old CS flies were collected and maintained on
standard food with or without 5-HT7 antagonist (3.0 mM SB258719) for
48 hrs prior to testing for olfactory avoidance in a large 64 ounce commercial
juice bottle with the large end cut off and replaced with fine plastic mesh.
Flies were then transferred to the choice point of t-maze device (a standard
olfactory learning and memory apparatus), where they were presented with an
aversive odor (either 3-methylcyclohexanol or benzaldehyde, at varying
concentrations) in one arm of the apparatus paired with fresh room air in the
opposite arm of the apparatus for 120 seconds following established protocols
used in olfactory learning and memory assays
Male flies were collected less than 72 hours post eclosion and anesthetization on ice. Individual flies were then placed into 5 mm diameter glass capillary tubes with an agar plug at one end consisting of 1% agarose, 10% sucrose and 3 mM drug (where appropriate), and then plugged at the other end with cotton. Tubes were then placed into Trikinetics (Waltham, MA) activity monitor arrays, which were subsequently placed into a humidified incubator at 25°C with a 12 h light-dark cycle. Infrared beam breaks, as a measure of activity, were monitored with the Trikinetics Drosophila Activity Monitor System (DAMS). Sixteen males were used in each experiment for each treatment and monitored for seven days. Only activity data for days 3 and greater were used for analysis, omitting the first two days to allow for acclimation to the environment and to build up steady state drug levels.
Genomic DNA was prepared by homogenizing 25 wild type Oregon-R flies in 400 ml lysis buffer (30 mM Tris (pH 9), 100 mM EDTA, 0.6% SDS, 0.5% sucrose) followed by heat inactivation for 15 min at 70°C; proteins were precipitated out by addition of 80 ml 6M KOAc on ice for 30 minutes followed by centrifugation at 4°C at maximum speed in a microcentrifuge. The aqueous supernatant was extracted with an equal volume of phenol, phenol-chloroform, then chloroform. Nucleotides was precipitated by addition of 2 volumes of ethanol, incubation at room temperature for 5 minutes, and centrifugation for 10 minutes in a microcentrifuge at room temperature. The pellet was washed in 75% ethanol, resuspended in 200 ml TE buffer +1 ml RNAse (Epicentre, Madison, WI), and incubated at 37°C for one hour. DNA was precipitated by addition of 0.1 volumes of 3M NaOAc, 2.5 volumes of ethanol, incubation at –20°C overnight, and centrifugation for 15 minutes at maximum speed in a microcentrifuge at 4°C. The pellet was washed with 75% ethanol, allowed to air dry for 5 minutes, and resuspended in 25 ml sterile H2O.
To isolate putative 5′ enhancer regions, which are normally contained
within the first few kb of genomic DNA upstream of the RNA transcription
start site, 5 kb of genomic DNA immediately upstream of the ATG start codon
within the 5-HT7Dro locus was amplified from 1 µl of
genomic DNA using the Expand High Fidelity PCR System from Roche
(Indianapolis, IN) following manufacturers instructions (
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.
The pCaSpeR4 plasmid (Dr. Bih-Hwa Sheih, Vanderbilt University, Nashville, TN) was digested with Kpn I (Promega, Madison, WI) and blunt ended using the End-IT DNA End Repair Kit (Epicentre, Madison, WI). The GAL4-hsp70 fragment from the pGaTB vector (Dr. Norbert Perrimon, Harvard University, Boston, MA) was excised by digesting with Not I and Bam HI (Promega), followed by gel purification, and blunt ending. The GAL4-hsp70 fragment was ligated into the blunt ended Kpn I cut pCaSpeR4 vector using the Fast-Link DNA Ligation Kit from Epicentre following manufacturers directions. The resulting product, pERGP (‘Enhancer-Ready Gal4-P element’), contains a unique Not I restriction site 5′ of the GAL4 region, and a unique Eco RI restriction site 3′ of the hsp70 terminator region for the subcloning of enhancer elements into either, or both, unique restriction sites. The 3′ Eco RI site may be useful for inclusion of intronic enhancers when generating GAL4 expression constructs.
