The present study characterized the effects of the novel, selective,
and potent 5-hydroxytryptamine1A (serotonin)
(5-HT1A) receptor agonist, alnespirone [S-20499,
(S)-N-4-[5-methoxychroman-3-yl)propylamino)butyl-8-azaspiro-(4,5)-diacetamide, hydrochloride] on offensive and defensive resident-intruder aggression in wild-type rats and compared its actions with those of the
prototypical full 5-HT1A agonist
8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT), the partial
5-HT1A agonists ipsapirone and buspirone, and the mixed
5-HT1A/1B agonist eltoprazine. All five agonists exerted effective dose-dependent decreases of offensive aggressive behavior in
resident rats; 8-OH-DPAT was the most potent (ID50 = 0.074 mg/kg), followed by eltoprazine (0.24), buspirone (0.72), ipsapirone (1.08), and alnespirone (1.24). However, in terms of selectivity of the
antiaggressive effects as determined by the absence of decrements in
social interest and general motor activity, alnespirone appeared to be
superior. In the defensive aggression test, neither alnespirone nor any
of the other four agonists changed defensive behaviors in the intruder
rats. The involvement of 5-HT1A receptors in the
antiaggressive actions of these drugs was confirmed by showing that the
selective 5-HT1A receptor antagonist WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride), which was inactive alone, fully prevented the antiaggressive effects of alnespirone, 8-OH-DPAT, and buspirone and
partly reversed those of ipsapirone and eltoprazine. The data clearly
indicate that alnespirone effectively suppresses offensive aggression
with an advantageous profile of action compared with other full or
partial 5-HT1A agonists. These selective antiaggressive actions of alnespirone are mediated by stimulating 5-HT1A
receptors, presumably the somatodendritic autoreceptors at the raphe
nuclei. Furthermore, the data provide evidence for a major involvement of these 5-HT1A receptors in the modulation of aggressive
behavior by 8-OH-DPAT, ipsapirone, buspirone, and eltoprazine.
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Introduction |
Alnespirone
[S-20499,
(S)-N-4-[5-methoxychroman-3-yl)propylamino)butyl-8-azaspiro-(4,5)-diacetamide,
hydrochloride] is a novel amino chroman derivative with potent and
selective agonist properties at central
5-hydroxytryptamine1A (serotonin)
(5-HT1A) receptors (Kidd et al., 1993
; Scott et
al., 1994; Casanovas et al., 1997; Fabre et al., 1997). Accordingly,
because brain 5-HT1A receptors are implicated in
the pathophysiology of anxiety and depression (Coplan et al., 1995),
alnespirone has reliably been reported to display anxiolytic and
antidepressant properties in a variety of animal behavioral models
(Griebel et al., 1992; Porsolt et al., 1992; Barrett et al., 1994;
Curle et al., 1994; File and Andrews, 1994; MacSweeney et al., 1998).
Besides the involvement in anxiety and depression, serotoninergic
receptors of the 5-HT1A type are particularly
implicated in aggressive behavior. Drugs that selectively activate this
receptor site are among the most promising novel therapeutic agents for
altering excessive violent and aggressive behavior (Ratey et al., 1991;
Miczek et al., 1995; Olivier et al., 1995). Stimulation of the
5-HT1A receptor by more or less selective partial
and full agonists, such as buspirone, gepirone, ipsapirone,
8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT), fluprazine, eltoprazine,
and flesinoxan results in potent antiaggressive effects across a range
of animal species and test situations (Tompkins et al., 1980; Flannely
et al., 1985; Lindgren and Kantak, 1987; Blanchard et al., 1988;
McMillen et al., 1988; Nikulina, 1991; Bell and Hobson, 1994; Bonson et
al., 1994; Olivier et al., 1994; Sanchez et al., 1996; Sanchez
and Hyttel, 1994; Miczek et al., 1995; Muehlenkamp et al., 1995, 1996).
However, with the noticeable exception of eltoprazine and fluprazine,
the profound antiaggressive effects of these drugs coincide with and
probably are secondary to the behaviorally nonspecific incapacitating
effects these drugs exert, such as sedation, motor debilitation, and
inhibition of the entire social behavioral repertoire (i.e., the
"5-HT syndrome": Tricklebank, 1985). After a comparison of a
variety of 5-HT receptor agonists in a number of aggression studies
(Mos et al., 1992, 1993; Sijbesma et al., 1991), Olivier et al.
(1995) concluded that the specific reduction of offensive
aggression (or "serenic activity") by eltoprazine and fluprazine is
mediated via postsynaptically located 5-HT1B
binding sites. However, in all these studies, a contribution of the
5-HT1A receptor could not be ruled out because of
the lack of good selective 5-HT1A receptor
antagonists at the time.
