Haloperidol and sulpiride are clinically proven antipsychotic and sedative
agents both being dopaminergic antagonists (Jenner, Clow, Reavill, Theodorov
and Mardsen, 1978; Jenner and Marsden, 1979; Kebabian and Calne, 1979). Haloperidol
has well-known antiaggressive properties in males (Poshivalov, 1982; Olivier,
Van Aken, Jaarsma, Van Oorschot, Zethof, and Bradford,1984; Miñarro,
Castaño, Brain, and Simón,1990). Sulpiride also markedty decreases
attack behaviour shown by male mice in a social encounter test (Simón,
Miñarro, Redolat, and Garmendia, 1989; Redolat, Brain and Simón,
-in press-): Most studies on the effects of haloperidol and sulpiride on social
and agonistic behaviours have involved male mice. Indeed, three are relatively
few studies on females in the general area of behavioural pharmacology (see
exceptions in Benton, Smoothy and Brain, 1985 and in Smoothy, Brain, Berry and
Haug,1986). It was consequently thought desirable to attempt to provide a detailed
analysis of the behavioural actions of haloperidol and sulpiride in female mice.
Resident-intruder tests employing male mice with docile 'standard opponents'
have proved useful in studies assessing the anti-aggressive properties of particular
compounds. This is so because such situations reliably generate reasonable levels
of threat and attack (Brain and Al-Maliki, 1978, Brain, Smoothy and Benton,
1985) with little indication of pronounced defensive or submissive behaviour
by the test animals in such situations. Conversely, resident-intruder tests
employing non-reproductive female subjects do not generally produce much fighting
or threat but they do generate higher base-lines of defensive-submissive and
also exploratory behaviour (Benton et al, 1985; Smoothy et al. 1986). For this
reason, it was thought that this experimental paradigm might prove appropriate
for the assessment of effects of three drugs on defensive behaviour. In the
present study, the effects of haloperidol and sulpiride were assessed on social
and defensive behaviour by examining the responses of female mice to the presence
of a nonfamiliar anosmic male intruder, in two different experiments.
MATERIALS AND METHODS
Ninety-six naive female mice of the Alderley Park strain (from a stock supplied
by I.C.I. Pharmaceuticals, Alderley Park, Cheshire, England) were used as experimental
subjects (48 for each drug study). They were bred and housed under highly controlled
conditions in the Animal Facility of the University College of Swansea. Subjects
were weaned at 19-23 days of age and placed in opaque polypropylene cages, measuring
30 x 12 x 11 cm (North Kent Plastics). The Subjects were housed in groups of
six from weaning until one week before testing. Animals were maintained at 21°-
C., with a reversed lighting schedule (white lights on 22:30 - 10:30 hrs. G.M.T.),
dim red lighting was used at all other times. Food and water were available
ad lib, except during behavioural trials. An equal number of male mice were
housed in groups of six and used as "standard opponents" after being rendered
temporally anosmic by intranasal lavage with 4 % zinc sulphate solution, both
three days and one day before testing(see Smoothy et al., 1986). Such animals
elicit attack by males but do not initiate it. In spite of their lack of threat
and attack they stimulate timid behaviour in females (Benton et al., 1985; Smoothy
et al., 1986).
Drug treatment: Animals were injected i.p. with 0.01, 0.1 or 0.2 mg/kg of haloperidol
(Haloperidol®, Latino Laboratories, Spain) or with physiological saline
(control group) in a first study. In a second study female mice received acute
treatment i.p. with one of the following injections: 31.25 , 62.5 or 125 mg/kg
of sulpiride (Dogmatil®, Delagrange, Spain) or physiological saline (control
group). Each female was given a single injection.
Social encounters: Twenty or thirty minutes respectively, after haloperidol
or sulpiride injection, an anosmic standard opponent (marked with fur dye) was
introduced into the female's cage. The interaction between female and the opponent
lasted 10 minutes and was videotaped using a camera sensitive to red light (National
Panasonic, model 1350A). During the test, the wiremesh lid of the cage was replaced
with a perforated transparent Perspex cover to facilitate observation. All tests
were carried out under dim red illumination between the second and fifth hour
of the dark phase of the light/dark cycle on thee successive days.
Behavioural Analysis: The tapes were analyzed using a microprocessor (Commodore
64 computer) and a custom-developed program (Brain, McAllister and Walmsley,
1989) which facilitated estimation of times allocated to eleven broad functional
behavioural categories. Each category includes a collection of different behavioural
postures and elements. The names of categories and their constituent elements
are the following: 1) Body care (abbreviated groom, self groom, wash, shake,
scratch); 2) Digging (dig, kick dig, push dig); 3)Non social exploration (explore,
rear, supported rear, scan); 4) Explore from a distance (approach, attend, circle,
head orient, stretched attention); 5) Social Investigation (crawl over, crawl
under, follow, groom, head groom, investigate, nose sniff, sniff, push past,
walk around); 6) Threat (aggressive groom, sideways offensive, upright offensive,
tail rattle); 7) Attack (charge, lunge, attack, chase); 8) Avoidance/Flee (evade,
flinch, retreat, ricochet, wheel, startle, jump, leave, wall clutch); 9)Defensive/Submissive
(upright defensive, uprigth submissive, sideways defensive); 10) Sexual (attempted
mount, mount) and 11) Immobility (squat, cringe). A detailed description of
all elements can be found in Martinez, Castaño, Simon and Brain (1986)
and Martinez, Miñarro, and Simon (1991). This ethopharmacological method
allows a complete quantification of the behavioural elements shown by the animal
during the social encounters.