Both the purified 5-HT7Dro PCR product and pERGP were digested with Not I and gel purified. Digested pERGP was dephosphorylated using Apex Heat-Labile Alkaline Phosphatase (Epicentre) following manufacturers directions. The 5-HT7Dro promoter fragment was ligated into the Not I site of the pERGP vector using the Fast-Link DNA Ligation Kit following manufacturers directions. The final construct was verified using a panel of restriction enzymes, as well by sequence analysis of the cloning site junctions. Five independent transgenic lines were generated from this final product using the services of BestGene Inc. (Chino Hills, CA).
To generate the sym-p5-HT7RNAi plasmid, we used the full length 5-HT7Dro cDNA (DGRC #4507) as template for a PCR reaction with the forward primer = 5′-ataagaattcCGCAGGACTTTAATAGCAGTAGC -3′ (with the restriction sequence for Eco RI added to the 5′ end of the primer) and the reverse primer = 5′-CTTCTCTTTGGCCAGTTGA - 3′ (Integrated DNA Technologies) using the Expand High Fidelity kit from Roche. The PCR product was digested with BglII and EcoRI (Promega), and the 800 bp fragment was gel purified using the Zymo Gel DNA Recovery Kit (Orange, CA) per the manufacturers instructions.
The sym-pUAST vector contains two regions, each containing five UAS activating
sequences in opposite orientations
The prepared 5-HT7Dro cDNA fragment was ligated into the sym-pUAST vector using the Fast-Link DNA Ligation Kit following manufacturers directions. The construct was verified using a panel of restriction enzymes, and by sequence analysis of the cloning site junctions. Independent transgenic lines were generated from this final product using the services of BestGene Inc. (Chino Hills, CA).
QPCR was performed on the RNA isolated from the heads of F1 crosses and their
parentals between the 5-HT7Dro-GAL4 and sym-pUAST-5-HT7Dro
flies to determine knockdown efficiency. Total RNA from 20 combined heads was
isolated by Tri-Reagent (Molecular Research Center, Cincinnati, OH) following
manufacturers protocols. First strand cDNA synthesis was performed using the
Improm-II kit from Promega using 0.3 µg total RNA per reaction following
manufactures directions using random hexamer primers. Quantitative real-time PCR
assays were designed using the Universal ProbeLibrary system (Roche,
Indianapolis, IN;
Larva and adult brains were dissected in 0.1 M sodium phosphate buffer (pH 7.4)
and fixed in PLP (2% paraformaldehyde, 75 mM lysine, 10 mM sodium
periodate, pH 7.4) for 90 mins, permeabilized in 0.1 M sodium phosphate buffer
(pH 7.4), with 0.1% saponin and 0.4% NP40 for 30 mins at room
temperature, followed by incubation overnight at 4°C with primary antibody
in 0.1 M sodium phosphate buffer (pH 7.4), with 0.1% saponin and
0.4% NP40. The primary antibodies used were rabbit-anti-5-HT
(1∶750; Sigma), and mouse-anti-PDF (1∶50; Developmental Studies
Hybridoma Bank, Iowa City, Iowa). After three washes in 0.1 M sodium phosphate
buffer (pH 7.4), tissues were incubated in secondary antibody for one hour at
room temperature. Secondary antibodies used were mouse Alexa 633 conjugated
anti-rabbit (1∶750) (Invitrogen), and goat Texas Red conjugated anti-mouse
(1∶150) (Santa Cruz Biotechnology, Santa Cruz, CA). After staining, brains
were washed in 0.1 M sodium phosphate buffer for 3×20 min, then cleared
through a series of glycerol (25%, 50%, 75%, 90%),
and mounted in 90% glycerol. To visualize the 5-HT7Dro
circuitry in 5-HT7Dro-gal4/UAS-mCD8::GFP flies, as well as conjugated
secondary antibodies, optical sections of whole brains were acquired on a Leica
TCS-SP2 confocal microscope (Leica Microsystems, Exton, PA, USA) at a thickness
of 0.25–0.5 µm. For the detailed analysis of serotonin, fruitless
and 5-HT7Dro-GAL4 expression in the central adult brain and ventral
nerve cord, 5-HT7Dro-gal4/UAS-mCD8::GFP tissues were fixed in
4% paraformaldehyde in 0.1 M sodium phosphate buffer. After thorough
washes in buffer, dissected brains were incubated in a mouse monoclonal antibody
to serotonin (Clone 5HT-H209; Dako, Copenhagen, Denmark) at dilution of
1∶80. For visualization of the male form of the Fruitless protein,
FruM, expression we utilized a rabbit antiserum to
FruM
Heterozygous flies carrying both the 5-HT7Dro-GAL4 and UAS-mCD8::GFP
constructs were used to examine the expression patterns of the
5-HT7Dro-GAL4 element. GFP expression, representing putative
5-HT7Dro expression, is observed in discreet populations of cells
in the hemispheres of the 3rd instar larva brain, as well as in
neurons in the ventral ganglia (
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.