A first experiment by File and Andrews (1994) using the social
interaction test in male Lister rats provided some indications that
alnespirone, in addition to increasing social investigation (indicating
anxiolytic-like actions), had the effect of reducing aggressive
behaviors. Because the social interaction test is not specifically
aimed at demonstrating antiaggressive effects of drugs, a more
extensive ethological analysis in an animal model for agonistic
behavior is required to determine how selectively alnespirone affects
aggressive behavior in the rat (see, for example, Miczek et al., 1995).
Therefore, in this study, the antiaggressive effects of alnespirone
were studied in a resident-intruder paradigm, which has the possibility
to test the effects of the drug on offensive aggression (in the
resident initiated by the intrusion of an unfamiliar male into it home
territory) as well as on defensive aggression (in the intruder).
Moreover, by making an extensive recording of the behavior in this
test, it was possible to determine the selectivity of the drug effects
for aggression or nonspecific (i.e., sedative) effects (see Olivier et
al., 1995; Miczek et al., 1989, for reviews of the various kinds of
aggressive behavior and use of different animal models). The effects of
alnespirone in this paradigm were compared with those of the
prototypical full 5-HT1A agonist 8-OH-DPAT, the
partial 5-HT1A agonists ipsapirone and buspirone,
and the mixed 5-HT1A/1B agonist eltoprazine.
Furthermore, the involvement of 5-HT1A receptors
in the putative antiaggressive actions of these drugs was assessed by
using the selective and silent 5-HT1A antagonist
WAY-100635
(N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride) (Forster et al., 1995; Fletcher et al., 1996).
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Materials and Methods |
Subjects and Housing.
A total of 527 male Wild Type
Groningen (WTG) rats (Rattus norvegicus; originally
wild-trapped animals and bred for approximately 17 generations in our
own laboratory under specific-pathogen-free conditions) that were 3.5 months old were used as experimental subjects. This strain is preferred
for agonistic studies because they exhibit an easily to evoke and rich
natural repertoire of intraspecific aggressive and social behaviors.
They were housed in clear Plexiglas cages (60 × 60 × 20 cm)
in groups of five or six from weaning (23 days after birth) until the
start (at age 140 days) of the experiments. The cages were placed in a
temperature-controlled room (22 ± 2°C) with a fixed 12-h
light/dark photoperiod (lights off at 1:00 pm). The animals were
allowed free access to water and food (Hope Farms Lab Chow).
Experimental Procedures.
A resident-intruder agonistic
paradigm was used to monitor either offensive behavior (experimental
resident) or defensive behavior (experimental intruder) that strongly
resembles the natural patterns of wild rats to establish and defend
their territory (Koolhaas et al., 1980). In the resident-intruder
offensive model, the animals were housed individually in observation
cages (80 × 55 × 50 cm), each with a sterilized female to
avoid social isolation and to facilitate territorial behavior. After 1 week, the baseline level of offensive behavior was tested on 3 consecutive days during a 10-min confrontation with an unfamiliar male
conspecific in the home territory of the experimental (resident) rat.
These naive intruder-rats were socially housed in groups of five or six
animals in clear Plexiglas cages (60 × 60 × 20 cm).
Approximately 1 h before the start of the confrontation, the
female of the experimental rat was removed from the observation cage.
Experimental groups were balanced on the basis of offensive behavior
performed during the third baseline test, during which the full range
of behavioral elements was recorded (see below). Animals that showed
less than 10% offensive behavior (ALT greater than 500 s) were
not included in the drug treatment tests (approximately 15% of the animals).
On the next day, 30 min before the 10-min confrontation with an
intruder, the experimental resident rats received one dose of one of
the following s.c. injections (i.e., animals were tested only once):
vehicle (1 ml/kg distilled water) alnespirone (0.5, 1, 5.0, and 10 mg/kg), 8-OH-DPAT (0.05, 0.1, 0.25, 0.5, and 1.0 mg/kg), buspirone
(0.25, 0.5, 1.0, and 5.0 mg/kg), ipsapirone (0.5, 1.0, and 5.0 mg/kg),
or eltoprazine (0.1, 0.25, 0.5, 1.0, and 2.5 mg/kg). During the 10-min
confrontation with an unfamiliar and undrugged conspecific intruder,
the full range of behaviors was recorded again.
In case of the antagonism studies, vehicle (distilled water) or
WAY-100635 (0.01, 0.1, or 1.0 mg/kg) was administered 15 min before
single challenge doses of either vehicle, alnespirone (5.0 mg/kg),
8-OH-DPAT (0.1 mg/kg), buspirone (2.5 mg/kg), ipsapirone (5 mg/kg), and
eltoprazine (1 mg/kg), and 30 min later, the agonistic behavior of the
drugged resident rats was examined by ethological procedures during a
10-min social encounter with an undrugged intruder. The selected doses
of the 5-HT1A agonists were based on submaximal
effective dosages to inhibit aggressive behavior found previously in
the dose-response study.
