Drosophila as a new model organism for the neurobiology of aggression?
Lehrstuhl für Genetik und Neurobiologie, Biozentrum, Am Hubland,
D-97074 Würzburg, Germany
* These authors contributed equally to this work
Author for correspondence at present address: Department of Neurobiology and
Anatomy, W. M. Keck Center for the Neurobiology of Learning and Memory,
University of Texas-Houston Medical School, Houston, Texas 77030, USA (e-mail:
bjoern{at}brembs.net
)
Accepted 22 February 2002
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Summary |
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Key words: Drosophila melanogaster, aggression, fighting behaviour, amine, mushroom body
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Introduction |
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To our knowledge, only two genetic factors have been reported to affect
aggressive behaviour in Drosophila: the sexdetermination hierarchy
(SDH) and the ß-alanine pathway. fruitless (fru) and
dissatisfaction (dsf) mutants have been described as more
aggressive than wild-type controls (Lee
and Hall, 2000). Both genes are part of the SDH. Flies carrying
mutant alleles of the black (b) gene appear less aggressive,
whereas ebony (e) mutants appear more aggressive
(Jacobs, 1978
). The enzymes
encoded by the two genes regulate ß-alanine levels (b flies have
reduced and e flies elevated levels).
It is straightforward to expect genes of the SDH to affect sex-specific
behaviours, but the pathways by which they modulate that behaviour are largely
unknown. One possibility could be via the regulation of small
neuroactive molecules (such as ß-alanine and the biogenic amines) and
their receptors. Biogenic amines play a key role in the regulation of
aggressive behaviour, not only in vertebrates, but also in arthropods (e.g.
Edwards and Kravitz, 1997;
Heinrich et al., 1999
,
2000
; Huber et al.,
1997a
,
b
;
Kravitz, 2000
;
Schneider et al., 1996
;
Stevenson et al., 2000
). The
biogenic amine system in flies is well described (see
Monastirioti, 1999
). Most
serotonin and dopamine mutants in Drosophila are either lethal or
affect both serotonin and dopamine, due to their shared pathways of synthesis
(e.g. Johnson and Hirsh, 1990
;
Lundell and Hirsh, 1994
;
Shen et al., 1993
;
Shen and Hirsh, 1994
).
However, established protocols are commonly used to manipulate the levels of
these amines individually in the adult fly
(Neckameyer, 1998
;
Vaysse et al., 1988
).
Octopamine null mutants have been generated and characterized
(Monastirioti et al., 1996
).
Interestingly, certain octopamine and dopamine receptors are preferentially
expressed in a prominent neuropil in the Drosophila brain called the
mushroom bodies (Han et al.,
1996
,
1998
). Thus, all of the
prerequisites for a systematic analysis of the neurobiological factors
involved in the expression of aggressive behaviour are available: (1) a
considerable body of knowledge about the behaviour and its ecological context,
(2) circumstantial evidence about possible neurobiological factors involved in
regulating the behaviour, and (3) methods for manipulating these factors and
for quantifying the behaviour.
As a first attempt to characterize the effects of various possible neurobiological factors that might regulate aggression, we report here the results of a competition experiment. Six male flies competed for a food patch and three mated females. The experimental males were manipulated in one of various ways: by a classical mutation affecting ß-alanine levels, a P-element mutation affecting octopamine levels, or insertion of transgenes affecting synaptic output from the mushroom bodies, or by pharmacological treatment affecting serotonin or dopamine levels, and then tested for their aggressive behaviour.
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Materials and methods |
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Mutants
Black1 and ebony1 mutant strains
from the laboratory's 18°C stock collection (provided by S. Benzer in
1970) were kept at 25°C for at least two generations. The M18 P-element
octopamine mutant and control stocks
(Monastirioti et al., 1996)
were kept at 25°C for two generations after arrival.
Transgenes
Sweeney et al. (1995)
developed a method that constitutively blocks synaptic transmission by
expressing the catalytic subunit of bacterial tetanus toxin (Cnt-E) in target
neurons in the Drosophila brain using the P[GAL4] technique
(Brand and Perrimon, 1993
).
Inspired by the preferential expression of certain dopamine and octopamine
receptors in the mushroom bodies (Han et al.,
1996
,
1998
), we used the Cnt-E
transgene to block synaptic output from the mushroom bodies
(Sweeney et al., 1995
).
