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Differential Roles of Two Major Brain Structures,

Mushroom Bodies and Central Complex, for

Drosophila Male Courtship Behavior

Takaomi Sakai, Toshihiro Kitamoto

Department of Anesthesia, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242

Received 12 August 2005; accepted 4 January 2006

ABSTRACT: Drosophila male courtship is a com- ment affects neither initiation nor maintenance of court-
plex and robust behavior, the potential for which is ge- ship toward young males that release courtship-stimu-
netically built into specific neural circuits in the central lating pheromones different from those of virgin
nervous system. Previous studies using male-female females. In contrast, blocking of synaptic transmission
mosaics and the flies with defects in particular brain in a central complex substructure, the fan-shaped body,
structures implicated the critical central regions in- slightly but significantly reduces courtship activity to-
volved in male courtship behavior. However, their acute ward both virgin females and young males with little
physiological roles in courtship regulation still largely effect on courtship initiation. Taken together, our
remain unknown. Using the temperature-sensitive results indicate that the neuronal activity in the mush-
Dynamin mutation, shibirets1, here we demonstrate the room bodies plays an important role in responding to
significance of two major brain structures, the mush- female-specific sex pheromones that stimulate initiation
room bodies and the central complex, in experience-in- and maintenance of male courtship behavior, whereas
dependent aspects of male courtship. We show that the fan-shaped body neurons are involved in mainte-
blocking of synaptic transmission in the mushroom body nance of male courtship regardless of the nature of
intrinsic neurons significantly delays courtship initiation courtship-stimulating cues. ' 2006 Wiley Periodicals, Inc. J
and reduces the courtship activity by shortening the Neurobiol 66: 821–834, 2006
courtship bout length when virgin females are used as a Keywords: Drosophila melanogaster; courtship; mush-
sexual target. Interestingly, however, the same treat- room body; central complex; shibire

INTRODUCTION terns, composed of orientation, tapping, wing exten-

sion and vibration, licking, attempted copulation, and
Male courtship is arguably the most complex behav- copulation (Bastock and Manning, 1955; Cobb et al.,
ior performed by the fruit fly Drosophila melano- 1986; Welbergen et al., 1987). Multiple sensory
gaster. It is a stereotyped sequence of behavioral pat- modalities, in particular chemical perception and
vision, play important roles in the regulation of dif-
Correspondence to: T. Kitamoto (toshi-kitamoto@uiowa.edu).
ferent behavioral elements in male courtship (Hall,
Contract grant sponsor: Uehara Memorial Foundation (T.S.). 1994; Greenspan and Ferveur, 2000). The main pher-
Contract grant sponsor: NIH; contract grant number: MH62684 omone components of male courtship are cuticular
hydrocarbons (Jallon, 1984; Ferveur and Jallon,
This article includes Supplementary Material available via the
Internet at http://www.interscience.wiley.com/jpages/0022-3034/ 1996), which are mainly nonvolatile and detected by
suppmat specific gustatory neurons (Bray and Amrein, 2003).
' 2006 Wiley Periodicals, Inc. Volatile pheromones emitted by females also stimu-
Published online 3 May 2006 in Wiley InterScience (www.interscience.
wiley.com). late male courtship (Tompkins et al., 1980; Tomp-
DOI 10.1002/neu.20262 kins, 1984). In addition, dynamic as well as static vis-
822 Sakai and Kitamoto

ual stimuli are needed for efficient courtship in D.

melanogaster (Markow and Manning, 1980; Tomp-
kins, 1984; Hall, 1994; Sakai et al., 1997, 2002).
Because a male fly kept in social isolation is fully
capable of carrying out the entire courtship in
response to the aphrodisiac sensory cues, the potential
for this behavior must be genetically built in the cen-
tral nervous system as the particular neural circuits
and the molecular machinery regulating their physiol-
ogy (Hotta and Benzer, 1976; Hall, 1979; Baker
et al., 2001). To understand how courtship behavior
is controlled by the nervous system, we need to iden-
tify the components of the relevant neural circuits in
the brain and elucidate how they interpret the multi-
ple sensory cues and regulate the highly coordinated
motor outputs.
The mushroom bodies (MBs) and the central com-
plex (CC) are two prominent structures in the Dro-
sophila brain [Fig. 1(A)] that have been extensively
studied in terms of their roles in a variety of behav-
iors. The MBs are paired structures composed of
thousands of intrinsic neurons called Kenyon cells
whose cell bodies are localized in the dorso-posterior
region of the brain. They receive multimodal sensory
information via the dendritic calyx mainly from the
olfactory centers of the antennal lobes (ALs) and
from other brain regions including the gustatory cen-
ters of the subesophageal ganglion (SOG) (Strausfeld
et al., 1998; Ito et al., 1998). The Kenyon cells send
axonal projections to the anterior part, where they
bifurcate to form the medial and vertical lobes. The
MBs are considered to be the ‘‘memory center’’, play-
ing critical roles in various learning and memory
paradigms such as olfactory conditioning, experi-
ence-dependent courtship suppression, and context
generalization in visual learning (Dubnau et al., Figure 1 GAL4 expression patterns in the adult brain of
2001; McGuire et al., 2001; McBride et al., 1999; Liu eight GAL4 lines. (A) Schematic representation of mush-
et al., 1999). In addition, nonlearning functions in room bodies (MBs), ellipsoid body (EB), and fan-shaped
walking (Martin et al., 1998) and centrophobism/thig- body (FB) in adult brain. (B–J) GAL4 expression patterns
motaxis (Besson and Martin, 2005) are also attributed in the brain of eight GAL4 lines visualized by GFP reporter
gene. F1 males between GAL4 females and UAS-GFP
to the MBs. Another significant structure is the CC,
males were used. We observed at least four males in each
which is located centrally in the brain. It consists of
line. (B) OK107. Arrow, pars intercerebralis (PI); arrow-
four characteristic neuropils, the protocerebral- heads, antennal lobes (ALs); asterisk, optic lobe. (C) 30Y.
bridge, the fan-shaped body [FB; Fig. 1(A)], the ellip- Arrow, subesophageal ganglion (SOG). (D) c772. (E,F)
soid body [EB; Fig. 1(A)], and the noduli. The CC J183. (G) OK348. (H) 104Y. Arrowhead, nerve bundle in
forms elaborate connections to a variety of brain lateral areas. (I) c232. (J) c41.
regions (Hanesch et al., 1989). Previous studies have
indicated the involvement of the CC in the olfactory
learning task (Heisenberg et al., 1985), as well as in ment of the CC in the CaM kinase-mediated memory
different features of motor outputs such as mainte- formation induced by courtship conditioning.
nance or temporal organization of locomotion Compared to the aforementioned functions of the
(Strauss and Heisenberg, 1993; Martin et al., 1999) MBs and the CC, their acute physiological roles in
and visual flight control (Ilius et al., 1994). Further- experience-independent aspects of male courtship
more, Joiner and Griffith (1999) revealed the involve- behavior are relatively unknown or still controversial.
Journal of Neurobiology. DOI 10.1002/neu
Central Control of Male Courtship 823