In the resident-intruder defensive model, another group of experimental
animals served as naive intruders into the home territory of a well
trained (5-10 consecutive successful winning experiences) aggressive
resident counterpart. The ensuing agonistic interaction was accompanied
by a variety of defensive body postures and escape behaviors of the
experimental intruder rat that can be recorded. Therefore, this model
gives the opportunity to assess the effects of the
5-HT1A agonists on the complete natural defensive
behavioral repertoire. Alnespirone (1, 5, and 10 mg/kg),
8-OH-DPAT (0.1, 0.25, and 0.5 mg/kg), buspirone (0.5 and 5 mg/kg),
ipsapirone (0.5 and 5 mg/kg), eltoprazine (0.5 and 1 mg/kg), or vehicle
(distilled water) was administered s.c. 30 min before placement of the
experimental animal into the home territory of an aggressive male
resident for 10 min. The selected doses of the
5-HT1A agonists were based on effective dosages
to inhibit aggressive behavior found previously in the dose-response study.
During the agonistic confrontations, the full range of behaviors of
either the experimental resident rat (offensive aggression test) or the
experimental intruder rat (defensive aggression test) was recorded on
videotape and manually scored on a keyboard processor. An extensive
description of the different behavioral elements displayed during
agonistic interactions has been reported previously (Koolhaas et al.,
1980). Briefly, the following behavioral elements were distinguished:
1) lateral threat; 2) keep down; 3) clinch; 4) chase/flight; 5)
offensive/defensive upright; 6) investigating opponent/moving toward;
7) ambulation, scanning, digging; 8) rearing; 9) grooming; 10) social
grooming/crawl over; 11) ano-genital sniffing; 12) inactivity; 13)
freeze/crouch; 14) submissive posture; 15) (attempt to) mounting; and
16) keep off. The duration of the different behavioral elements was
computed and expressed as a percentage of the total duration (10 min)
of the confrontation. To promote a clear representation of the data,
the elements lateral threat, offensive upright, keep down, clinch, and
chase were taken as one behavioral category (i.e., "offensive
behavior"), whereas the term "defensive behavior" is used for
submissive posture, defensive upright, flight, freeze, and keep off.
Also, the latency time to the first attack ("attack latency time")
by the resident was taken as a measure of aggressiveness. The term
"social explorative behavior" is used for the elements
investigating opponent and anogenital sniffing, whereas "social
contact behavior" encompasses the elements social grooming, crawl
over, and mounting. "Nonsocial explorative behavior" is used for
the elements ambulation and rearing.
Drugs.
Alnespirone [(+)-S-20499-2 hydrochloride; lot no.
45109; molecular weight 479) was provided by Institut de Recherches
Internationales (Servier, France). WAY-100635
[N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride; lot no. A-05; molecular weight 513] was a gift from
Wyeth Research (UK) Ltd. (±)-8-OH-DPAT hydrobromide (molecular weight
328) and buspirone hydrochloride (molecular weight 422) were obtained
from Research Biochemicals International (Natick, MA). Ipsapirone
hydrochloride (molecular weight 438) and eltoprazine dihydrochloride
(molecular weight 258) were kindly provided by Solvay Duphar
Pharmaceuticals (Weesp, The Netherlands). All drugs were freshly
dissolved in sterile distilled water (vehicle solution) and pH adjusted
to as close to neutrality as possible. Solutions were prepared
approximately 1 h before the start of the experiments. The
injections were given s.c. in the flank region in a volume of 1 ml/kg
b.wt.
Data Analysis.
Data are expressed as mean ± S.E.M. The
dose-effect curves for each behavioral catagory and attack latency time
were analyzed by a one-way ANOVA, with drug dose as between-subject
factor. In the dose-response studies, the drug effects on each
behavioral catagory were also computed as a percentage of the
respective vehicle control values to enable a comparison between the
various drugs. Least-squares linear regression analysis was used to
estimate the dose (mg/kg) that would elicit 50% aggression reduction
(ED50) and the corresponding 95% confidence
limits. ED50 values with 95% confidence limits
that did not overlap were considered to be statistically different. In
the antagonist study, the drug-effect histograms for each behavioral
category and attack latency time were analyzed by a two-way ANOVA, with
pretreatment as between-subject factor 1 (two levels: vehicle and WAY
0.1) and drug as between-subject factor 2 (six levels). Further
analyses were made by Duncan's new multiple-range test
(between-subject effects) to determine the source of detected
significance in the ANOVAs. The criterion of significance was set at
P < .05.
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Results |
Offensive Aggression Test: General Aspects and Dose-Response
Effects.