In the adult brain, 5-HT7Dro-GFP is expressed at high levels in
large-field R neurons that innervate the ellipsoid body, and within discreet
populations of cells between the central brain and the optic lobes (
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).
We performed serotonin-immunolabeling on brains of adult flies expressing
5-HT7Dro-GAL4 driven GFP to study spatial relations between
ligand and receptor. Whereas the distribution of serotonin-immunoreactive
neuronal processes is abundant throughout most of the major brain neuropils,
5-HT7Dro-GAL4 mediated GFP expression is most prominent in part
of the central complex and in the antennal lobe (
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.
Two large centrifugal neurons with processes in most of the glomeruli of the
antennal lobe are known to produce serotonin
Expression of 5-HT7Dro-GAL4 is also detected in several neurons in the
ventral ganglion of the adult. The Gal4-driven GFP was seen in paired neurons in
all thoracic and abdominal neuromeres of the ganglion (
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.
In Drosophila, complex behaviors such as learning and memory, aggression,
locomotor reactivity, circadian rhythm, olfaction, and sleep are mediated by
higher brain centers such as the mushroom body, ellipsoid body, and central
complex
The effects of the 5-HT7 receptor antagonist, SB258719 (SB), on
courtship behavior were assessed after maintaining flies for five days on food
containing various amounts of drug ranging from 0.01 mM to 3.0 mM (
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.
Decreases in licking frequency were observed at levels greater than 0.5 mM SB,
with an IC50 = 0.47 mM (
To determine if the observed impairments in mating behavior in SB treated flies
were due to dysfunction in the male, the female, or both, SB fed females were
paired with control males, and SB fed males and control females were paired. We
chose a dose of 3.0 mM to test, because this dose produced a maximal effect
across all behaviors. In the SB female and control male pairs, orient, wing
vibration, and lick, latencies were significantly increased (
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
Behavior | Control | SB-F | SB-M |
Orientation | 100% | 90% | 50% |
Wing Vibration | 100% | 80% | 20% * |
Licking | 100% | 70% | 20% * |
Curling | 100% | 20% * | 20% * |
Successful Copulations | 100% | 0% * | 0% * |
The frequency of courtship behaviors and successful copulations was measured in pairs where either the male (SB–M) or female (SB–F) was fed 3.0 mM of the antagonist and paired with an untreated partner. The frequency of all early courtship behaviors (orient and wing vibrations) was reduced only slightly, but later behaviors (licking, curling and copulation attempts) was significantly decreased in these pairs when compared to untreated control flies. In both experimental sets, successful copulation was never observed. (n = 10 observed pairs for each behavior, *p<0.001 by Fischer's Exact test).
In experiments paring SB males and control females, orient, wing vibration, and
lick latencies were not statistically different from control pairs. Curl latency
was significantly reduced (
Serotonin has previously been implicated in courtship behaviors, specifically
male-male courtship. For example, ectopic expression of the
We did observe moderate expression of 5-HT7Dro-GAL4 GFP in the
antennal lobes of the adult brain. To ensure that the deficits in courtship and
mating induced by SB treatment were not simply due to general problems with
olfaction induced by the receptor antagonist, flies were checked for olfactory
avoidance and sensitivity using different concentrations of the odors
methylcyclohexanol (MCH) and benzaldehyde (BA). The SB treated flies had
performance indices equivalent to control flies at all concentrations of odor,
indicating that SB258719 does not impair general olfaction, or sensitivity to
odors (
MCH | Control PI | SB PI |
1∶1000 | 47±2 | 41±1 |
1∶750 | 49±3 | 47±2 |
1∶250 | 59±7 | 55±1 |
1∶100 | 83±3 | 79±4 |
BA | ||
1∶1000 | 42±3 | 41±1 |
1∶750 | 43±4 | 45±7 |
1∶250 | 47±3 | 47±5 |
1∶100 | 71±4 | 71±3 |
Untreated and SB-treated (3.0 mM) flies were assayed for olfactory avoidance and sensitivity at different concentrations of the odors methylcyclohexanhol (MCH) and benzaldehyde (BA). The performance indices (PI) of the SB-treated flies are equivalent to control flies at all concentrations.