Previous studies reported that partial feminization of brum (LPR), a higher-order neuropil region located
the MBs by ectopically expressing the female form of in the lateral part of the brain, plays a critical role in
the sex-determining gene transformer (traF) resulted courtship initiation.
in ‘‘bisexual’’ courtship, suggesting that mate dis- Here we further expanded the usage of the UAS-
crimination is dependent on subsets of the MB intrin- shits1 transgene and specifically focused on the acute
sic neurons (Ferveur et al., 1995; O’Dell et al., 1995). physiological functions of the MBs and the CC in
The MB’s role in mate discrimination was to be male courtship behavior. We found that blocking of
reconsidered, however, because based on their large- synaptic transmission in the MBs significantly af-
scale analysis of flies expressing traF in different fected courtship initiation as well as maintenance
regions of the brain, Kido and Ito (2002) found that when virgin females were used as a sexual target. In-
bisexual behavior was not correlated with the femini- terestingly, the same treatment did not change their
zation of the MBs. Besides mate discrimination, the courtship behavior toward immature males that have
involvement of the MBs in other aspects of experi- different pheromone profiles from virgin females and
ence-independent courtship behavior also still re- elicit strong courtship behavior from sexually ma-
mains elusive. According to Kido and Ito (2002), ab- tured males (Tompkins et al., 1980; Hall, 1994). In con-
lation of the MBs by feeding newly hatched larvae trast, blocking of synaptic transmission in a CC sub-
with hydroxyurea resulted in significant reduction in structure, the FB, reduces courtship activity slightly
the male courtship activity toward virgin females, but significantly toward both virgin females and
while the similar hydroxyurea treatment did not affect young males, with little effect on courtship initiation.
the amount of their courtship in the study of McBride These results suggest that synaptic transmission in
et al. (1999). As for the CC, the functional study re- the MBs plays a significant role in responding to
garding experience-independent aspects of male female-specific pheromones that stimulate courtship
courtship is much limited. The involvement of the initiation and maintenance, whereas neurotransmis-
CC in male courtship was indicated by the mutants sion in the FB is involved in the maintenance of
having structural defects in the CC. These mutants courtship regardless of the nature of courtship-stimu-
were reported to start courtship with the same latent lating pheromones.
period as wild-type flies, but to show significantly
lower copulation success rates, which could be attrib-
uted to their abnormal courtship song (Popov et al.,
Studies of flies with the partially feminized or
structurally damaged brain have provided useful in-
formation about central regulation of male courtship A wild-type strain Canton-S (CS), eight enhancer-trap Gal4
behavior. Approaches using these flies, however, are lines, and a UAS-shits1 line were used in this study. OK348,
not sufficient to define the neural circuits for court- J183, c232, 30Y, OK107, and c772 were outcrossed for at
ship, because the neurons playing a key role in court- least five generations to white flies with Canton-S genetic
ship are not necessarily sexually dimorphic. In addi- background. The origin of genetic background in c41 and
104Y is unknown. All flies were raised on glucose-yeast-
tion, irreversible developmental defects in particular
cornmeal medium at 23 6 18C in a 12:12 h light/dark (LD)
brain regions, induced chemically or genetically, may
cycle (lights on at 8:00 am). Virgin males and females were
cause functional abnormalities in other brain regions collected within 6 h after eclosion and maintained in vials
that directly or indirectly interact with the damaged until experiments. All experiments were carried out during
neurons, which makes it difficult to attribute the daytime between 9:00 and 14:00 hours at 24.5 6 0.5 or 30
observed behavioral defects to the initially damaged 6 0.58C. The mating activity of D. melanogaster does not
brain regions. To circumvent these potential prob- change during the period between Zeitgeber Time (ZT) 0
lems, the temperature-sensitive Dynamin mutation and ZT 9 (Sakai and Ishida, 2001).
shibirets1 (shits1) can be used in combination with
the Gal4/UAS binary expression system to inhibit
neurotransmission in a spatially specific and tempe- Microscopy
rature-dependent manner (Kitamoto, 2001, 2002).
Adult male brains and appendages were dissected in PBS
Broughton et al. (2004) have recently utilized the and fixed with PBS containing 3.7% formaldehyde for
UAS-shits1 transgene with various Gal4 lines and ana- 15 min at room temperature. GFP fluorescence was
lyzed the function of different brain regions in acute observed with a confocal microscope (Zeiss 510). Z sec-
physiological control of male courtship. They have tions were collected at 2 m intervals and processed to con-
found that a cluster of cells in the lateral protocere- struct projections through an extended depth of focus.
Journal of Neurobiology. DOI 10.1002/neu
824 Sakai and Kitamoto