Social confrontation initiated by the intrusion of an
unfamiliar male rat into the home cage of the territorial experimental male counterpart resulted in a typical offensive aggressive behavioral pattern of the resident, consisting of an approach to and pursuing of
the intruder, followed by anogenital sniffing (sometimes followed by
mounting attempts), and a threaten/attack sequence resulting in
clinching, biting, chasing, and forcing the intruder into submission. The latency time to the first attack (clinch) in undrugged residents (n = 223) ranged from 6 to 461 s with a mean of
125 ± 8.2 s. These fights always resulted in defeat of the
intruder rat, which exhibited a variety of defensive/submissive body
postures and escape responses. Characteristically, several bouts of
fighting alternate with periods of no agonistic interactions during the
observation trial. During the 10-min agonistic encounters on day 1, undrugged resident rats spent 43.1 ± 1.7% of the time on
offensive aggressive behavior and 12.9 ± 0.7% on total social
explorative behavior, thus spending 56.1 ± 1.5% on total social
interaction. In the remainder of the 10-min observation period, animals
spent 33.9 ± 1.3% of the time on nonsocial exploration, 2.5 ± 0.3% on grooming, and 7.0 ± 0.7% on inactivity. A roughly
similar behavioral pattern was observed the next day in the group of
rats injected with vehicle (Fig. 1), but
compared with the undrugged condition, the vehicle-injected animals
spent significantly less time on offensive and social explorative
behavior and more time on inactive and grooming behaviors.
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Fig. 1.
Mean ± S.D. percentages of time spent on six
distinct behavioral catagories and mean attack latency scores (inset)
during offensive aggression test on day 1 (undrugged condition; gray
bars) and day 2 (vehicle-treated; black bars). *Values are
significantly (at least p < .05; Student's
t test) different from undrugged condition value
(n = 58).
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Compared with vehicle treatment, alnespirone-treated rats showed a
significant, dose-dependent delay in the latency time to attack (Fig.
2, inset) and reduction in the amount of
offensive behavior (Fig. 2) toward the intruder rat. This reduction in
offensive behavior was accompanied by a significant increase in social
explorative behavior, thereby leaving total social interaction time in
the alnespirone-drugged rats as the same. In addition, the drug did not
significantly modify the nonsocial activities of exploration, grooming,
or inactivity (Fig. 2).
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Fig. 2.
Effect of alnespirone on ALT and behavior of resident
rats in offensive aggression test. One-way ANOVA revealed significant
effects of dose for ALT (F4,37 = 9.4;
p < .0001), offensive behavior
(F4,37 = 6.64; p < .0005), social exploration (F4,37 = 15.72;
P < .0001), and social interaction
(F4,37 = 2.74; p < .05). *Values are significantly (at least p < .05;
Duncan's) different from vehicle (dose 0) value.
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8-OH-DPAT-treated rats showed a pronounced, dose-dependent reduction in
offensive aggressive behavior (Fig. 3) as
well. However, this antiaggressive effect was accompanied by a
pronounced increase in behavioral inactivity: Starting from the 0.1 mg/kg dose, 8-OH-DPAT induced clear signs of the full serotonergic
syndrome, especially characterized by flat body-posture, head-waving,
forepaw treading, and hindlimb abduction, leading to increased
behavioral inactivity scores. Because social exploration was not
affected by 8-OH-DPAT, total social interaction decreased significantly
after 8-OH-DPAT treatment.
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Fig. 3.
Effect of 8-OH-DPAT on ALT and behavior of resident
rats in offensive aggression test. One-way ANOVA revealed significant
effects of dose for ALT (F5,36 = 27.4;
p < .0001), offensive behavior
(F5,36 = 15.74; p < .0001), social interaction (F5,36 = 8.78;
p < .0001), nonsocial exploration
(F5,36 = 4.33; p < .005), and immobility (F5,36 = 14.39;
p < .0001). *Values are significantly (at least
p < .05; Duncan's) different from vehicle (dose
0) value.
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Similar to the behavioral profile induced by 8-OH-DPAT, buspirone-
(Fig. 4) and ipsapirone- (Fig.
5) treated
animals showed a potent, dose-dependent reduction in aggression that
was accompanied by significant increases in behavioral inactivity. The
highest dose of each drug (5.0 mg/kg) produced some of the signs of the serotonergic syndrome (flat body posture).
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Fig. 4.
Effect of buspirone on ALT and behavior of resident
rats in offensive aggression test. One-way ANOVA revealed significant
effects of dose for ALT (F4,35 = 29.1;
p < .0001), offensive behavior
(F4,35 = 9.12; p < .0001), social interaction (F4,35 = 10.71;
p < .0001), nonsocial exploration
(F4,35 = 3.61; p < .02), and immobility (F4,35 = 21.51;
p < .0001). *Values are significantly (at least
p < .05; Duncan's) different from vehicle (dose
0) value.
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Fig. 5.
Effect of ipsapirone on ALT and behavior of resident
rats in offensive aggression test. One-way ANOVA revealed significant
effects of dose for ALT (F3,39 = 10.81;
p < .0001), offensive behavior
(F3,39 = 12.98; p < .0001), social interaction (F3,39 = 9.04;
p < .0001), and immobility
(F3,39 = 5.35; p < .005). *Values are significantly (at least p < .05; Duncan's) different from vehicle (dose 0) value.
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Fig. 6.
