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Comparison of the effects of yohimbine and clonidine on the behaviour of female mice during social encounters in an “approach-avoidance” situation
NeuropharmacologyVol. 32, No. 5, pp. 411-417, 1993
0028-3908/93$6.00+ 0.00 Copyright © 1993PergamonPress Ltd
Printed in Great Britain.All rights reserved
COMPARISON OF THE EFFECTS OF YOHIMBINE A N D C L O N I D I N E ON THE BEHAVIOUR OF FEMALE MICE D U R I N G SOCIAL ENCOUNTERS IN AN " A P P R O A C H - A V O I D A N C E " SITUATION MARGARETG. CUTLER Department of Biological Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 0BA, U.K.
(Accepted 4 February 1993) Summary--Effects of yohimbine (2 and 5mg/kg, i.p.) and clonidine (10 and 50/zg/kg, i.p.) on the behaviour of adult female CDI mice during 5 min encounters in a neutral cage with unfamiliar male partners have been examined by ethological procedures at 30 min after injection. Yohimbine induced dose-related increases in the frequency, bout length and duration of the immobile postures, "sit" and "social crouch", while decreasing the frequency of "explore". "scan", "attend" and "investigate", and increasing their bout lengths in a dose-related manner. These results suggest that yohimbine decreased the rate of switching from one behavioural act to another. Pausing between acts was increased by yohimbine to a similar extent at both of the tested dose levels. The act "wash" was increased in duration by yohimbine, whereas the strenuous activity of "digging" showed a dose-related decrease in frequency, duration and bout length. It is proposed that these effects are induced by the known interactions of yohimbine with receptors for dopamine as well as with ~t2-adrenoceptors. Clonidine reduced motor activity, evident as a dose-related increase in the frequency and duration of "sitting" coupled with decreased frequency and increased bout length of the act, "explore" (significant at 50/~g/kg). Clonidine also dose-dependently reduced the frequency and duration of substrate "sniffing". Clonidine decreased occurrence of the specific social acts, "'attend" and "investigate", as well as reducing frequency although not duration of overall social investigation. These findings have parallels with reported clinical effects of clonidine, such as sedation and impairments of attention, which must limit its clinical usefulness.
Key words--yohimbine, clonidine, female mouse, social behaviour, ct2-adrenoceptors,dopamine receptors.
There has been considerable research into the neurochemical mechanisms associated with stress and its behavioural correlates in humans and experimental animals. The noradrenergic system and especially the locus coeruleus is intimately involved in the manifestations of fear-related behaviour (Hoehn-Saric, 1982). Indeed, electrical stimulation of the locus coeruleus in rodents has been shown to induce a pattern of behavioural effects resembling those produped by the ~t2-noradrenergic antagonist, yohimbine (Charney, Heninger and Redmond, 1983). Effects of yohimbine have been classified as anxiogenie, since it is known, from several studies, to induce fearfulness and feelings of panic in humans (Charney et al., 1983; Mattila, Seppala and Mattila, 1988). In laboratory animals, yohimbine has been found to induce "behavioural agitation" in dogs and sheep (Gershon and Land, 1962) and increased "fearfulness" of monkeys in response to threat (Redmond and Huang, 1979). In rats and mice, yohimbine produces locomotor depression and hypothermia (Papeschi, Sourkes and Youdim, 1971; Sanghvi and Gershon, 1974). It has been found to decrease openarm entries in the elevated plus maze (Johnston and File, 1989) and the number of transitions between light and dark compartments in the light--dark box
(Lapin, 1990). It also decreases exploration activity in the dark compartment of this apparatus (Merlo Pich and Samanin, 1989). In the place conditioning paradigrn, yohimbine was found to produce a conditioned aversion (File, 1986). Effects of yohimbine have been reviewed by Costa and Marino (1991). Early studies indicated that the ~t2-noradrenergic agonist, clonidine, may attenuate human anxiety (Hoehn-Saric, Merchant, Keyser and Smith, 1981). More recently, clonidine was found to be effective in the short-term control of panic disorder (Uhde, Stein, Vittone, Siver, Boulenger, Klein and Mellman, 1989). In rodents, clonidine can reduce the potentiated startle response (Davis, Cedarbaum, Aghajanian and Gendelman, 1977) and increase open-arm entries in the elevated plus-maze (Handley and Mithani, 1984). Clonidine has been found to show reinforcing properties in the conditioned place preference test (Asin and Wirkshafter, 1985) and in tests involving drug self-administration (Davis and Smith, 1977; Shearman, Hynes and Lal, 1981), as well as to be active in conflict tests (Kruse, Dunn, Theurer, Novick and Shearman, 1981; Soderpalm, 1989). Additionally, clonidine has been found to reduce the signs of fear induced in monkeys by electrical stimulation of the locus coeruleus (Redmond and Huang, 1979).