Statistical Analysis: Non parametric Kruskal-Wallis tests were performed to
assess the variance over different treatment groups. Subsequently two-tailed
Mann-Whitney U tests were used to contrast times allocated to each behavioural
category by experimental and control groups.
The detailed behavioural results are presented in tables 1 and 2 for haloperidol
and sulpiride, respectively.
Kruskal-Wallis analysis showed that there was no significant increase in Immobility
in groups treated with different doses of haloperidol and sulpiride i.e. none
of the doses used here were ataxic. In spite of this, both drugs significantly
changed aspects of exploratory behaviour of female mice during interaction with
an anosmic opponent. Kruskal-Wallis revealed significant variance across the
categories of Social Investigation in experiment with haloperidol and in Non
Social Exploration and Explore-from-a distance in experiment with sulpiride.
Haloperidol and sulpiride increased explore from a distance (tentative exploration
directed by female mica towards the partner during encounter test). This increment
was statistically significant with all doses of sulpiride. However, these neuroleptics
affect non social exploration (exploration around the cage) and social investigation
(directed towards the opponent) in opposite directions. Non social exploration
was unchanged by haloperidol but time allocated to this behavioural category
significantly diminished with the lowest and intermediate doses of sulpiride.
Social investigation decreased in the animals treated with the two higher doses
of haloperidol and was increased (although not significantly) in animals treated
Times allocated lo threat and attack were, as expected, minimal in all categories
used here and these were no statistical differences between animals treated
with sulpiride or haloperidol and their control groups. Neither haloperidol
nor sulpiride significantly reduced defensive or submissive behaviours.
Haloperidol and sulpiride increased the time devoted to body tare, although
this increase only was statistically significant for the higher dose of haloperidol.
Neither haloperidol nor sulpiride changed significantly digging behaviour.
Ethopharmacological assessment of the behaviour of female mica when confronted
with 'standard opponents' showed that exploratory behaviour were specifically
changed, although differently by the two drugs: sulpiride diminished non-social
exploration and increased explore-from-a-distance and social investigation.
These results may reflect an increased "interest" by female mica towards the
males. Because of this increase in time spent exploring the opponent, time devoted
to non social exploration was reduced. Conversely, haloperidol significantly
decreased time devoted to social investigation but did not significantly change
time allocated to non-social exploration by female mice. This differential effect
could be attributed to the well known motor effects of haloperidol. One could
argue that social investigation is an activity that requires more energy and
coordination than non social exploration. Consequently, social investigation
(interest towards the opponent) would be more sensitive to sedative actions
of haloperidol. Effects of these drugs on social exploratory behaviour are not
specific for female mice. In previous studies we found that haloperidol diminishes
social investigation in males whereas sulpiride increases this behaviour (Miñarro
et al., 1990; Redolat et al. -in press-).
This differential effect of haloperidol and sulpiride on exploratory behaviour
could be related with the selective effects of these drugs in neural dopaminergic
system. Both drugs are dopaminergic antagonists, having more afinity for the
D2 than for the D1 receptors (Hyttel and Amt, 1986), although they differentially
alter dopamine turnover. Experimental measurements of regional levels of Homovanillic
acid (HVA), the main metabolite of Dopamine, after acute administration of haloperidol
and sulpiride showed that haloperidol induces larger concentrations of HVA in
the striatum than in the limbic areas (Bartholini, 1976) whereas sulpiride increases
dopamine turnover more in the limbic system than in the striatum (Bartholini
and Lloyd, 1980). These differences could explain the fact that haloperidol
has greater effect than sulpiride on activities with greater motor components
(i.e. social investigation).
In agreement with previous studies (Benton et al., 1985; Smoothy et al., 1986),
moderate levels of "avoidance-flee" and "defensive-submissive" behaviour and
an absence of aggressive postures were evident although the males were anosmic
and did not attack. Some female mice very frequently showed defensive postures
when males approached them. Defensive behaviour was uninfluenced by any of the
administered doses of haloperidol and sulpiride.
In the present study, haloperidol increased significantly body care while previous
data had shown that this parameter significantly diminished in male mice treated
with haloperidol (Miñarro et al., 1989).
One of the most remarkable results was that neither haloperidol nor sulpiride
significantly increased immobility. This fact is specially salient with regard
to haloperidol. Previous data showed that haloperidol strongly increased immobility
in male mice. This has been observed when male mice are confronted with standard
opponents in a neutral arena (Miñarro et al., 1990) or in their own cage
(Miñarro, Brain and Simon,1989).
Comparing present results with previous studies (Miñarro et al., 1990;
Redolat et al., -in press-) obvious differences in social behaviour appear between
males and females. These results support previous data about sex differences
in the behavioural effects of neuroleptic drugs in animals (Dalton, Vickers
and Roberts, 1986) and man (Swet, 1975).