Effect of eltoprazine on ALT and behavior of resident
rats in offensive aggression test. One-way ANOVA revealed significant
effects of dose for ALT (F5,52 = 4.86;
p = 0.001), offensive behavior
(F5,52 = 9.68; p < .0001), social explore (F5,52 = 2.78;
p < .05), social interaction
(F5,52 = 3.45; p < .01), and immobility (F5,52 = 2.86;
p < .0025). *Values are significantly (at least
p < .05; Duncan's) different from vehicle (dose
0) value.
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Eltoprazine treatment (Fig. 6) also produced a potent dose-dependent
inhibition of aggressive behavior, which was accompanied by an increase
in both social exploration and inactivity. Although there was a
significant enhancement of social exploration, this did not fully
compensate for the decrease in aggression, and thus total social
interaction decreased significantly with the two highest doses of
eltoprazine. It was noted that the two lowest doses (0.25 and 0.5 mg/kg) of eltoprazine that significantly decreased aggression were not
accompanied by a significant increase in behavioral inactivity,
indicating a more specific profile of action compared with buspirone,
ipsapirone, and 8-OH-DPAT. Figure
7 shows the comparative
dose-response relationships of the five compounds to inhibit offensive
aggression and to enhance behavioral inactivity. Clearly, all five
agonists exerted a qualitatively similar dose-response pattern to
decrease offensive aggressive behavior. The antiaggressive potencies of
8-OH-DPAT (ID50 = 0.074 mg/kg) and eltoprazine
(ID50 = 0.24 mg/kg) were significantly different
from each other as well as from buspirone (ID50 = 0.72 mg/kg), ipsapirone (ID50 = 1.08 mg/kg), and
alnespirone (ID50 = 1.24 mg/kg), whereas the potencies between alnespirone, ipsapirone, and buspirone were not
significantly different. As noted before, Fig. 7 also more clearly
shows the different qualitative and quantitative abilities of the five
agonists to increase behavioral inactivity: a potent and pronounced
dose-dependent increase after 8-OH-DPAT, followed by buspirone,
ipsapirone, and eltoprazine, but no increase after any dose of
alnespirone.
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Fig. 7.
Comparative potency of alnespirone, 8-OH-DPAT,
buspirone, ipsapirone, and eltoprazine to inhibit offensive aggression
(bottom) and to enhance behavioral inactivity (top). Antiaggressive
ID50 values (95% confidence limits) are for alnespirone,
1.24 mg/kg (0.86-1.56); 8-OH-DPAT, 0.074 mg/kg (0.051-0.17);
buspirone, 0.72 mg/kg (0.57-1.06); ipsapirone, 1.08 mg/kg
(0.82-1.55); and eltoprazine, 0.24 mg/kg (0.18-0.54).
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Defensive Aggression Test.
The introduction of a
vehicle-treated experimental rat into the home territory of an
undrugged, trained aggressive resident counterpart resulted in an
agonistic interaction (mean attack latency time of the resident = 88.8 s; see Table 1), leading to a
rapid defeat of the experimental intruder. The socially defeated intruder exhibited immediately a variety of defensive body postures and
escape behaviors, including flight, freezing, defensive upright, keep
off, and submission. Vehicle-treated rats spent 64.2 ± 4.3% of
the observation time on defensive behavior (submissive posture, defensive upright, flight, freeze, and keep off), 9.7 ± 1.0% on clinching, and the remaining part on social (2.5 ± 1%) and
nonsocial (19.4 ± 3.3%) exploration. Within the dose-ranges
tested, alnespirone-, 8-OH-DPAT-, buspirone-, ipsapirone-, and
eltoprazine-treated rats showed a similar defensive behavioral pattern
as vehicle-treated rats when exposed to an aggressive resident (Table
1). In addition, the drug-treated intruders were not attacked more
often or vigorously than the vehicle-treated counterparts, nor was
their other behavioral repertoire affected by the drugs. The observed
unchanged aggressive behavior of the resident attacker toward
drug-treated intruders also indicates that the behavior of the latter
is not dramatically changed. With the highest dose of 8-OH-DPAT,
buspirone, and ipsapirone, however, clear signs of flat body posture
appeared, but once defeated by the resident rat, this behavioral
response was indistinguishable from the exhibited submissive and
freezing behavior.
WAY-100635 Pretreatment.
Pretreatment with the selective
5-HT1A antagonist WAY-100635 (0.01, 0.1, and 1.0 mg/kg) did not result in overall significant behavioral changes of the
vehicle-treated group of rats (Fig. 8).
One-way ANOVA with drug dose as between-subject factor did not reveal
significant effects for any of the behavioral parameters. Nevertheless,
at the individual level, the drug seems to decrease offensive behavior
in animals with very high (greater than 50%) levels of aggressiveness
and to increase it in animals with low (less than 15%) levels of
aggressiveness. WAY-100635 (0.1 mg/kg) almost completely blocked the
antiaggressive effects (enhanced ALT, decreased offensive behavior)
induced by submaximal doses of alnespirone, 8-OH-DPAT, and buspirone
and partly antagonized the antiaggressive effects of ipsapirone and
eltoprazine (Fig. 8). A higher dose of WAY-100635 (1 mg/kg) also was
not able to fully block the antiaggressive effects of ipsapirone and
eltoprazine. Interestingly, however, the significantly enhanced
behavioral inactivity observed after treatment with 8-OH-DPAT,
buspirone, ipsapirone, and eltoprazine was reversed with the WAY-100635
pretreatment.