411
412
MARGARETG. CUTLER
However, clonidine also possesses hypothermic, analgesic and dose-dependent sedative actions (Drew, Gower and Marriott, 1979; Durcan, Campbell and Chitkara, 1985; Woolverton, Wessinger and Balster, 1982), and therefore it may influence social behaviour in a manner that differs from that of most anxiolytic agents. In an earlier study, we found that the "timid female paradigm", in which drug-treated female mice in a neutral cage, were confronted with an unfamiliar male, was highly sensitive in detecting anxiolytic effects produced by the novel anxiolytic agents, buspirone and a 5-HT 3 receptor antagonist (Cutler, 1991). In this test situation, characterized by "approach- avoidance conflict", both compounds increased social investigation and reduced flight in drug-treated females. The sensitivity of this paradigm to the effects of anxiogenic compounds has not as yet been examined. Indeed, there have been comparatively few studies of the effects of yohimbine and clonidine on social behaviour in animals. Most of the reported studies have concentrated on the effects of yohimbine. Thus, in the rat social interaction test, Pellow, Chopin and File (1985) found yohimbine to reduce the number and duration of social contacts between animals. Kemble, Behrens, Rawleigh and Gibson (1991) found that yohimbine inhibited isolation-induced attack in mice and increased social distances between the animals. Using ethopharmacological procedures, we found that yohimbine in the resident-intruder paradigm increased immobility in intruder males and decreased several social and non-social acts (Aitken and Cutler, 1990). The present study, using ethological techniques, compares effects of yohimbine with those of clonidine on the behaviour of female mice when encountering unfamiliar male partners.
METHODS
Animals Adult CDI female mice (weighing 40-46g) and male mice (weighing 50---60g) were employed in these studies. The mice had been bred in our laboratory from a stock originally purchased from B. and K. Universal Ltd, Hull. All animals were housed in groups of 5 per cage prior to the experiments and were given an ad libitum supply of pelleted stock cubes (RM3 expanded diet, SDS, Wetham, Essex) and tap water. Mice were maintained at a temperature of 21 + 2°C. A 24 hr cycle of reversed lighting was employed (lights on from 18.00 to 6.00 hr), so that behaviour of the animals should be examined at a time when they were normally most active. Experimental procedures Oestrous female mice were randomly distributed between the five groups shown in Tables 1 and 3. Yohimbine hydrochloride (Sigma) and clonidine
hydrochloride (a gift from Reckitt & Colman Ltd) were dissolved in physiological saline. Yohimbine was given by intraperitoneal (i.p.) injection at 2.0 and 5.0mg/kg, respectively, to mice of two groups (n = 15/group), while clonidine was injected i.p. to mice of a further two groups (n = 15/group) at 10 and 50 pg/kg, respectively. These doses were selected as being within the range known to induce anxiogenic-like or anxiolytic-like behavioural changes in mice and rats (Goldberg and Roberston, 1983; Handley and Mithani, 1984; Johnston and File, 1989; Oluyomi and Hart, 1990). Control mice (n = 18)were given a similar volume of physiological saline (0.1 ml/ 25 g mouse) by i.p. injection. Observations of behaviour shown by each of the mice commenced at 30 min followed injection. Behavioural tests Effects of the treatments on behavioural responsiveness of the females during 5 min social encounters with an unfamiliar group-housed male partner in an illuminated unfamiliar neutral cage (60 x 25 x 25 cm; light intensity 38 cd/m 2) were assessed by ethopharmacological procedures. All behaviourai observations were made during the dark phase of the 24 hr lighting regime, when mice were most active. Acts and posture of behaviour shown by each of the injected mice during the social interactions were recorded as a spoken commentary by tape recorder, using the check list of 49 elements employed in similar previous studies (Cutler, 1991). These are derived from the ethological profile described by Dixon, Fisch and McAllister (1990). Data from the spoken commentaries were transcribed from audiotape onto a floppy disc through keyboard input for analysis by computer of the frequency and duration of each behavioural element and category. The duration of each category of behaviour shown by the partner animals was simultaneously recorded on an electronic machine designed for that purpose, as employed previously (Donald, Cutler and Moore, 1987). As in preceding studies, data have