Expression of another transgene, an inactive form of the tetanus toxin light
chain (imp-tntQ), controlled for deleterious effects of protein overexpression
(Sweeney et al., 1995
). The
P[GAL4] line mb247 (Schulz et al.,
1996
) served as a mushroom body-specific GAL4 driver
(Zars et al., 2000
) for both
toxins. The trans-heterozygote offspring from the GAL4 driver strain and the
two UASGAL4 reporter strains (Cnt-E and imp-tntQ) entered the
study.
Pharmacological treatments
Drosophila from the wild-type strain Berlin (wtb) were treated as
described by Neckameyer (1998)
and Vaysse et al. (1988
).
Briefly, the animals were fed a sucrose solution containing either 10 mg
ml-1 of the serotonin precursor 5HTP (5-hydroxy-tryptophan) or 10
mg ml-1 of the serotonin synthesis inhibitor pCPA
(para-chlorophenylalanine) to manipulate serotonin levels. Effectiveness of
the treatment was verified qualitatively with standard immunohistochemical
techniques using rabbit serotonin antisera (data not shown; Buchner et al.,
1986
,
1988
). Alternatively, the
animals were treated with 1 mg ml-1 of the dopamine precursor
L-DOPA (L-3,4-dihydroxyphenylalanine) or 10 mg ml-1 of the dopamine
synthesis inhibitor 3IY (3-iodo-tyrosine) to manipulate dopamine levels.
Effectiveness of the treatment was verified by observation of cuticle tanning.
A dose of 10 mg ml-1 L-DOPA was lethal, confirming unpublished data
from Wendy Neckameyer (St Louis University School of Medicine).
Experimental groups
Using the different stocks described above, we arranged six different
groups of `low' versus `high' males, such that the respective amine
or the amount of synaptic output from the mushroom bodies was manipulated to
produce relative high-and low-level subgroups.
Thus, we arranged four experimental groups and two control groups. For each group, the two subgroups (`high' and `low') compete against each other in one recording chamber. Each group was tested twice with different animals.
Recording chambers
Aggression was studied in cylindrical cages similar to those used by
Hoffmann (1987), i.e. 100 mm
Petri dishes, top and bottom separated by a 40 mm high spacer (i.e. a
cylindrical chamber of 100 mm diameter and 40 mm height). The bottom of the
chamber was filled with 2 % agar to moisturize the chamber. Flies were
introduced by gentle aspiration through a small hole in the spacer. A food
patch (10 mm diameter, 12 mm high) was positioned in the centre of the
chamber, containing a mixture of minced 2 % agar, apple juice, syrup and a
live yeast suspension (after Reif,
1998
), filled to the level of the rim of the containing vial. The
chamber was placed in a Styrofoam box (used to ship biochemical reagents on
dry ice; outer measurements: 275x275 mm, height, 250 mm; inner
measurements: 215x215 mm, height, 125 mm) to standardize lighting
conditions and to shield the chambers from movements by the experimenters. Two
Styrofoam boxes with one chamber each were arranged underneath video cameras,
focused on the food patch in a darkened room at 25 °C. Ring-shaped
neon-lights (Osram L32W21C, power supply Philips BRC406) on top of the boxes
provided homogenous illumination throughout the experiment.
Experimental time course
The stocks were treated completely in parallel (see
Table 1). A 5 % sucrose
solution (in Drosophila ringer) with or without added treatment was
pipetted onto 5 pieces of filter paper snugly fitting in cylindrical
(12x40 mm) vials before transferring newly eclosed (0-24 h) male flies
into the vials. The flies were transferred into new vials with new solution
and new filter paper on a daily basis for 5 days. Each group was treated in
two replicates, starting with new flies on different days (see
Table 1). On the fifth day, 4-6
flies per subgroup were briefly immobilised on a cold plate and marked with
one small dot on the thorax in either green or white acrylic paint. At 08.00 h
(1 h after lights-on) on the sixth day, the animals of the two groups treated
in parallel were transferred into the recording chambers (three mated, but
otherwise untreated, Canton S females, and six males, three from each paired
subgroup) and placed underneath the video cameras under identical conditions
to those used during the recording time, except that the video recorders
(VCRs) were turned off. Continuing the parallel treatment of two groups per
day, two video set-ups were used simultaneously (`left' and `right'). After an
acclimatisation period of 2 h, the VCRs were set to record. For each group, we
recorded 4 h of fly behaviour, once in each location (yielding the two
replicates for each group), resulting in 12 video tapes (see
Table 2). Data from both
replicates were pooled. Since each group was measured twice with six (3+3)
experimental animals (males) for each recording, the total number of observed
males was 6 animalsx2 replicatesx6 groups=72. Recording of the
experiments was randomised across days.
|
|
Behavioural scoring
Only malemale interactions were counted. Mated females lose their
receptivity to male advances and the males cease courting quickly, refraining
from courting for a number of hours (courtship conditioning; e.g.