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Fig. 8.
Antagonism of behavioral effects of alnespirone (5 mg/kg), 8-OH-DPAT (0.1 mg/kg), buspirone (2.5 mg/kg), ipsapirone (5 mg/kg), and eltoprazine (1 mg/kg) by pretreatment with
5-HT1A receptor antagonist WAY-100635 (0.01, 0.1, and 1.0 mg/kg). Data are mean ± S.E.M. (n = 7-15/value). Top, effects on behavioral inactivity. Two-way ANOVA
revealed significant effects of drug (F5,96 = 6.09; p < 0.001), pretreatment
(F1,96 = 11.32; p < .001), and drug × pretreatment interaction
(F5,96 = 4.09; p < .002). Middle, effects on ALT. Two-way ANOVA revealed significant
effects of drug (F5,96 = 8.32;
p < .001), pretreatment
(F1,96 = 5.34; p < .001), and drug × pretreatment interaction
(F5,96 = 3.45; p < .002). Bottom, effects on offensive aggressive behavior. Two-way ANOVA
revealed significant effects of drug (F5,96 = 7.75; p < .001), pretreatment
(F1,96 = 22.24; P < .001), and drug × pretreatment interaction
(F5,96 = 6.50; p < .001). *Significant differences from vehicle/vehicle values.
#Significant differences from respective vehicle/drug values.
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Discussion |
The present findings clearly demonstrate that 1) the selective
5-HT1A receptor agonist alnespirone
dose-dependently suppresses offensive aggressive behavior without the
disruptive effects on (social) exploration and motor activity typically
occurring after 8-OH-DPAT, buspirone, and ipsapirone and 2) the
antiaggressive effects are prevented by the selective
5-HT1A antagonist WAY-100635, providing evidence
that 5-HT1A receptors are mediating this effect of alnespirone, 8-OH-DPAT, buspirone, ipsapirone, and eltoprazine.
The antiaggressive profile of alnespirone was characterized by a strong
reduction (both in frequency and duration) in resident rat attacks and
pursuits toward an intruder and accompanied by a compensatory increase
in normal social explorative and contact behavior (mainly mounting
attempts), thereby not affecting total social interaction scores. This
observation is in agreement with the previously reported lack of effect
of alnespirone on social anxiety in the social interaction test using
Lister rats (File and Andrews,1994), where alnespirone increased social
investigation but at the same time decreased aggression. These
dose-dependent changes in the final consummatory parts of the offensive
behavioral repertoire are not secondary to any general depressant or
motor-incapacitating effects of alnespirone because neither nonsocial
exploration and behavioral inactivity (sitting, lying, immobility) nor
the defensive behavioral repertoire was modified by this drug. Thus,
the antiaggressive effect of alnespirone seems to be very specific in
that it is dose dependent, does not impair normal social interactions
and defense/flight abilities, and is without any unwanted motor effects like general sedation and muscle relaxation.
This specific antiaggressive profile of alnespirone is profoundly
different from that of all the other tested full or partial 5-HT1A agonists such as 8-OH-DPAT, buspirone, and
ipsapirone, which potently decrease offensive aggression at doses while
strongly reducing social exploration and motor activity. These
observations of rather unselective antiaggressive effects of 8-OH-DPAT,
buspirone, and ipsapirone are in line with what has been reported
before by several authors using various aggression paradigms (Tompkins et al., 1980; Flannely et al., 1985; Lindgren and Kantak, 1987; Blanchard et al., 1988; McMillen et al., 1988; Nikulina, 1991; Bell and
Hobson, 1994; Olivier et al., 1994, 1995; Sanchez and Hyttel, 1994;
Miczek et al., 1995; Muehlenkamp et al., 1995). The reduction in
offensive aggression by the full 5-HT1A agonist 8-OH-DPAT and partial agonists buspirone and ipsapirone are generally explained by the primary sedating/motor-incapacitating effects these
compounds induce as part of the well described 5-HT behavioral syndrome
(Tricklebank, 1985; Scott et al., 1994; Millan et al., 1994).