been recorded as the means for each group and probability values for the significance of the differences between control and drug-treated groups determined by the nonparametric Mann-Whitney U-test and the KruskalWallis one-way analysis of variance (ANOVA). Changes having a two-tailed probability of 0.05 or less were deemed to be statistically significant. RESULTS Behaviour of the female mice treated with yohimbine Table l shows that yohimbine induced dose-related increases in the frequency and duration of immobility of the mice and that this was associated with doserelated reductions in the frequency of social investigation and in the frequency and duration of non-social activity. Yohimbine had no significant
Yohimbine and clonidine: their effects on behaviour of female mice
413
Table 1. Changes to catgories of behaviour induced by yohimbine in female mice when encountering male partners Yohimbine Group n
Controls 18
2 mg/kg 15
5 mg/kg 15
Kruskal-Wallis H-test
Non-social activity Immobility Social investigation Aggression Aggressive defence Flight
Behavioural categories Mean duration, sec ( 4- SEM)/observation 229.1 4- 9.1 216.0 4- 6.3 187.0 + 7.6** I.I + 0.6 13.7 4- 4.3** 31.4 4- 7.3** 56.3 4- 4.9 58.2 4- 3.5 66.3 + 4.5 0.2 4- 0.2 0.4 4- 0.3 0.1 + 0.1 4.2 4- 1.6 2.2 4- 0.8 2.3 4- 1.0 10.1 4-4.4 10.24-3.4 13.5+6.0
14.8tt 25.7tt 5.4 0.6 1.0 7.3
Non-social activity Immobility Social investigation Aggression Aggressive defence Flight
Mean frequency ( 4- SEM)/observation 86.4 4- 6.0 55.5 4- 5.4** 34.5 + 3.4** 0.7 4- 0.3 5.7 + 1.3"* 9.2 + 1.3"* 31.3 + 2.1 24.5 4- 1.2" 21.1 + 1.8"* 0.2 4- 0. I 0.2 + 0. I 0.1 4- 0.1 2.4 4- 0.9 1.5 4- 0.6 1.5 + 0.6 4.8 + 2.0 3.9 4- 1.4 4.0 4- 1.4
29.1$t 26.9tt I I.Itt 2.8 0.3 0.3
*P < 0.05, **P < 0.01, between drug-treated and control mice by the Mann-Whitney U-test. ~ ' P < 0.01, between drug-treated and control groups by the KruskaI-Wallis test.
effects on the occurrence of aggression, aggressive defence or flight. The significant effects of yohimbine on the occurrence of individual elements within each of the categories of behaviour can be seen from Table 2. This shows dose-related increases in the frequency and duration of the immobile acts, "sit", "pause" and "social crouch" after yohimbine administration. Yohimbine also significantly increased the bout lengths of "sit" (mean + SEM; controls: 0.3 + 0.2; yohimbine: 2 mg/kg, 1.3 ___0.5; 5 mg/kg, 3.8 ___0.6; P < 0.01) and of "social crouch" (mean + SEM; controls: 1.1 ___0.5; yohimbine: 2 mg/kg, 0.8 ___0.8; 5 mg/kg, 3.0 + 0.5; P < 0.05).
Yohimbine had significant effects on the occurrence of four acts of non-social activity. The duration and frequency of "scan" and "dig" were dose-dependently decreased by yohimbine. However, the bout length of "scan" was increased by yohimbine (mean + SEM; controls: 2.5 + 0.1; yohimbine: 2mg/kg, 3.1+0.2, P<0.05; 5mg/kg, 3.7+0.2, P <0.01), while that of "dig" was reduced (mean + SEM; controls 2.2 + 0.4; yohimbine: 2 mg/kg, 2.2 + 0.5; 5 mg/kg, 0.3 + 0.2; P < 0.01). The frequency of "explore" also was decreased in a dose-dependent manner by yohimbine, yet the bout length of "explore" was dose-dependently raised (mean + SEM; controls: 3.1 +0.3; yohimbine:
Table 2. Significant effects of yohimbine on the elements of behaviour shown by female mice when encountering male partners Yohimbine Group n
ControLs 18
2 mg/kg 15
5 mg/kg 15
KruskaI-Wanis H-test
Behavioural elements Mean duration, set' ( 4- SEM)/obsert, ation Non-social activity Explore Scan Dig Wash Immobility Sit Pause Social investigation Attend Investigate Social crouch
119.74-6.8 86.1 4- 7.3 11.3 + 2.7 2.0 4- 0.7
143.5+_5.3" 53.7 4- 5.1"* 5.6 + 1.7 2.5 4- 0.8
135.24-7.3 34.9 4- 5.6** 0.7 4- 0.5** 7.4 +_ 1.6"
9.5t 29.6tt 16.7tt 7.1t
0.3 4- 0.2 0.8 4- 0.5
8.9 4- 4.3 4.8 4- 1.1"*
26.8 + 7.4** 4.6 4- 0.6**
22.3tt 26.4t
8.9 _+ I.I 23.3 4- 2.7 8.6 + 2.3**
5.6 0.2 13.2t~
8.3 4- 0.9 25.2 4- 2.6 1.3 4- 0.6
6.2 _+ 0.6 25.2 + 2.3 4.3 +_ 3.0
Mean ]?equency ( 4- SEM)/obsert, ation Non-social activity Explore Scan Dig Immobility Sit Pause Social investigation Attend Investigate Social crouch
41.9 + 2.7 36.2 + 2.9 4.1 4- 0.9
28.5 + 1.4"* 18.3 4- 1.9"* 1.8 + 0.4
20.9 + 1.6"* 9.7 + 1.8"* 0.3 4- 0.2**
28.4tf 35.1tf 16.8tq
0.2 4- 0.1 0.6 + 0.3
2.4 4- I.I 3.5 + 0.7**
5.8 _+ 1.4"* 3.5 +_ 0.5**
22.0t+ 26.8t~"
8.5 4- 0.8 I 1.2 4- 1.1 0.3 4- 0.1
5.7 4- 0.4* 7.7 4- 0.6* 1.3 _4-0.7
5.3 4- 0.5* 5.7 __.0.5** 2.2 +_ 0.5**
13.7t 19.0tt 12.2t
*P < 0.05, **P < 0.01, between drug-treated and control mice by the Mann-Whitney U-test. t t P < 0.01, t P < 0.05, between drug-treated and control groups by the Kruskal-Wallis test.