Recording and Analysis of RESULTS

Male Courtship
Analysis of Gal4 Expression Patterns
A male and a target male or female were placed in an
acrylic plastic observation chamber (15 mm in diameter
Using the Green Fluorescent Protein
 3 mm in depth) using a manual aspirator. Courtship (GFP) Reporter Gene
behavior was video recorded (DCR-PC300; Sony, Japan) In order to examine the physiological functions of the
and the courtship latency (CL) and courtship index (CI)
MBs and the CC in male courtship, we used the tem-
were determined. The CL was defined as the period from
the initial presentation of a target object to the initiation of
perature-sensitive Dynamin mutation shits1 in combi-
courtship behavior, and the CI was defined as the percent- nation with eight Gal4 lines that have been reported
age of time the male spent performing courtship behavior in to specifically mark the MBs or the CC. To confirm
a given observation period. We observed male courtship for and document expression patterns of these Gal4 lines,
10 min or until the moment of copulation. we visualized Gal4-positive cells in the adult nervous
In the present study, we used three types of target ani- system by driving GFP.
mals: sexually mature CS females (3 days old), sexually Three Gal4 lines, OK107, 30Y, and c772, showed
immature CS females (0–24 h old), and newly emerged strong GFP expression in the MBs as reported previ-
white males (0–4 h old). GAL4-line females were mated to ously (Connolly et al., 1996; Zars et al., 2000;
UAS-shits1 males and F1 male progeny (3–4 days old) were
McGuire et al., 2001) [Fig. 1(B–D)]. Besides the
used in the experiment. F1 males between GAL4-line
females and CS males (3–4 days old) or between CS MBs they showed scattered GFP expression in the
females and UAS-shits1 males (3–4 days old) were the con- optic lobes [an asterisk in Fig. 1(B)] and restricted,
trol. We observed 32–56 males in each genotype and dis- apparently nonoverlapping expression in the ALs
carded the data for males that did not show any courtship [arrowheads in Fig. 1(B)] as well as the SOG [an
behavior during the observation period. Experiments were arrow in Fig. 1(C)]. In these three lines GFP expres-
carried out either at 25 or 308C. For the 308C experiments, sion was also observed in the pars intercerebralis (PI)
experimental males were pretreated at the temperature for located in the median part of the brain [arrow in
30–50 min. Fig. 1(B)]. Although weak, GFP expression was de-
A courtship bout is defined as a male’s uninterrupted
tected in the EB within the CC for c772 (data not
display of any courtship elements. We measured frequency
of courtship bout and courtship bout duration by observing
courtship of 30 males in each genotype. We confirmed that three ‘‘FB’’ Gal4 lines, J183,
OK348, and 104Y (Joiner and Griffith, 1999; Con-
nolly et al., 1996; Rodan et al., 2002), directed GFP
expression in the FB, but not in the EB [Fig. 1(E–H)].
Measurement of the Locomotor Activity
Expression outside the FB was most obvious in J183
The locomotor activity was measured with a Drosophila [Fig. 1(F)], where the restricted subsets of the MB
Activity Monitor IV (TRIKINETICS, Waltham, MA). A neurons as well as scattered cells in the optic lobes
male fly (3–4 days old) was introduced into a glass tube were GFP-positive [Fig. 1(E)]. OK348 is most spe-
(4 mm in diameter and 65 mm in length) through which an cific to the FB among these three Gal4 lines [Fig.
infrared beam crossed the path of the fly’s movement. The
1(G)], although faint expression was detected in the
activity monitor was shaken for 3 s and kept at 25 or 308C
for 30 min, followed by the measurement of the locomotor
MBs. In addition, a cluster of cells in the lateral
activity for 20 min. The number of incidences at which the region of the brain showed prominent expression
fly crossed the infrared beam was counted every 1 min. [Fig. 1(G)]. In 104Y, GFP expression was also seen
in cells in the optic lobes and the SOG as well as
nerve bundles in the lateral regions of the brain [an
Statistics arrowhead in Fig. 1(H)]. GFP expression was not
observed in the MBs for this line [Fig. 1(H)].
In some cases values of the CL, CI, locomotor activity, and GFP expression was highly restricted in the EB for
courtship bout duration were not distributed normally. c232 [Fig. 1(I)] as reported by O’Dell et al. (1995).
Thus, we carried out the arcsine transformation of the data c41 showed strong GFP-expression in the EB and weak
of CI, and the log transformation of the data of the other pa-
expression in subsets of the MB neurons [Fig. 1(J)].
rameter. When the transformed data distributed normally
and homoscedasticity was evident, such data were analyzed
GFP-positive cells were also seen in the PI as well as
using a two-sample t-test. When the data were not normally the SOG for c41 [arrow and arrowhead in Fig. 1(J)].
distributed after the transformation, they were analyzed We found that a few Gal4 lines showed GFP
using the Mann-Whitney U test. We used computer soft- expression in the limited groups of putative sensory
ware (SPSS 11.5J Base System for Windows) in these tests. neurons associated with gustatory receptors in the
Journal of Neurobiology. DOI 10.1002/neu
Central Control of Male Courtship 825

Figure 2 Effects of temperature on male courtship behavior. The CL (A,C) and the CI (B,D)
were measured at 25 (open bars) and 308C (solid bars). Mature or immature virgin CS females were
used as a sexual target. We observed 24 CS males toward mature and immature females, 32 104Y/
þ males toward mature females, and 48 104Y/þ males toward immature females. We discarded
the data obtained from males that did not show courtship behavior during the observation period.
Error bars show SEM. *p < 0.05. NS, not significant. (A) Mean courtship latency in CS males. n ¼
23–24 in each bar. (B) Mean courtship index in CS males. n ¼ 23–24 in each bar. (C) Mean court-
ship latency in 104Y/þ males (104Y/þ; F1 progeny between 104Y females and CS males). n ¼
31–43 in each bar. (D) Mean courtship index in 104Y/þ males. n ¼ 31–43 in each bar.

proboscis and the leg (see Supplementary Material). for mean courtship latency and courtship index at
In labial palps of 30Y males, in addition to strong 25 and 308C. Disruption of Dynamin function in
GFP expression in non-neuronal structures called the neurons most probably results in acute disruption of
labellar glands, a subset of gustatory neurons showed neurotransmission. We used immature virgins, instead
positive signals. GFP expression was also observed in of mature virgins, as a female target throughout this
restricted putative sensory neurons in the tarsus of the study because under the conditions we used, the CIs
legs for 30Y, OK348, and J183 males. Apparently, of control males (CS and 104Y/þ) toward mature vir-
the sporadic PNS expression observed in these Gal4 gin females (3–4 days old) were slightly but signi-
lines did not overlap each other. ficantly higher at 30 than at 258C [Fig. 2(B,D)]. This
is apparently because mature virgins were more
receptive at 258C and copulated with males in shorter
time with less courtship than at 308C. The CI and
Effects of Disrupted Neurotransmission
CL toward immature virgin females (0–24 h old),
in the MBs and the CC on Male Courtship
which stimulate male courtship but do not copulate
Behavior toward Virgin Females
(Gailey et al., 1984), were not affected by tempera-
To investigate the physiological functions of the ture change.
MBs and the CC in male courtship, we expressed When shits1 expression was directed by OK107,
shits1 in these structures using the eight Gal4 lines 30Y, and c772 and temperature was raised, the male
and examined the effects of conditional disruption flies showed a common phenotype in courtship to-
of Dynamin function on courtship activity toward ward immature virgins. The latency to initiate court-
virgin females by comparing the relative numbers ship was significantly lengthened and the courtship
Journal of Neurobiology. DOI 10.1002/neu
826 Sakai and Kitamoto