Greenspan and Ferveur, 2000).
Little courtship behaviour was thus observed after the acclimatisation
period.
Behavioural scoring was done blind, before the colour codes on the flies'
thoraces were decoded into `high' and `low'. An interaction
between two males was classified as either aggressive or non-aggressive as
defined by Hoffmann (1987).
Briefly, we classified encounters that contained the previously described
boxing, head-butting, lunging, wrestling, tussling, charging and chasing
behaviours (Dow and von Schilcher,
1975
; Hoffmann,
1987
,
1988
,
1989
,
1994
; Hoffmann and Cacoyianni,
1989
,
1990
;
Jacobs, 1978
;
Skrzipek et al., 1979
) as
aggressive. Encounters that only contained approach, leg contact, wing
vibration or wing flapping were classified as non-aggressive. If the encounter
was classified as aggressive, it was straightforward to discern the aggressor
as one animal attacking and/or chasing the other. Non-aggressive encounters
could usually not be classified directionally. Thus, with three
`high' and three `low' animals in the recording chamber, any
interaction between them falls into seven categories, listed below:
This design thus yielded seven values, one for each of the respective interaction categories, giving each of the six groups a characteristic aggression profile (Fig. 1A).
|
Data analysis
A loglinear analysis (delta=0.005, criterion for convergence=0.0005,
maximum iterations 500) was performed over the 6x7 table of observed
behavioural frequencies to determine the effect of the treatments on the
distribution of behavioural classes. To normalize for the total number of
encounters, two derived parameters were computed from the raw data. The first
is the likelihood that an individual of one subgroup will attack during an
encounter (attack probability, PA). It is calculated as
the fraction of all encounters in that group involving a `high' (or
`low', respectively) animal, where such an animal was the aggressor:
![]() | (1) |
![]() | (2) |
![]() | (3) |
Thus, PA describes the probability that a given
individual will act aggressively against any other individual it encounters.
The second derived parameter assesses the representation of each subgroup in
the total number of encounters (encounter probability,
PE). It is calculated analogously to the first parameter
as the fraction of all encounters in a group, where an animal of a specific
subgroup (i.e. `high' or `low') participated:
![]() | (4) |
![]() | (5) |
![]() | (6) |
Thus, PE describes the probability that an individual of one subgroup will be a participant in an encounter.
While PA can be said to describe the level of
aggression of a certain subgroup, PE can be perceived as a
control measure for the overall number of interactions in that subgroup, as
influenced by, for example, general activity, visual acuity, etc. After the
data transformation, the resulting probabilities were tested against random
distribution using 2 tests.
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Results |
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The raw data (Fig. 1A), reveal that the two control groups behaved according to our expectations. The wtb negative control shows a uniform distribution of aggressive encounters, whereas the ß-alanine positive control is skewed towards the mutants with high levels of ß-alanine (Fig. 1Ai).
The clearest effects among experimental groups were obtained from the octopamine mutants and the mb group. Both octopamine null mutants (oa-) and animals with inhibited mushroom bodies (mb-) are virtually non-aggressive (Fig. 1A). In Fig. 1Aii, the octopamine group seems similar to the wild-type control except for the missing values for 6ag and 7ag. However, while the oa+ animals appear to show a wildtype level of aggression, the mb+ animals show elevated levels of aggression compared to all other groups (Fig. 1A).
It also appears that our serotonin treatment had little effect on aggression (Fig. 1A).
The dopamine treatment appears to be somewhat effective in decreasing the number of aggressive encounters in animals with high levels of dopamine, while the animals with low levels of dopamine seem to have numbers of aggressive encounters similar to, if not slightly higher than, the wild-type controls. Obviously, the number of non-aggressive encounters in the dopamine-treated animals is strongly elevated (Fig. 1A). Interestingly, the two subgroups show inverted profiles for intra- and inter-subgroup aggression (i.e. 1ag/2ag and 6ag/7ag).
The total number of encounters also varies considerably between the different treatments (Fig. 1Aii).