414
MARGARET G . CUTLER Table 3. Changes to catgories of behaviour induced by clonidine in female mice when encountering male partners Clonidine Group n
Controls 18
0.01 mg/kg 15
0.05 mg/kg 15
Kruskal-Wallis H-test
Non-social activity Immobility Social investigation Aggression Aggressive defence Flight
Behavioural categories Mean duration, sec ( + SEM)/observation 229.1 _+ 9.1 205.7 _+ 13.8 195.0 _+ 18.5 I.I _+ 0.6 24.9 _+ 11.2"* 36,4 _+ 14.6"* 56.3 _+ 4.9 50.7 _+ 5.5 59.3 _+ 6.1 0.2 _+ 0.2 0.4 _+ 0.2 0,1 + 0.1 4.2 _+ 1.6 4.5 _+ 2.2 1.4 + 0.9 12.1 -+ 4.4 14.0 _+ 6.3 8.3 _+ 4.9
8.3 13.4t't 3.8 0.7 1.5 0.9
Non-social activity Immobility Social investigation Aggression Aggressive defence Flight
Mean frequency ( ± SEM)/observation 86.4 _+ 6.0 66.7 _+ 8.4 60.1 _+ 8.1" 0.7 _+ 0.3 3.3 _+ 0.9** 4.6 _+ 0.9** 31.3 _+ 2.1 23.9 + 2.3* 23.9 + 2.4* 0.2 + 0.1 0.1 + 0.1 0.1 -+ 0.1 2.4 -+ 0.9 1.4 _+ 0.7 1.8 _+ 0.5 4.8 _+ 2.0 3.7 _+ 1.4 2.7 _+ 1.4
6.5"t 14.8'I"'I" 5.3"I" 0.5 0.9 1.9
*P < 0.05, **P < 0.01, between drug-treated and control mice by the Mann-Whitney U-test. t t P < 0.01, t P < 0.05, between drug-treated and control groups by the Kruskal-Wallis test.
2 mg/kg, 5.3 _ 0.4; 5 mg/kg, 6.9 + 0.6; P < 0.01). As a result of these contrasting influences, the duration of exploration was slightly increased in the mice given yohimbine at 5 mg/kg and significantly raised in the group given yohimbine at 2 mg/kg. Duration of the act, "wash", although not its frequency, was raised by yohimbine at the higher dose of 5 mg/kg. Significant effects of yohimbine on elements of social investigation, included a dose-related decrease in the frequency of "attend" and "investigate" (Table 2). There were, however, no significant changes to the duration of these elements. The bout length of "investigate" was increased by yohimbine at both dose levels (mean + SEM; controls: 2.3 + 0.2; yohimbine: 2 mg/kg, 3.4_+ 0.3, P < 0.05; 5 mg/kg, 4.2 _ 0.4, P < 0.01), and that of "attend" was raised by yohimbine at 5 mg/kg (mean + SEM; controls: i.0 __.0.1 ; yohimbine: 2 mg/kg, 1.1 ___0.1 ; 5 mg/kg 1.7 ___0.2, P < 0.05).
Behaviour of the female mice treated with clonidine Table 3 shows that clonidine induced a doserelated increase in the frequency and duration of immobility. Although clonidine had no significant effects on the duration of other categories of behaviour, it reduced the frequency of social investigation at 0.01 and 0.05mg/kg and decreased the frequency of non-social activity at 0.05mg/kg. Clonidine did not alter the frequency of aggressive behaviour, flight or aggressive defence. Significant effects of cionidine on the occurrence of individual elements within the behavioural categories are shown in Table 4. Clonidine increased frequency and duration of the element, "sit". Within the category of social investigation, "investigate" was significantly reduced in frequency and duration by clonidine at 0.01 and 0.5 mg/kg. Cionidine had significant effects on two elements of non-social activity.