The behavioral profile of alnespirone closely resembles that observed
in a similar test situation by Olivier et al. (1989) for the
"serenics" eltoprazine (mixed 5-HT1A/B
agonist), fluprazine, and TFMPP (weak 5-HT1A/B/C
and 5-HT2c agonists). In an extensive and well
conducted series of experiments, it was reported that these drugs
specifically and effectively reduce offensive aggression, leaving
defense and other activities intact. In this study, eltoprazine in the
lower dose-range also exerted very potent antiaggressive effects
without clear signs of sedation/motor dysfunctions, but with higher
dosages, significant increases of behavioral inactivity did occur. The
fact that the antiaggressive effect of eltoprazine at higher doses was
also associated with a significant increase of behavioral inactivity
seems, at first sight, to contrast with data from Olivier and
associates. However, with scrutinization of the results of a number of
their studies, they clearly show that the reduction in aggressive
behavior by eltoprazine is accompanied by enhanced inactivity scores,
which after the highest doses of eltoprazine even reach statistical
significance (Mos et al., 1993; Olivier et al., 1994). Furthermore,
after an extensive series of experiments aimed at determining the 5-HT
site and mechanism of action, it was concluded by Olivier et al. (1995)
that the specific antiaggressive properties of eltoprazine are most
likely mediated via activation of postsynaptic
5-HT1B binding sites (Sijbesma et al., 1991; Mos
et al., 1992, 1993), although a contribution from the
5-HT1A site could not be ruled out. The view of a
predominant involvement of the postsynaptic
5-HT1B site in selectively inhibiting aggression
is on the one hand supported by the enhancement of aggressive behavior
in 5-HT1B "knockout" mice
(Ramboz et al., 1996) but on the other hand is not
supported by the anecdotal report that eltoprazine remains effective in
decreasing aggressive behavior in these 5-HT1B
mutant mice, suggesting involvement of the 5-HT1A
receptor (Miczek et al., 1995).
In vivo, alnespirone has a very high and selective affinity
(Ki = 0.19 nM) for the
5-HT1A receptor site, shows a comparatively low
affinity for dopaminergic D2 receptors (20 nM),
and exhibits only very low (in the micromolar range) affinity for the
5-HT1B, 5-HT2,
alpha and beta adrenergic,
D1 dopaminergic, H1
histaminergic, and GABA/benzodiazepine receptor binding sites (Porsolt
et al., 1992; Kidd et al., 1993). Given these properties, it already
seems most likely that the specific reduction in aggression is mediated through an interaction of this compound at 5-HT1A
sites rather than any of the other receptors. Conclusive evidence that
the 5-HT1A site is indeed important for the
modulation of offensive aggressive behavior comes from the current
antagonism studies with the selective 5-HT1A
receptor antagonist WAY-100635. Blockade of the
5-HT1A receptors by WAY-100635 not only fully
counteracted the effects of alnespirone and the other
5-HT1A agonists but also antagonized the
antiaggressive effects of the mixed 5-HT1A/1B agonist eltoprazine. This result provides convincing evidence that the
5-HT1A receptor is prominently involved in the
antiaggressive effects of these compounds. This is an important
observation regarding the aforementioned question of the particular
roles the 1A and 1B subtypes play in the modulation of offensive
intermale aggression. However, the fact that WAY-100635 was not able to
fully block the antiaggressive effect of eltoprazine indicates that the
the 5-HT1B receptor is involved in modulating
aggressive behavior as well. Future studies using selective
5-HT1B antagonists should provide conclusive
evidence for this involvement. The potent effects of WAY-100635 to
reverse the behavioral effects of 5-HT1A agonists agree with its reported selective and efficient antagonistic properties at this site (Fletcher et al., 1996). Recently, it was also reported, similar to our findings, that the antiaggressive effects of 8-OH-DPAT in socially isolated mice were potently blocked by WAY-100635 (Sanchez
et al., 1996). Given these previous findings together with our current
observations, it seems that the 5-HT1A site is indeed important for the specific modulation of offensive behavior.