With significant effects of our treatments on the distribution of the
behaviours within each group, we can process the data in order to determine
the effect of our treatments on the propensity of the animals to become
aggressive. The fraction of all encounters involving a `subgroup'
animal, where such an animal was the aggressor, is calculated
(Fig. 1Bi;
PA, see Materials and methods). The PA
value allows us to estimate the effects of the treatments on aggression.
2 tests can be computed on PA values to
test the null hypothesis that our treatments had no effect on the probability
of the fly being aggressive. Table
3 summarizes the
2 results for all six groups. The
statistics confirm the effects already visible in the raw data
(Fig. 1A): the two control
groups (wtb and b/e) were consistent with our expectations. The obvious effect
of octopamine null mutants being completely non-aggressive is corroborated by
our statistical analysis, as are the extreme effects of expressing active and
inactive tetanus toxin, respectively, in the flies' mushroom bodies
(Fig. 1Bi). The serotonin
treatment had no significant effect on the probability of the flies becoming
aggressive during an encounter, despite the fact that we could verify the
effectiveness of the treatment immunohistochemically (data not shown). The
group in which the dopamine levels were manipulated shows a moderate, but
statistically reliable, effect of high dopamine levels leading to a higher
probability to attack in an encounter.
|
Despite the fact that most of our treatments have a record of influencing
aggression in other animals, the possibility exists that the different
treatments may have altered the number of aggressive encounters indirectly by
altering the total number of encounters, through other factors such as general
activity, visual acuity, etc. The distribution of encounters over the
subgroups, PE, should reveal such candidate variables. For
instance, if the treatment rendered the animals of one subgroup inactive, the
PE of that subgroup should be smaller than the
PE of the other subgroup. If the obtained aggression
scores were but a reflection of asymmetric PE values, they
should follow the pattern of PE asymmetry.
Fig. 1Bii depicts the
distribution of encounters over the two subgroups, independently of encounter
classification. Again, 2 statistics were performed and
summarized in Table 2. All
treatments led to a significant asymmetry in PE between
subgroups, with the exception of the negative wtb controls. However, the
pattern of asymmetry does not seem to match the pattern of asymmetry in the
level of aggression (see Discussion).
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Discussion |
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Octopamine null mutants exhibit strongly reduced aggression, as do flies
with low levels of synaptic output from their mushroom bodies. Interestingly,
certain types of octopamine and dopamine receptors are preferentially
expressed in the mushroom bodies of wild-type flies (Han et al.,
1996,
1998
). It is tempting to
interpret this phenocopy of the octopamine mutants as resulting from Kenyon
cells being the major regulators of octopamine- (and/or dopamine-) mediated
aggression. Recently, temperature sensitive shibirets1 constructs
have been developed to conditionally block synaptic transmission (e.g.
Dubnau et al., 2001
;
Kitamoto, 2001
;
McGuire et al., 2001
;
Waddell et al., 2000
).
Unfortunately, at the time of our experiments, the shibirets1
constructs were not yet available. Future experiments definitely should
include shibirets1 constructs in order to replicate our mb-
results, examine the high levels of aggression in the mb+ flies and look for
other brain areas involved in aggression. Replication of our results using the
shibirets1 constructs would also eliminate the possible explanation
that the expression of tetanus toxin anywhere in the fly's brain abolishes
aggressive behaviour and solve the problem of UAS promoter leakiness. The
octopamine result is conspicuous in another respect: it is consistent with
studies in crickets, where depletion of octopamine and dopamine decreases
aggressiveness (Stevenson et al.,
2000
), but contrasts with studies in crustaceans, where high
octopamine levels tend to bias behaviour towards submissiveness
(Antonsen and Paul, 1997
;
Heinrich et al., 2000
;
Huber et al., 1997a
).
The high aggression observed in the mb+ animals is difficult to interpret. In principle, the inactive toxin should not have any effect on the secretion of neurotransmitter at the synapse. More likely is an insertion effect of the P-element containing the imptntQ transgene. In that case it would be extremely interesting to characterize the genetic environment within which the P-element lies in order to find the gene responsible for such aggressiveness. One may argue that high aggressiveness by flies of one subgroup may produce low aggression in the respective other subgroup. In the case of the mb group, this is unlikely, because there still should be at least some aggression between mb- animals, even if mb+ animals attacked every other male they encountered. Moreover, mb- animals seemed unaffected by the repeated attacks from mb+ males and kept coming back to the patch soon after an mb+ male chased it off (the reason for the high 2ag value in Fig. 1). However, mb- animals were never observed to be the aggressor. It thus seems more likely that the high frequency of attacks by mb+ males is due to a combination of high levels of aggression due to insertion effects of the imp-tntQ transgene and returning mb- males repeatedly eliciting aggressive behaviours in the mb+ males.