Table 4. Significant effects of clonidine on the elements of behaviour shown by female mice when encountering male partners Clonidine Group n
Controls 18
0.01 mg/kg 15
0.05 mg/kg 15
Kruskal-Wallis H-test
Oehavioural elements Mean duration, sec ( +_SEM)/observation Non-social activity Explore Suhstrate sniff Immobility Sit Social investigation Investigate
119.7_+6.8 9.4 +_ 2.2
107.9_+6.3 5.0 -+ 1.3
104.9_+9.8 1.5 +_ 1.0"*
2.6 13.8"t't
0.3_+_0.2
22.5-+ I1.1"
35.3_+ 14.7"*
I 1.4"tt
25.2 _+ 2.6
17.9 _+ 2.0*
15.8 _+ 3.7**
I 1.5"i"l"
Mean [requeno" ( ± SEM)/obserration Non-social activity Explore Substrate sniff Immobility Sit Social investigation Investigate
41.9 ± 2.7 2.9 _+ 0.5
32.2 _+ 4.3 1.8 _+ 0.4
30.5 _+ 3.9* 0.7 _+ 0.3**
6.6 18.3tt
0.2 _+ 0.1
2. I _+ 0.8*
3.7 +_ 1.0"*
14.9~t
11.2 ± 1.1
6.6 ± 0.9**
6.6 _+ 1.0"
12.5tt
*P < 0.05, **P < 0.01, between drug-treated and control mice by the Mann-Whitney U-test. t'['P < 0.01, between drug-treated and control groups by the Kruskal-Wallis test.
Yohimbine and clonidine: their effects on behaviour of female mice It reduced the frequency and duration of substrate sniffing at 0.05mg/kg, and decreased frequency although not duration of the act, "explore" at 0.5 mg/kg. Clonidine increased the bout length of "explore" at 0.05 mg/kg (mean + SEM; controls: 3.1 +__0.31; clonidine; 0.01 mg/kg, 3.9 _ 0.41; 0.05 mg/kg, 4.0 +__0.1, P <0.05). Behaviour o f the male partners
There were no significant differences in the duration of non-social behaviour, social investigation or aggression between the groups of partner males. None of the partner males showed flight or aggressive defence during the social encounters. DISCUSSION
The anxiogenic effects of yohimbine in rodents at pharmacological dose levels involve an increase of arousal (Sanger, 1988) coupled with inhibition of locomotor activity (Lapin, 1990; Merlo Pich and Samanin, 1989; Papeschi et al., 1971; Sanghvi and Gershon, 1974). Gray (1987) interpreted these effects as activation of the behavioural inhibition system in the locus coeruleus and dorsal noradrenergic bundle. It appears that the antagonist effects of yohimbine on presynaptic ct2-adrenoceptors in the locus coeruleus reduce negative feedback, increase the firing rate of coeruleus neurons and thus enhance central noradrenergic activity (Goldberg and Robertson, 1983; Starke, Borowski and Endo, 1975). The present ethological study has shown that yohimbine induced dose-related increases in the frequency, duration and bout length of the immobile postures, "sit" and "social crouch" in female mice when engaged in encounters with male partners. Yohimbine also reduced the frequency of social investigation and induced a dose-related decrease in the frequency and duration of non-social activity. There appeared to be a decreased rate of"switching" from one behavioural activity to another since the bout lengths of the behavioural acts, "explore", "scan", "attend" and "investigate" were significantly increased by yohimbine and "pausing" between each of the behavioural activities was enhanced. Interestingly, yohimbine was found to reduce bout lengths of the strenuous activity of sawdust digging. Yohimbine had no significant effects on aggression or flight during the present test, but this may arise from the fact that urine excreted by female mice contains an olfactory principle with aggressioninhibiting properties (Dixon and Mackintosh, 1971), so that females are not subjected to attack from male partners. In male mice, it has been shown that yohimbine inhibits isolation-induced attack (Kemble et al., 1991) and acts to reduce social investigation and increase social distances (Aitken and Cutler, 1990; Kemble et al., 1991). Thus, in male mice, the behavioural actions of yohimbine bear some resem-
415
blance to those of anxiogenic benzodiazepine inverse agonists and partial inverse agonists, which reduce the aggression of dominant animals and, in some tests, decrease social investigation (Beck and Cooper, 1986; Cutler and Aitken, 1991; Piret, Depaulis and Vergnes, 1991; Sulcova, Krsiak and Donat, 1992). Yohimbine appears to differ from the benzodiazepine inverse agonists in its marked enhancement of immobility and lack of significant effect on defensive and submissive behaviour. It is possible that the immobility and reduction of motor activity induced by yohimbine may in part be related to its interaction at other receptor sites. Although the behavioural and biochemical responses to yohimbine are consistent with presynaptic ~t2adrenoceptor blockade, yohimbine does bind with lower affinity to other receptors, including the ~ttadrenoceptors (Doxey, Lane, Roach and Virdee, 1984), serotonin (Dwoskin, Neal and Sparber, 1988) and dopamine D2 (van Oene, de Vries and Horn, 1984) receptors. The experiments of Johnston and File (1989) indicate that yohimbine may be producing its anxiogenic responses by activity on both the noradrenergic and dopaminergic systems. Dopamine is well known to play a vital role in the initiation of movement (Waddington, 1989), and D2 dopamine agonists, such as quinpirole, reduce motor activity and induce immobility of mice when given at small doses (Gao and