activity was reduced at 308C, compared to those at

258C [Fig. 3(A,B)]. Control males heterozygous for
either UAS-shits1 [CL at 258C ¼ 60.1 6 11.1 s (n
¼ 32), CL at 308C ¼ 70.0 6 13.3 s (n ¼ 26), Mann-
Whitney U ¼ 376, p ¼ 0.53; CI at 258C ¼ 85.1
6 2.2% (n ¼ 32), CI at 308C ¼ 81.6 6 2.6% (n ¼
26), Mann-Whitney U ¼ 327.5, p ¼ 0.17] or Gal4
insertions showed neither the lengthened CL nor
reduced CI at 308C [Fig. 3(A,B)]. Because OK107,
30Y, and c772 shared strong expression in the MBs,
but not in other brain regions or PNS neurons, the
results suggest that the disruption of synaptic trans-
mission in the MBs causes defects in courtship initia-
tion and maintenance. The reporter gene expression
was observed in PI neurons for all these lines. It is
not likely, however, that the PI is responsible for the
observed temperature-dependent phenotype, because
c41 with expression in the PI did not show the court-
ship defects [Fig. 3(E,F)].
Expression of shits1 using three Gal4 lines, J183,
OK348 and 104Y resulted in the same temperature-
dependent courtship phenotype. The CIs showed the
slight but statistically significant reduction at 30
compared to at 258C, whereas the CL was not
affected by the temperature change [Fig. 3(C,D)].
As shown in Figure 1 these Gal4 lines all showed
marked expression in the FB, suggesting that dis-
ruption of neurotransmission in the FB results in
reduction of courtship activity, but leaves courtship
initiation intact. J183 also showed reporter gene
expression in the MBs, but contrary to other MB
expressing Gal4 lines, the CL was not extended at
308C. The MB expression in J183 was relatively
weak and restricted to small subsets of the MB neu-
rons. It is possible that J183 does not direct gene
expression in the MB neurons that play a role in
courtship initiation. Alternatively, the expression
level of shits1 in the pertinent MB neurons was too
Figure 3 Effects of shits1 expression in eight GAL4 low to block synaptic transmission in J183/ UAS-
lines on courtship behavior toward immature virgin shits1. Similarly, OK348 showed only faint expres-
females at permissive and restrictive temperatures. Male sion in the MBs, therefore the effect of shits1 in the
courtship behavior was observed at 25 (open bars) and MBs might be disregarded.
308C (solid bars). Error bars show SEM. NS, not signifi-
In contrast to other Gal4 lines, c232/UAS-shits1
cant; *p < 0.05; **p < 0.01; ***p < 0.001. (A) Mean
and c41/UAS-shits1 males did not show any temp-
courtship latency in three Gal4 lines (OK107, 30Y, and
c772) with strong expression in the MBs. n ¼ 32–40 in erature-dependent abnormality in courtship [Fig. 3(E,
each bar. (B) Mean courtship index in OK107, 30Y, and F)]. In these lines, distinct GAL4 expression was
c772. n ¼ 32–40 in each bar. (C) Mean courtship la- seen in the EB but not in the FB (Fig. 1), implying
tency in three Gal4 lines (J183, OK348, and 104Y) with that disruption of the EB function does not affect
expression in the FB. n ¼ 35–56 in each bar. (D) Mean male courtship. Although c41 directed Gal4 expres-
courtship index in J183, OK348, and 104Y. n ¼ 35–56 sion in the MBs, the level of expression was very low
in each bar. (E) Mean courtship latency in two Gal4 [Fig. 1(J)], which might explain why c41/UAS-shits1
lines (c232 and c41) with expression in the EB. n ¼ 33– males did not show defects in the CL and the CI as
40 in each bar. (F) Mean courtship index in c232 and seen in the Gal4 lines with marked expression in the
c41. n ¼ 33–40 in each bar.
Journal of Neurobiology. DOI 10.1002/neu
Central Control of Male Courtship 827

Figure 4 Effects of shits1 expression on courtship behavior toward newly emerged males at
permissive and restrictive temperatures. Male courtship behavior was observed at 25 (open bars)
and 308C (solid bars). Four Gal4 lines, c772, OK107, J183, and 104Y, were used in the exp-
eriments. Error bars show SEM. NS, not significant; *p < 0.05. (A) Courtship latency toward
immature males. n ¼ 30–39 in each bar. (B) Courtship index toward immature males. n ¼ 27–39 in
each bar.

Effects of Disrupted Neurotransmission UAS-shits1 and OK107/UAS-shits1 males. This indi-

in the MBs and the CC on Male Courtship cates that the activity of the MBs neurons is not re-
Behavior toward Immature Males quired for responding to courtship-stimulating cues re-
leased from immature males. On the other hand, the
In Drosophila, newly emerged immature males have
temperature-dependent courtship phenotype toward
different pheromone profiles from virgin females or
immature males was similar to that toward virgin
mature males, and elicit strong courtship behavior
females when two lines (J183 and 104Y) driving shits1
from sexually matured males (Tompkins et al., 1980;
Hall, 1994). It is not well known whether courtship expression in the FB were used. In these flies the
behaviors toward virgin females and immature young higher temperature slightly reduced the courtship ac-
males require the same critical brain regions. We tivity with no effect on courtship initiation (Fig. 4).
therefore examined if male courtship toward imma- Although J183 expresses the reporter gene in the re-
ture males was affected when neurotransmission in stricted subsets of MB neurons [Fig. 1(E)], the charac-
the MBs and the CC was disrupted. teristic phenotype of J183/UAS-shits1 toward immature
In contrast to the earlier result obtained with virgin males is not likely due to the inactivation of these neu-
females as a sexual target [Fig. 3(A,B)], courtship to- rons because courtship toward immature males did not
ward immature males was not affected by tempera- show any temperature-dependent phenotype in c772
ture change for the males expressing shits1 in the MB and OK107 directing extensive expression in the MBs.
neurons using two independent Gal4 lines, c772 and This temperature-dependent courtship phenotype of
OK107. As shown in Figure 4(A) and (B), the levels J183/UAS-shits1 and 104Y/UAS-shits1 is thus likely to
of the CL and the CI toward immature males did not be due to the disruption of neurotransmission in the
show significant differences at 25 and 308C for c772/ FB, where J183 and 104Y share expression.
Journal of Neurobiology. DOI 10.1002/neu
828 Sakai and Kitamoto