Pharmacological and genetic knockdown experiments can provide complementary data
regarding the function of a receptor. With pharmacological studies we have
measured the effect of different levels of inactivation using different doses of
antagonist to show that 5-HT7Dro receptor function is potentially
required more at the later stages of courtship than the earlier stages. In order
to validate our pharmacological results, we created a UAS-dsRNA strain to knock
down 5-HT7Dro mRNA expression. Quantitative RT-PCR was performed to
examine the levels of 5-HT7Dro transcript in F1 flies carrying both
the 5-HT7Dro-GAL4 and the UAS-sym-p5-HT7RNAi. Flies
carrying both transgenes show an approximately 80% decrease in transcript
levels when compared to flies carrying only one trangene (
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.
If the pharmacological agents (e.g. the SB antagonists) were specifically acting
at 5-HT7Dro receptors, as anticipated, then knockdown of
5-HT7Dro mRNA in putative 5-HT7Dro expressing cells
would be anticipated to recapitulate major aspects of the behavioral phenotypes
of the antagonist. Indeed, expression of double-stranded 5-HT7Dro
mRNA in cells defined by our 5-HT7Dro-GAL4 driver produced courting
and mating deficits consistent with the antagonist 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).
Behavior | Gal4 | UAS | F1 |
Orientation | 100% | 100% | 90% |
Wing Vibration | 100% | 100% | 60% |
Licking | 100% | 100% | 20% * |
Curling | 100% | 100% | 20% * |
Successful Copulations | 100% | 100% | 0% * |
The frequency of courtship behaviors was measured in transgenic F1 lines expressing the 5-HT7-dsRNA under control of the 5-HT7-gal4 promoter (F1). When compared with the parental lines 5-HT7-Gal4 (Gal4) and UAS-sym-5-HT7-dsRNA (UAS), the frequency of licking, curling and copulation attempts was significantly decreased. In the F1 lines, flies were not observed to successfully copulate. (n = 10 observed pairs for each behavior, p<0.001 by Fischer's Exact test).
If either the GAL4 driver strain was not a valid representation of 5-HT7Dro expression, or the SB antagonist was not acting at this receptor to inhibit courtship and mating, or the RNAi studies were producing their effects through off target effects or not producing efficient knockdown, expression of 5-HT7Dro double stranded RNA to produce RNA interference within the circuits defined by our 5-HT7Dro-GAL4 driver would not produce behavioral effects consistent with the pharmacological studies. Therefore, we believe that these methods cross validate each other, as well as the driver strain, to demonstrate that we are indeed examining 5-HT7Dro receptor function within the brain.
To explore the role of the 5-HT7Dro receptor in the fly, we have created
an enhancer GAL4 driver strain and used it to characterized the putative CNS
expression and function of the receptor. Using our 5-HT7Dro-GAL4 driver
to drive expression of a UAS-mCD8::GFP transgene, we have found GFP expression in
third larval instar brain localized to discreet circuits within the brain
hemispheres, as well as to specific neurons in the ventral ganglion. In the adult
brain, there is a high level of 5-HT7Dro-GAL4 expression in large-field R
neurons that innervate the ellipsoid body, as well as in neurons in the brain that
tightly cluster with the PDF-positive LNv clock neurons and innervate the optic
lobes. There is moderate expression detected in the olfactory and gustatory regions
of the brain, and weak expression in other central complex structures like the fan
shaped body. 5-HT7Dro-GAL4 expression appears to be post-synaptic both in
larvae and adults. This is consistent with the observed post-synaptic expression for
the 5-HT7 receptor in vertebrate CNS
Treatment with the 5-HT7 receptor antagonist SB258719 interferes with
courtship and mating behaviors. Interestingly, the IC50 values for
inhibition of courtship behavior frequency decreases as the courtship and mating
process progresses from hardly any disruptive effect with respect to early behaviors
involving sensory cues (orient and wing vibration), to more pronounced effects for
intermediate behaviors (licking, curling, attempts), to complete loss of successful
copulation at higher levels of the drug. In corroboration with the pharmacological
studies, knockdown of 5-HT7Dro message within 5-HT7Dro-GAL4
expressing neurons produces behavioral changes consistent with our pharmacological
results. The early behaviors of orientation and wing song are not affected much, the
intermediate behaviors of licking and curling are significantly disrupted, and
successful copulation is eliminated. There are some subtle differences between
methods, however, like for curling latencies, which could be due to the nature of
receptor inactivation (more acute pharmacological methods