Cutler, 1993). This most probably arises from activation of autoreceptors in the nigros-" triatal and mesocorticolimbic dopamine systems, so that neuronal firing rate is suppressed. Clonidine, despite its antagonism of the anxiogenic actions of yohimbine (Goldberg and Robertson, 1983), shows a profile of behavioural effects in rodents which differ from those of many anxiolytic agents due to its motor-depressant, sedative, hypotensive and analgesic actions (Drew et al., 1979; Durcan et al., 1985; Goldberg and Robertson, 1983). Its agonist activity at ct2-adrenoceptors results in a decrease of central sympathetic activity (Charney et al., 1983), and synchronization of EEG rhythm that is associated with the sedative action (Parale, Nayar and Kulkarni, 1986). In the present ethopharmacological experiments, clonidine was found to dose-dependently reduce non-social activity and increase "sitting". At the highest dose tested, clonidine reduced the frequency of exploration but increased its bout length, indicating that reduction of motor activity was a major behavioural effect of clonidine in this paradigm. An additional behavioural action of clonidine was to dose-dependently reduce sniffing of the sawdust substrate, which may have arisen from reduced attention to olfactory stimuli in the cage. Furthermore, clonidine reduced the frequency and duration of the specific social act, "investigate", although it did not significantly change the time spent by the animals in overall social investigation. The profile of effects of clonidine in this test differed markedly from those noted previously in female mice
416
MARGARETG. CUTLER
treated with the anxiolytic agents, b u s p i r o n e a n d granisetron, b o t h o f which increased social investigation a n d reduced defensive b e h a v i o u r (Cutler, 1991). In conclusion, the present results indicate t h a t the sedative a n d o t h e r p s y c h o p h a r m a c o l o g i c a l actions o f clonidine m a y complicate its use as a n anxiolytic agent. Such a conclusion supports the findings of several clinical studies ( R e d m o n d , 1982; U h d e et al., 1989). The fact t h a t clonidine impairs a t t e n t i o n a n d p e r f o r m a n c e provides a further limit to its clinical usefulness (Clark, Geffen a n d Geffen, 1986). Y o h i m b i n e , in contrast, provides a useful preclinical model for studying " a l t e r n a t i v e " m o d e s o f anxiogenic action from those o f the benzodiazepine inverse agonists. Acknowledgements--I wish to express my thanks to Mrs V. Graham and Mr B. Leiper for technical assistance. REFERENCES
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Yohimbine and clonidine: their effects on behaviour of female mice behaviour and body temperature in the rat. Eur. J. Pharmac. 15: 318-326. Parale M. P., Nayar U. and Kulkarni S. K. (1986) Modification by tricyclic antidepressants of cortical EEG changes induced by clonidine in conscious rats. Indian J. Physiol. Pharmac. 30: 70-78. Pellow S., Chopin P. and File S. E. (1985) Are the anxiogenic effects of yohimbine mediated by its action at benzodiazepine receptors? Neurosci. Left. 55: 5-9. Piret B., Depaulis A. and Vergnes M. (1991) Opposite effects of agonist and inverse agonist ligands of benzodiazepine receptor on self-defensive and submissive postures. Psychopharmacology 103: 5~61. Redmond D. E. (1982) Does clonidine alter anxiety in humans? Trends Pharmac. Sci. 3: 477~,80. Redmond D. E, and Huang Y. H. (1979) Current concepts II: New evidence for a locus coeruleus-norepinephrine connection with anxiety. Life Sci. 25: 2149-2162. Sanger D. J. (1988) Behavioural effects of the alpha 2adrenoceptor antagonists idazoxan and yohimbine in rats: comparisons with amphetamine. Psychopharmacology 96: 243-249. Sanghvi I. and Gershon S. (1974) Yohimbine: behavioral and biochemical effects in mice. Archs Int. Pharmacodyn. Ther. 210: 108-120. Shearman G. T., Hynes M. and Lal H. (1981) Self-adminis-
417
tration of clondine by the rat. In" Psychopharmacology of Clonidine (Lal H. and Fielding S., Eds), pp. 53-74. Liss, New York. Soderpalm B. (1989) The SHR exhibits less "anxiety" but increased sensitivity to the anticonflict effect of clonidine compared to normotensive controls. Pharmac. Toxic. 65: 381-386. Starke K., Borowski E. and Endo T. 0975) Preferential blockade of presynaptic ~t-adrenoceptors by yohimbine. Eur. J. Pharmac. 34: 385-388. Sulcova A., Krsiak M. and Donat P, (1982) Beta-CCE and FG 7142 increase defensiveness during intraspecies encounters in mice. Psychopharmacology 108: 205-209. Uhde T. W., Stein M. B., Vittone B. J., Siver L. J., Boulenger J. P., Klein E. and MeUman T. A. (1989) Behavioral and physiologic effects of short-term and long-term administration of clonidine in panic disorder. Archs Gen. Psychiat. 46: 170-177. Waddington J. L. (1989) Functional interactions between D-1 and D-2 dopamine receptor systems: their role in the regulation of psychomotor behaviour, putative mechanisms and clinical relevance. J. Psychopharmac. 3: 54--63. Woolverton W. L., Wessinger W. D. and Balster R. L. (1982) Reinforcing properties of clonidine in Rhesus monkeys. Psychopharmacology 77: 17-23.