Effects of Disrupted Neurotransmission shits1 controls [Fig. 6(A,B)], indicating that the activ-
in the MBs and the CC on Courtship Bout ity of the MB neurons normally suppresses general
Duration locomotor activity. This result is consistent with the
report by Martin et al. (1998) demonstrating that
In observations of courtship performed by Gal4 lines chemical ablation of the MBs, a mutation affecting
expressing shits1 in the MBs or the CC, we noticed Kenyon cell differentiation (mushroom body minia-
that it was frequently interrupted and did not last ture1), and the disrupted MB neurotransmission all
long. To quantify this defect in courtship, we re- induced the elevated locomotion. As for the Gal4
corded the duration of each courtship bout performed lines directing expression in the FB, OK348/UAS-
by 30 males in each genotype at 25 and 308C. The shits1 males showed the higher locomotor activity at
mean duration of courtship bout was calculated from 30 than at 258C, whereas the levels of the locomotor
total courtship bouts observed for this analysis. activity at 30 and 258C were similar in J183/UAS-
When neurotransmission in the MBs was disrupted shits1 and 104Y/UAS-shits1 males [Fig. 6(C,D)]. In
at 308C in c772/UAS-shits1 males, the frequency of c232 and c41, disruption of neurotransmission in the
short courtship bouts (<60 s) toward virgin females EB did not affect locomotor activity [Fig. 6(E,F)]. In
was strikingly increased and the mean duration of any case, the locomotor activity at 308C was not
courtship bouts was greatly reduced [Fig. 5(A)]. In lower than that measured at 258C for all GAL4 lines
contrast, temperatures did not affect mean duration of expressing shits1. Therefore, it is not likely that the
courtship bouts toward newly emerged males in reduced locomotor activity is a cause of temperature-
c772/UAS-shits1 [Fig. 5(B)], showing that the effect dependent courtship defects observed in Gal4 lines
is specific to courtship toward virgin females. In both expressing shits1 in the MBs or the FB.
c772/þ and þ/UAS-shits1 control males, temperature
did not significantly affect the mean duration of
courtship bouts regardless of the sexual targets [Fig. DISCUSSION
5(A,B)]. These results indicate that the on-going ac-
tivity of the MB neurons is required for maintaining In this study we focused on two major structures in
courtship activity when virgin females are used as a the Drosophila brain, the MBs and the CC, and exam-
sexual target. J183/UAS-shits1 males showed the ined their physiological roles in experience-independ-
reduced mean duration of courtship bouts at 308C ent aspects of male courtship behavior using the shits1
both toward virgin females and newly emerged transgene in combination with various Gal4 lines.
immature males [Fig. 5(C,D)], which resulted in Since GAL4 expression is not absolutely limited to
reduction of the overall courtship activity regardless the MB or CC in ‘‘MB lines’’ or ‘‘CC lines’’ used in
of the sexual targets [Figs. 3(D) and 4(B)]. this study, we cannot formally rule out the possibility
that neurons outside the MB or CC somehow contrib-
ute to their shits1-dependent courtship phenotypes.
Locomotor Activity at Permissive and
However, it is most likely that suppression of MB or
Restrictive Temperatures
CC functions is the main cause of the observed court-
The general locomotor activity in flies plays an im- ship phenotypes, because: (1) these GAL4 lines show
portant role in male courtship behavior. Kyriacou strong, overlapping GAL4 expression in the MB or
(1981) reported that high locomotor activity levels CC; (2) all ‘‘MB lines’’ or ‘‘CC lines’’ show, in
were positively correlated with superiority in male essence, the same tendency in shits1 -dependent court-
mating performance in D. melanogaster. It is possible ship phenotypes; (3) there are no GAL4-positive
that the courtship defects observed in males with structures in the brain that are common to the all
functionally disrupted MB or FB neurons are simply ‘‘MB lines’’ or ‘‘CC lines’’ other than the MB or CC
due to their low locomotor activity. To address this (except for the PI neurons in the MB lines); and (4)
issue we measured their locomotor activity at 25 and involvement of the MB and CC in male courtship
308C every 1 min for 20 min (see Methods). was suggested in other studies.
The locomotor activity was higher at 30 than at We found that the acute disruption of Dynamin
258C in all control males (Gal4/þ and þ/UAS-shits1) function using the shits1 transgene, most probably
except for c41/þ and 30Y/þ (Fig. 6, solid lines or resulting in blockage of synaptic neurotransmission,
bars). When shits1 expression was directed to the in the MBs leads to two clear defects in male court-
MBs using OK107 and c772, the temperature- ship toward virgin females [Fig. 3(A,B)]. First, the
induced increase in the locomotor activity was further length of the period from the initial presentation of a
enhanced in comparison with Gal4/þ and þ/UAS- virgin female to the initiation of courtship behavior is
Journal of Neurobiology. DOI 10.1002/neu
Central Control of Male Courtship 829

Figure 5 Effects of shits1 expression in c772 and J183 on courtship bout duration toward imma-
ture virgin females and newly emerged males at permissive and restrictive temperatures. NS, not
significant; *p < 0.05; ***p < 0.001. Gal4/þ and þ/UAS-shits1 males were used as a control. (A)
Histograms and mean duration of courtship bout toward virgin females in c772. (B) Histograms
and mean duration of courtship bout toward newly emerged males in c772. (C) Histograms and
mean duration of courtship bout toward virgin females in J183. (D) Histograms and mean duration
of courtship bout toward newly emerged males in J183.