From our results, there appears to be little 5-HT7Dro receptor involvement in the early stages, where we observed that antagonist treated males are receptive to females present in the mating chamber, and are able to initiate the first elements of the mating process. During the intermediate stages, involving licking and curling, there is likely more involvement of 5-HT7Dro receptor signaling. A decrease in licking behavior may result in decreased curling, and decreased curling may result in decreased successful copulation attempts, with the effects compounding at each successive behavior leading to an overall failure at successful copulation. These effects could arise from alterations in physical performance and coordination, sensory perception, olfaction, or to decreases in receptivity or interest, or a lack of ‘motivation’ to perform the higher intensity physical behaviors. We believe these effects are not due to deficits in overt locomotor activity because the drug treated flies demonstrate normal levels of measured activity, or in coordination because at drug levels where mating frequency is decreased the flies that do perform, perform well with latencies not significantly different than control pairs. General alteration of sensory perception is also not a likely reason because males recognize females and readily perform behaviors related to visual and acoustic cues (orienting and wing vibration) despite administration of antagonist or knockdown of message. Whereas olfaction is necessary for receptivity, it is unlikely impeded following antagonist administration because flies fed 3 mM SB as well as the knockdown flies exhibit normal aversion and sensitivity to odors in olfactory tests. Nevertheless, there still may be an olfactory or pheromone component if the limited 5-HT7Dro-GAL4 expression detected in the olfactory lobes correlates with select neurons necessary for reception of specific courtship related pheromones, or the expression does not correlate appropriately with native 5-HT7Dro expression and localization in the olfactory lobes.
A key gene known to be significantly involved in courtship behaviors is
Ectopic expression of the
What then is the role that 5-HT7 receptor signaling plays in reducing receptivity at each stage? One possibility is that pheromone release from the female may be disrupted, resulting in a decrease of courtship. We believe that this is unlikely, however, because control males paired with SB fed females continue to attempt copulation with SB fed females that are not receptive, and will continually chase these females in the mating chamber, which remain uninterested despite repeated attempts by the males. Furthermore, when males fed SB were paired with control females, we often observed females that seemed to seek out the male, which would then run away from the female. Therefore, it would appear that because the control fly of the pair seeks out and attempts to initiate courtship behaviors, SB treatment and manipulation of 5-HT7 receptor function does not interfere with pheromone release, or potentially other sensory cues, related to receptivity. If the default behavior is to initiate courtship in the absence of pheromones, however, then it may still be possible that SB is interfering with pheromone reception at later stages if they are required for the maintenance and intensification of courtship. In the SB/control pairing experiments, the SB fed male + control female pairs were the least successful at courtship behavior. One interpretation of these results are that males are essential to initiating certain elements of mating that are regulated by 5-HT7 receptor function, and when these do not occur it may contribute to further lack of receptivity by females. Alternatively, males and females may be only responding to the SB drug differently.
Because of the high level of expression of the 5-HT7Dro-GAL4 driver in the
large-field R field neurons that innervate the ellipsoid body, we hypothesize that
receptor expression in these neurons is relevant for normal courtship and mating.
Furthermore, that the 5-HT7 receptors expressed by these neurons regulate
receptivity/interest of one fly for its partner. Little is known regarding the exact
function of the ellipsoid body, and the neurons that feed into it. The overall
structure is believed to be involved in mediating higher order behaviors, including
aspects of learning and memory
Courtship and mating has been shown by others to involve additional neurotransmitter
systems including dopamine, which has been shown to play a role in both female
receptivity
In summary, we have generated a 5-HT7Dro-GAL4 reporter and have used it to characterize the putative expression of the 5-HT7Dro receptor, and find it highly expressed in the brain in large-field R neurons in the adult, as well as in small groups of cells that cluster with PDF-positive LNvs neurons. Functional studies utilizing pharmacological and genetic methods indicate that this receptor is necessary for normal courtship and mating.
(TIF)
(TIF)
(TIF)
We thank Dr. Stephen F. Goodwin (Oxford, UK) for supplying the FruM antiserum.