0028-3908/93$6.00+ 0.00 Copyright © 1993PergamonPress Ltd
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COMPARISON OF THE EFFECTS OF YOHIMBINE A N D C L O N I D I N E ON THE BEHAVIOUR OF FEMALE MICE D U R I N G SOCIAL ENCOUNTERS IN AN " A P P R O A C H - A V O I D A N C E " SITUATION MARGARETG. CUTLER Department of Biological Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 0BA, U.K.
(Accepted 4 February 1993) Summary--Effects of yohimbine (2 and 5mg/kg, i.p.) and clonidine (10 and 50/zg/kg, i.p.) on the behaviour of adult female CDI mice during 5 min encounters in a neutral cage with unfamiliar male partners have been examined by ethological procedures at 30 min after injection. Yohimbine induced dose-related increases in the frequency, bout length and duration of the immobile postures, "sit" and "social crouch", while decreasing the frequency of "explore". "scan", "attend" and "investigate", and increasing their bout lengths in a dose-related manner. These results suggest that yohimbine decreased the rate of switching from one behavioural act to another. Pausing between acts was increased by yohimbine to a similar extent at both of the tested dose levels. The act "wash" was increased in duration by yohimbine, whereas the strenuous activity of "digging" showed a dose-related decrease in frequency, duration and bout length. It is proposed that these effects are induced by the known interactions of yohimbine with receptors for dopamine as well as with ~t2-adrenoceptors. Clonidine reduced motor activity, evident as a dose-related increase in the frequency and duration of "sitting" coupled with decreased frequency and increased bout length of the act, "explore" (significant at 50/~g/kg). Clonidine also dose-dependently reduced the frequency and duration of substrate "sniffing". Clonidine decreased occurrence of the specific social acts, "'attend" and "investigate", as well as reducing frequency although not duration of overall social investigation. These findings have parallels with reported clinical effects of clonidine, such as sedation and impairments of attention, which must limit its clinical usefulness.
Key words--yohimbine, clonidine, female mouse, social behaviour, ct2-adrenoceptors,dopamine receptors.
There has been considerable research into the neurochemical mechanisms associated with stress and its behavioural correlates in humans and experimental animals. The noradrenergic system and especially the locus coeruleus is intimately involved in the manifestations of fear-related behaviour (Hoehn-Saric, 1982). Indeed, electrical stimulation of the locus coeruleus in rodents has been shown to induce a pattern of behavioural effects resembling those produped by the ~t2-noradrenergic antagonist, yohimbine (Charney, Heninger and Redmond, 1983). Effects of yohimbine have been classified as anxiogenie, since it is known, from several studies, to induce fearfulness and feelings of panic in humans (Charney et al., 1983; Mattila, Seppala and Mattila, 1988). In laboratory animals, yohimbine has been found to induce "behavioural agitation" in dogs and sheep (Gershon and Land, 1962) and increased "fearfulness" of monkeys in response to threat (Redmond and Huang, 1979). In rats and mice, yohimbine produces locomotor depression and hypothermia (Papeschi, Sourkes and Youdim, 1971; Sanghvi and Gershon, 1974). It has been found to decrease openarm entries in the elevated plus maze (Johnston and File, 1989) and the number of transitions between light and dark compartments in the light--dark box
(Lapin, 1990). It also decreases exploration activity in the dark compartment of this apparatus (Merlo Pich and Samanin, 1989). In the place conditioning paradigrn, yohimbine was found to produce a conditioned aversion (File, 1986). Effects of yohimbine have been reviewed by Costa and Marino (1991). Early studies indicated that the ~t2-noradrenergic agonist, clonidine, may attenuate human anxiety (Hoehn-Saric, Merchant, Keyser and Smith, 1981). More recently, clonidine was found to be effective in the short-term control of panic disorder (Uhde, Stein, Vittone, Siver, Boulenger, Klein and Mellman, 1989). In rodents, clonidine can reduce the potentiated startle response (Davis, Cedarbaum, Aghajanian and Gendelman, 1977) and increase open-arm entries in the elevated plus-maze (Handley and Mithani, 1984). Clonidine has been found to show reinforcing properties in the conditioned place preference test (Asin and Wirkshafter, 1985) and in tests involving drug self-administration (Davis and Smith, 1977; Shearman, Hynes and Lal, 1981), as well as to be active in conflict tests (Kruse, Dunn, Theurer, Novick and Shearman, 1981; Soderpalm, 1989). Additionally, clonidine has been found to reduce the signs of fear induced in monkeys by electrical stimulation of the locus coeruleus (Redmond and Huang, 1979).