Journal of Neurobiology. DOI 10.1002/neu

Figure 6

Journal of Neurobiology. DOI 10.1002/neu

Central Control of Male Courtship 831

significantly extended (initiation defect). Second, (de Belle and Heisenberg, 1994). As mentioned in the
courtship behavior is often interrupted and therefore Introduction, the MB ablation by the hydroxyurea
the length of each courtship bout is shortened (main- treatment was reported to cause a significant decrease
tenance defect). Interestingly, these defects are not in the activity of male courtship in one study (Kido
apparent in courtship toward immature males (Fig. 4). and Ito, 2002), while in the other study the MB abla-
Multiple sensory modalities are integrated to trigger tion was reported to have no effect on the quality and
male courtship behavior. Loss of one of the inputs quantity of male courtship (McBride et al., 1999).
does not abolish courtship, but reduces the intensity Although it is not known why the similar chemical
and quality of the behavior. Inactivation of the MB treatment leads to different behavioral consequences,
neurons is likely to deprive males, at least partially, the possibility exists that the extent of structural
of the chemical information from virgin females. Our defects in the MBs might be somehow different
finding suggests that MB-mediated information proc- between the two studies. Our observation that block-
essing of chemical cues from virgin females plays an ing of synaptic transmission in the MB intrinsic neu-
important role in courtship initiation as well as main- rons delays courtship initiation and reduces the court-
tenance. Although the MBs are not essential for ship activity rather fits in the result presented by Kido
males to accomplish copulation in laboratory court- and Ito (2002). On the other hand, a possibility cannot
ship settings, it is possible that male flies with defec- be ruled out at least formally that non-MB neurons
tive MBs would be much less fertile in nature could compensate for a role of the MBs in courtship
because of their subnormal courtship behavior. when MB neurons are removed irreversibly during
In support of our finding, Broughton et al. (2004) development. If that would be the case, our data
reported that the courtship activity toward virgin would not contradict the result presented by McBride
females was reduced when synaptic transmission was et al. (1999).
disrupted using UAS-shits1 and the Gal4 lines with Heimbeck et al. (2001) found that male courtship
strong expression in the MBs. In Broughton et al. was severely impaired when synaptic transmission
(2004), however, the courtship initiation defect was from the two-thirds of all projection neurons (PNs)
not detected in males with disrupted MB neurotrans- was blocked by ectopic expression of tetanus toxin
mission. This apparent discrepancy could be ex- light chain. PNs are the major elements of the central
plained by the different experimental conditions between olfactory pathway, which receive information from
the two studies. The chamber used for assaying court- olfactory receptor neurons in the first-order olfactory
ship in the current study was larger than the one in neuropils, the ALs (Stocker et al., 1990; Stocker,
Broughton et al. (2004) (15 mm in diameter  3 mm 1994). PNs transmit the information from the ALs to
in depth vs. 8  3 mm), resulting in relatively longer a higher order neuropil region, the LPR, through two
courtship latency. In a larger chamber, courtship ini- different pathways, either directly without passing
tiation might be more dependent on the sensory infor- through the MBs or indirectly via the MBs (Stocker
mation processed by the MB neurons, therefore the et al., 1990; Stoker, 1994). Heimbeck et al. (2001)
effect of MB dysfunctions could be more easily have concluded that the direct neuronal pathway from
detected. In addition, male flies were pretreated at ALs to the LPR, which is not mediated by the MBs,
308C for a longer period in this study (30–50 min vs. is necessary and sufficient to process male courtship.
10 min), which may lead to more severe inhibition of However, the argument was based on the assumption
the MB neurotransmission. that the MBs are not involved in experience-inde-
A short pulse of hydroxyurea administration to pendent courtship (McBride et al., 1999). Our results
newly hatched larvae results in ablation of the MBs indicate that the indirect neuronal pathway from ALs

Figure 6 Effects of shits1 expression in eight GAL4 lines on locomotor activity at permissive and
restrictive temperatures. The locomotor activity was measured for 20 min at 25 (dotted lines or
open bars) and 308C (solid lines or bars). Mean locomotor activity every 1 min and mean total loco-
motor activity for 20 min were calculated in each genotype. Gal4/þ and þ/UAS-shits1 males were
used as a control. Error bars show SEM. NS, not significant; **p < 0.01; ***p < 0.001. (A) Mean
locomotor activity every 1 min in UAS-shits1, OK107, 30Y, and c772. n ¼ 42–48. (B) Mean total
locomotor activity in UAS-shits1, OK107, 30Y, and c772. n ¼ 42–48. (C) Mean locomotor activity
every 1 min in J183, OK348, and 104Y. n ¼ 41–48. (D) Mean total locomotor activity in J183,
OK348, and 104Y. n ¼ 41–48. (E) Mean locomotor activity every 1 min in c232 and c41. n ¼ 40–
48. (F) Mean total locomotor activity in c232 and c41. n ¼ 40–48.