Our serotonin treatment has no significant effect on aggression, despite
the fact that we could verify the effectiveness of the treatment
immunohistochemically (data not shown). Also, Vaysse et al.
(1988) observed effects on
learning and memory after identical treatment, indicating that this
pharmacological manipulation of serotonin levels in principle can have
behavioural effects. Moreover, we observed a noticeable increase in activity
in the 5ht- flies, a subjective impression that is corroborated by the
significantly increased PE of this subgroup
(Fig. 1Bii). Nevertheless, the
possibility remains that the observed difference in serotonin immunoreactivity
was not high enough to generate significant differences in aggression,
although it was high enough to affect other behaviours. The lack of
serotonergic effect on aggression was also repeatedly observed in our pilot
studies (A. Baier, B. Wittek and B. Brembs, unpublished data). Lee and Hall
(2001
) have reported that the
pattern of serotonergic cells in the Drosophila brain is unaltered in
the more aggressive fru mutants, confirming the idea that serotonin
is not crucial for regulation of aggressive behaviour in the fly. The
serotonin results presented here are also consistent with data in crickets,
where serotonin depletion appears to have no effect
(Stevenson et al., 2000
); they
contrast with data in crustaceans, where injections of serotonin increase the
level of aggressive behaviour (Edwards and
Kravitz, 1997
; Huber et al.,
1997a
,b
;
Kravitz, 2000
). Our serotonin
data thus parallel our octopamine data in conforming with insect data but
contrasting with observations in crustaceans. Perhaps aminergic control of
aggression functions fundamentally differently in those two arthropod
groups?
Our dopamine treatment had complex effects. The absolute number of non-aggressive encounters appears elevated compared to the wild-type controls (Fig. 1A), reducing overall aggression probabilities (Fig. 1Bi; PA). Also, while the raw data indicate higher aggression scores in the animals with low dopamine (Fig. 1Ai), the PA is higher in animals with high dopamine levels (Fig. 1Bi). Taking the number of encounters that each subgroup experiences (Fig. 1Bii, PE) into account, it seems as if the higher raw scores for the `low' dopamine animals is generated by the higher PE in this subgroup. Once that factor is accounted for (Fig. 1Bi), the perceived difference between raw and derived data disappears.
A general point of concern is possible side effects of our treatments. Both
e and b flies exhibit varying degrees of visual impairment
(A. Baier, B. Wittek and B. Brembs, unpublished data; Heisenberg,
1971,
1972
;
Hovemann et al., 1998
;
Jacobs, 1978
), with e
flies showing more severe defects than b flies (A. Baier, B. Wittek
and B. Brembs, unpublished data; Jacobs,
1978
). Without screening pigments (i.e.
white-), the M18 octopamine jump-out mutants are expected
to have severely impaired vision compared with the control strain still
carrying the P-element. Also, the extent by which the treatments may affect
general activity is largely unknown (but see
Martin et al., 1998
). One may
assume that a subgroup's PE should reflect overall
activity. Not surprisingly, the more visually impaired e and oa-
flies have lower PE values than the b and oa+
subgroups, respectively (Fig.
1Bii). However, the probability to attack seems entirely
unaffected by this measure of general activity, as the relationships are
reversed. Moreover, both the dopamine and the mushroom body groups show a
higher probability to attack in the respective `high' subgroup
(Fig. 1Bi), but their
PE values are inverted with respect to their
PA values (Fig.
1Bii). Thus, while both vision and general activity may influence
aggression, those factors seem to have only marginal effects compared to the
determinants studied here.
Of course, this study is only a beginning. We did not examine encounter duration, behavioural composition or opponent identity/recognition, let alone investigate potential mechanisms as to how the identified factors might exert their effects. However, our method successfully reproduced published data (the e/b group) and yielded new insights into the neurobiological determinants of aggression in Drosophila melanogaster. Serotonin appears to have no effect, while dopamine, octopamine and the mushroom bodies could be linked to the promotion of aggressive behaviour. We hope that our work will inspire others to exploit Drosophila's numerous technical advantages for studying the neurobiology of aggression.
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Acknowledgments |
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