Brain 5-HT1A receptors are located
postsynaptically on the soma and dendrites of various neurons in the
limbic system and cortex (postsynaptic receptors), as well as on the
perikarya of serotoninergic neurons in the raphe nuclei
(somatodendritic autoreceptors). Activation of the postsynaptic sites
results in a reduction of neuronal activity (Andrade et al., 1986;
Stevens et al., 1992) and mediates a variety of functional responses,
like the induction of flattened body posture and forepaw treading
[e.g., symptoms of the 5-HT syndrome: Tricklebank, 1985; Millan et
al., 1994; Scott et al., 1994), hypothermia (Millan et al., 1993,
1994), and elevation of plasma adrenocorticotrophic hormone (Koenig et al., 1987)]. Activation of the somatodendritic autoreceptors inhibits the firing activity of 5-HT neurons themselves and, consequently, the
release of 5-HT from their axonal terminals (Gobert et al., 1995) and
is thought to underlie the anxiolytic effects of
5-HT1A agonists (Schreiber and De Vry, 1993; De
Vry, 1995; Millan et al., 1997). Among the various
5-HT1A agonists, differences exist not only
between their potency to stimulate somatodendritic and/or postsynaptic
5-HT1A receptors but also between their ability
to activate different subtypes of postsynaptic
5-HT1A sites mediating different functional
responses (Millan et al., 1993, 1994; Scott et al., 1994). As expected
of its agonist actions at somatodendritic 5-HT1A
autoreceptors, alnespirone produced a marked inhibition of the firing
of 5-HT neurons and a decrease in 5-HT release and turnover in their
projection areas (Kidd et al., 1993; Casanovas et al., 1997), probably
underlying its anxiolytic-like actions in a variety of behavioral
paradigms (Griebel et al., 1992; Porsolt et al., 1992; Barrett et al.,
1994; Curle et al., 1994; File and Andrews, 1994). These
somatodendritic 5-HT1A agonist properties of
alnespirone are similar (albeit with different potencies corresponding to its receptor affinity) to those of 8-OH-DPAT, buspirone, ipsapirone, and eltoprazine (Casanovas et al., 1997). Consistent with its agonist
actions at postsynaptic 5-HT1A receptors,
alnespirone induces hypothermia (Scott et al., 1994; S. F. de
Boer, M. Lesourd, E. Mocaer, and J. M. Koolhaas, unpublished
observations) and stimulates the release of adrenocorticotropin and
corticosterone (Levy et al., 1995), as has been reported for 8-OH-DPAT,
buspirone, ipsapirone, and eltoprazine (Millan et al., 1993; S. F. de Boer, M. Lesourd, E. Mocaer, and J. M. Koolhaas, unpublished
observations). However, in contrast to these other
5-HT1A agonists, alnespirone does not seem to
interact with the postsynaptic 5-HT1A receptors
that are responsible for inducing the 5-HT behavioral syndrome because alnespirone up to high doses does not cause any of the signs and symptoms of this behavior (Scott et al., 1994; Fabre et al., 1997). Furthermore, alnespirone differs from the other
5-HT1A agonists (and/or their metabolites) in
that it does not have (ant)agonistic properties at other receptor types
in vivo, particularly dopamine D2 receptors and
alpha-2 adrenergic receptors (Van Wijngaarden et al., 1990;
Kidd et al., 1993). Although such adrenergic and dopaminergic actions
of 5-HT1A ligands were reported to contribute to
the unfavorable motor-incapacitating effects seen after 8-OH-DPAT, buspirone, and ipsapirone, our results show that WAY-100635 completely antagonized the inactivity and increase in attack latency induced by
all of these compounds. This suggests that the
5-HT1A actions of these compounds are important
for these effects. Thus, the combination of very high selectivity for
5-HT1A receptors and differential agonist
efficacy at 5-HT1A receptors in postsynaptic target areas of serotoninergic projections appears to impart the specific antiaggressive properties of alnespirone.
The next important question to resolve then is whether the specific
antiaggressive effects of alnespirone are mediated via its actions at
5-HT1A autoreceptors, thereby inhibiting
(stress-activated) global serotonergic neurotransmission and/or via
actions at certain postsynaptic 5-HT1A receptors
located in (fore)brain structures important for modulating offensive
aggressive behavior. There are indications in the literature that favor
the hypothesis that the antiaggressive effects of
5-HT1A agonists are exerted via 5-HT1A autoreceptors in the raphe nuclei to
decrease serotonergic activity (see, for example, McMillen et al.,
1988), especially the recent observation that the novel
benzodioxopiperazine S-15535, which is an agonist and antagonist at
presynaptic and postsynaptic 5-HT1A receptors,
respectively, exerts antiaggressive actions (Millan et al., 1997)
supports this. Together with findings that the performance of
spontaneous or ethanol-enhanced aggressive behavior is associated with
marked increases in serotonergic activity in selected brain regions
(Daruna and Kent, 1976; Garris et al., 1984; Broderick et al., 1984;
Haney et al., 1990; Miczek et al., 1994), it is tempting to speculate
that activation of somatodendritic 5-HT1A
autoreceptors, resulting in a decreased (stress/intruder-activated) serotonergic neurotransmission, underlie the specific antiaggressive effects of 5-HT1A ligands. In line with this,
activation of terminal-localized 5-HT1B
autoreceptors, exerting a brake on the activated serotonergic neurotransmission as well, probably underlie the specific
antiaggressive effects of eltoprazine and other
5-HT1B agonists.
In conclusion, the present data clearly indicate that alnespirone very
effectively and specifically suppresses offensive aggression with an
advantageous profile of action compared with other full or partial
5-HT1A agonists. These selective antiaggressive
actions of alnespirone are mediated by stimulating
5-HT1A receptors, presumably the somatodendritic
autoreceptors at the raphe nuclei. Furthermore, the data also provide
evidence for the involvement of these 5-HT1A receptors in the modulation of aggressive behavior by 8-OH-DPAT, ipsapirone, buspirone, and eltoprazine.
We gratefully acknowledge the skillful technical assistance of
Ewold ter Veld and Auke Meinema in the behavioral observations and
animal care.
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Abstract
Introduction
)S 20500.
Psychopharmacology
116:
73-78[Medline].
-adrenoceptors.
Eur J Pharmacol
297:
1-8[Medline].