Journal of Neurobiology. DOI 10.1002/neu

832 Sakai and Kitamoto

to the LPR through the MBs also participates in at a higher level. In this regard, it should be pointed
courtship regulation. out that CaMKII activity in the FB is required for the
The fruitless gene (fru) plays a critical role in male experience-dependent modification of courtship
courtship behavior. Different fru mutant alleles and behavior called ‘‘courtship conditioning’’ or ‘‘condi-
their combinations variously disrupt each step of tioned courtship suppression’’, where a male fly dis-
male courtship (Gailey and Hall, 1989; Villella et al., plays a reduced amount of courtship toward virgin
1997). Recently Demir and Dickson (2005) and Man- females after being paired with a previously mated
oli et al. (2005) have shown that male-specific fru female (Siegel and Hall, 1979; Joiner and Griffith,
protein (FruM), which is expressed in 2% of neu- 1999). Courtship conditioning is considered to repre-
rons in the male nervous system, is both necessary sent an association of an attractive pheromone released
and sufficient to build the potential for male courtship by both virgin and mated females and an aversive
behavior into the Drosophila nervous system. Stock- pheromone released only by mated females (Tomp-
inger et al. (2005) and Manoli et al. (2005) have tar- kins et al., 1983). An interesting hypothesis is that ex-
geted the insertion of Gal4 into the fru locus and perience with a previously mated female causes stable
visualized the neurons expressing male-specific fru physiological changes in the FB of the conditioned
protein (FruM) and their projections. Many different male, which in turn leads to the reduction of courtship
synaptic neuropils in the brain are labeled for FruM, motivation or interest that lasts for hours and days.
including the /-lobe and -lobe of the MBs. Recently we have reported that a circadian clock gene,
Because most, if not all, FruM-expressing neurons are period, plays a key role in the formation of long-term
expected to be involved in male sexual behavior, this courtship memory (Sakai et al., 2004). Because the
is another piece of data indicating the involvement of period gene is expressed in the FB (Kaneko and Hall,
the MBs in courtship. 2000), per functions might be involved in the possi-
Compared to the functional disruption of the MBs, ble changes induced in the FB after a male has an ex-
that of the CC had different effects on male courtship perience with a mated female. The synaptic transmis-
(Figs. 3–5). Of distinct neuropil regions in the CC, our sion in the FB may play a role not only in experience-
results indicate that the FB, but not the EB, partici- independent aspects of courtship but also in its expe-
pates in the proper maintenance of male courtship. rience-dependent modification.
The CC has been proposed as a principal site of loco-
motor control (Strauss, 2002). Supporting this pro- We thank L. C. Griffith, U. Heberlein, T. Zars, R. L.
posal, Martin et al. (1999) showed that continuous Davis, M. Saitoe, and J. D. Armstrong for providing GAL4
blocking of the synaptic transmission by the targeted lines, and M. A. Joiner for useful discussion.
expression of tetanus toxin in the FB or the EB indu-
ces reduction of locomotor activity. However, the
slight reduction of the courtship activity observed in REFERENCES
male flies with acutely impaired FB neurotransmission
cannot be simply explained by a change in locomotor Baker BS, Taylor BJ, Hall JC. 2001. Are complex behav-
activity, because we could not detect significant iors specified by dedicated regulatory genes? Reasoning
from Drosophila. Cell 105:13–24.
reduction in general locomotor activity under the con-
Bastock M, Manning A. 1955. The courtship of Drosophila
ditions where courtship behavior was recorded (Fig. 6). melanogaster. Behaviour 15:85–111.
It is not known why the acute disruption of synaptic Besson M, Martin JR. 2005. Centrophobism/thigmotaxis, a
transmission in the FB leads to reduction of the male new role for the mushroom bodies in Drosophila. J Neu-
courtship activity. One possibility is that the fine reg- robiol 62:386–396.
ulation of motor outputs, other than the general loco- Bray S, Amrein H. 2003. A putative Drosophila pheromone
motor activity, is affected by the disruption of FB receptor expressed in male-specific taste neurons is re-
neurotransmission, which contributes to the frequent quired for efficient courtship. Neuron 39:881–883.
termination of courtship behavior. Alternatively, it is Broughton SJ, Kitamoto T, Greenspan RJ. 2004. Excitatory
also possible that courtship is defective in the FB- and inhibitory switches for courtship in the brain of Dro-
sophila melanogaster. Curr Biol 14:538–547.
impaired males because the FB neurons integrate sen-
Cobb M, Burnet B, Connolly K. 1986. The structure of
sory information and play a role in keeping courtship
courtship in the Drosophila melanogaster species sub-
motivation or interest at a certain state. The reduction group. Behaviour 97:182–212.
of the courtship activity in the FB-impaired males Connolly JB, Roberts IJ, Armstrong JD, Kaiser K, Forte M,
was observed for different sexual targets that release Tully T, O’Kane CJ. 1996. Associative learning dis-
different courtship-stimulating pheromones, implying rupted by impaired Gs signaling in Drosophila mush-
that the FB controls courtship motivation or interest room bodies. Science 274:2104–2107.
Journal of Neurobiology. DOI 10.1002/neu
Central Control of Male Courtship 833

De Belle JS, Heisenberg M. 1994. Associative odor learn- Kaneko M, Hall JC. 2000. Neuroanatomy of cells express-
ing in Drosophila abolished by chemical ablation of ing clock genes in Drosophila: transgenic manipulation
mushroom bodies. Science 263:692–695. of the period and timeless genes to mark the perikarya
Demir E, Dickson BJ. 2005. fruitless splicing specifies male of circadian pacemaker neurons and their projections.
courtship behavior in Drosophila. Cell 121:785–794. JComp Neurol 422:66–94.
Dubnau J, Grady L, Kitamoto T, Tully T. 2001. Disruption Kido A, Ito K. 2002. Mushroom bodies are not required for
of neurotransmission in Drosophila mushroom body courtship behavior by normal and sexually mosaic Dro-
blocks retrieval but not acquisition of memory. Nature sophila. J Neurobiol 52:302–311.
411:476–480. Kitamoto T. 2001. Conditional modification of behavior in
Ferveur JF, Jallon JM. 1996. Genetic control of male cutic- Drosophila by targeted expression of a temperature-sen-
ular hydrocarbon in Drosophila melanogaster. Genet Res sitive shibire allele in defined neurons. J Neurobiol 47:
67:211–218. 81–92.
Ferveur JF, Stortkuhl KF, Stocker RF, Greenspan RJ. 1995. Kitamoto T. 2002. Targeted expression of temperature-
Genetic feminization of brain structures and changed sensitive dynamin to study neural mechanisms of com-
sexual orientation in male Drosophila. Science 267:902– plex behavior in Drosophila. J Neurogenet 16:205–
905. 228.
Gailey DA, Hall JC. 1989. Behavior and cytogenetics of Kyriacou CP. 1981. The relationship between locomotor
fruitless in Drosophila melanogaster: different courtship activity and sexual behaviour in ebony strains of Dro-
defects caused by separate, closely linked lesions. Ge- sophila melanogaster. Anim Behav 29:462–471.
netics 121:773–785. Liu L, Wolf R, Ernst R, Heisenberg M. 1999. Context gen-
Gailey DA, Jackson FR, Siegel RW. 1984. Conditioning eralization in Drosophila visual learning requires the
mutations in Drosophila melanogaster affect an expe- mushroom bodies. Nature 400:753–756.
rience-dependent behavioral modification in courting Manoli DS, Foss M, Villella A, Taylor BJ, Hall JC, Baker
males. Genetics 106:613–623. BS. 2005. Male-specific fruitless specifies the neural sub-
Greenspan RJ, Ferveur JF. 2000. Courtship in Drosophila. strates of Drosophila courtship behaviour. Nature 436:
Annu Rev Genet 34:205–232. 395–400.
Hall JC. 1979. Control of male reproductive behavior by the Markow TA, Manning M. 1980. Mating success of photore-
central nervous system of Drosophila: dissection of a court- ceptor mutants of Drosophila melanogaster. Behav Neur
ship pathway by genetic mosaics. Genetics 92:437–457. Biol 29:276–280.
Hall JC. 1994. The mating of a fly. Science 264:1702– Martin JR, Ernst R, Heisenberg M. 1998. Mushroom bodies
1714. suppress locomotor activity in Drosophila melanogaster.
Hanesch U, Fischbach KF, Heisenberg M. 1989. Neuronal Learn Mem 5:179–191.
architecture of the central complex in Drosophila mela- Martin JR, Raabe T, Heisenberg M. 1999. Central complex
nogaster. Cell Tissue Res 257:343–366. substructures are required for the maintenance of loco-
Heimbeck G, Bugnon V, Gendre N, Keller A, Stocker RF. motor activity in Drosophila melanogaster. J Comp Phys-
2001. A central neural circuit for experience-independent iol [A] 185:277–288.
olfactory and courtship behavior in Drosophila mela- McBride SM, Giuliani G, Choi C, Krause P, Correale D,
nogaster. Proc Natl Acad Sci USA 98:15336–15341. Watson K, Baker G, et al. 1999. Mushroom body abla-
Heisenberg M, Borst A, Wagner S, Byers D. 1985. Dro- tion impairs short-term memory and long-term memory
sophila mushroom body mutants are deficient in olfac- of courtship conditioning in Drosophila melanogaster.
tory learning. J Neurogenet 2:1–30. Neuron 24:967–977.
Hotta Y, Benzer S. 1976. Courtship in Drosophila mosaics: McGuire SE, Le PT, Davis RL. 2001. The role of Drosoph-
Sex-specific foci for sequential action patterns. Proc Natl ila mushroom body signaling in olfactory memory. Sci-
Acad Sci USA 73:4154–4158. ence 293:1330–1333.
Ilius M, Wolf R, Heisenberg M. 1994. The central complex O’Dell KM, Armstrong JD, Yang MY, Kaiser K. 1995.
of Drosophila melanogaster is involved in flight control: Functional dissection of the Drosophila mushroom
studies on mutants and mosaics of the gene ellipsoid bodies by selective feminization of genetically defined
body open. J Neurogenet 9:189–206. subcompartments. Neuron 15:55–61.
Ito K, Suzuki K, Estes P, Ramaswami M, Yamamoto D, Popov AV, Sitnik NA, Savvateeva-Popova EV, Wolf R,
Strausfeld NJ. 1998. The organization of extrinsic neu- Heisenberg M. 2003. The role of central parts of the
rons and their implications in the functional roles of the brain in the control of sound production during courtship
mushroom bodies in Drosophila melanogaster Meigen. in Drosophila melanogaster. Neurosci Behav Physiol
Learn Mem 5:52–77. 33:53–65.
Jallon JM. 1984. A few chemical words exchange by Dro- Rodan AR, Kiger JA Jr, Heberlein U. 2002. Functional dis-
sophila during courtship and mating. Behav Genet 14: section of neuroanatomical loci regulating ethanol sensi-
441–478. tivity in Drosophila. J Neurosci 22:9490–9501.
Joiner MA, Griffith LC. 1999. Mapping of the anatomical Sakai T, Ishida N. 2001. Circadian rhythms of female mat-
circuit of CaM kinase-dependent courtship conditioning ing activity governed by clock genes in Drosophila. Proc
in Drosophila. Learn Mem 6:177–192. Natl Acad Sci USA 98:9221–9225.

Journal of Neurobiology. DOI 10.1002/neu

834 Sakai and Kitamoto

Sakai T, Isono K, Tomaru M, Fukatami A, Oguma Y. 2002. Strauss R. 2002. The central complex and the genetic dis-
Light wavelength dependency of mating activity in the section of locomotor behavior. Curr Opin Neurobiol 12:
Drosophila melanogaster species subgroup. Genes Genet 633–638.
Syst 77:187–195. Strauss R, Heisenberg M. 1993. A higher control center of
Sakai T, Isono K, Tomaru M, Oguma Y. 1997. Light-affected locomotor behavior in the Drosophila brain. J Neurosci
male following behavior is involved in light-dependent 13:1852–1861.
mating in Drosophila melanogaster. Genes Genet Syst 72: Tompkins L. 1984. Genetic analysis of sex appeal in Dro-
275–281. sophila. Behav Genet 14:411–440.
Sakai T, Tamura T, Kitamoto T, Kidokoro Y. 2004. A clock Tompkins L, Hall JC, Hall ML. 1980. Courtship-stimulat-
gene, period, plays a key role in long-term memory for- ing volatile compounds from normal and mutant Dro-
mation in Drosophila. Proc Natl Acad Sci USA 101: sophila. J Insect Physiol 26:689–697.
16058–16063. Tompkins L, Siegel RW, Gailey DA, Hall JC. 1983.
Siegel RW, Hall JC. 1979. Conditioned courtship responses Conditioned courtship in Drosophila and its mediation
in courtship behavior of normal and mutant Drosophila. by association of chemical cues. Behav Genet 13:565–
Proc Natl Acad Sci USA 76:3430–3434. 578.
Stocker RF. 1994. The organization of the chemosensory Villella A, Gailey DA, Berwald B, Ohshima S, Barnes PT,
system in Drosophila melanogaster: a review. Cell Tissue Hall JC. 1997. Extended reproductive roles of the fruit-
Res 275:3–26. less gene in Drosophila melanogaster revealed by behav-
Stocker RF, Lienhard MC, Borst A, Fischbach KF. 1990. ioral analysis of new fru mutants. Genetics 147:1107–
Neuronal architecture of the antennal lobe in Drosophila 1130.
melanogaster. Cell Tissue Res 262:9–34. Welbergen P, van Dijken FR, Scharloo W. 1987. Collation
Stockinger P, Kvitsiani D, Rotkopf S, Tirian L, Dickson BJ. of the courtship behaviour of the sympatric species Dro-
2005. Neural circuitry that governs Drosophila male sophila melanogaster and Drosophila simulans. Behav-
courtship behavior. Cell 121:795–807. iour 101:253–274.
Strausfeld NJ, Hansen L, Li Y, Gomez RS, Ito K. 1998. Zars T, Fischer M, Schulz R, Heisenberg M. 2000. Local-
Evolution, discovery, and interpretations of arthropod ization of a short-term memory in Drosophila. Science
mushroom bodies. Learn Mem 5:11–32. 288:672–675.

Journal of Neurobiology. DOI 10.1002/neu