411
412
MARGARETG. CUTLER
However, clonidine also possesses hypothermic, analgesic and dose-dependent sedative actions (Drew, Gower and Marriott, 1979; Durcan, Campbell and Chitkara, 1985; Woolverton, Wessinger and Balster, 1982), and therefore it may influence social behaviour in a manner that differs from that of most anxiolytic agents. In an earlier study, we found that the "timid female paradigm", in which drug-treated female mice in a neutral cage, were confronted with an unfamiliar male, was highly sensitive in detecting anxiolytic effects produced by the novel anxiolytic agents, buspirone and a 5-HT 3 receptor antagonist (Cutler, 1991). In this test situation, characterized by "approach- avoidance conflict", both compounds increased social investigation and reduced flight in drug-treated females. The sensitivity of this paradigm to the effects of anxiogenic compounds has not as yet been examined. Indeed, there have been comparatively few studies of the effects of yohimbine and clonidine on social behaviour in animals. Most of the reported studies have concentrated on the effects of yohimbine. Thus, in the rat social interaction test, Pellow, Chopin and File (1985) found yohimbine to reduce the number and duration of social contacts between animals. Kemble, Behrens, Rawleigh and Gibson (1991) found that yohimbine inhibited isolation-induced attack in mice and increased social distances between the animals. Using ethopharmacological procedures, we found that yohimbine in the resident-intruder paradigm increased immobility in intruder males and decreased several social and non-social acts (Aitken and Cutler, 1990). The present study, using ethological techniques, compares effects of yohimbine with those of clonidine on the behaviour of female mice when encountering unfamiliar male partners.
METHODS
Animals Adult CDI female mice (weighing 40-46g) and male mice (weighing 50---60g) were employed in these studies. The mice had been bred in our laboratory from a stock originally purchased from B. and K. Universal Ltd, Hull. All animals were housed in groups of 5 per cage prior to the experiments and were given an ad libitum supply of pelleted stock cubes (RM3 expanded diet, SDS, Wetham, Essex) and tap water. Mice were maintained at a temperature of 21 + 2°C. A 24 hr cycle of reversed lighting was employed (lights on from 18.00 to 6.00 hr), so that behaviour of the animals should be examined at a time when they were normally most active. Experimental procedures Oestrous female mice were randomly distributed between the five groups shown in Tables 1 and 3. Yohimbine hydrochloride (Sigma) and clonidine
hydrochloride (a gift from Reckitt & Colman Ltd) were dissolved in physiological saline. Yohimbine was given by intraperitoneal (i.p.) injection at 2.0 and 5.0mg/kg, respectively, to mice of two groups (n = 15/group), while clonidine was injected i.p. to mice of a further two groups (n = 15/group) at 10 and 50 pg/kg, respectively. These doses were selected as being within the range known to induce anxiogenic-like or anxiolytic-like behavioural changes in mice and rats (Goldberg and Roberston, 1983; Handley and Mithani, 1984; Johnston and File, 1989; Oluyomi and Hart, 1990). Control mice (n = 18)were given a similar volume of physiological saline (0.1 ml/ 25 g mouse) by i.p. injection. Observations of behaviour shown by each of the mice commenced at 30 min followed injection. Behavioural tests Effects of the treatments on behavioural responsiveness of the females during 5 min social encounters with an unfamiliar group-housed male partner in an illuminated unfamiliar neutral cage (60 x 25 x 25 cm; light intensity 38 cd/m 2) were assessed by ethopharmacological procedures. All behaviourai observations were made during the dark phase of the 24 hr lighting regime, when mice were most active. Acts and posture of behaviour shown by each of the injected mice during the social interactions were recorded as a spoken commentary by tape recorder, using the check list of 49 elements employed in similar previous studies (Cutler, 1991). These are derived from the ethological profile described by Dixon, Fisch and McAllister (1990). Data from the spoken commentaries were transcribed from audiotape onto a floppy disc through keyboard input for analysis by computer of the frequency and duration of each behavioural element and category. The duration of each category of behaviour shown by the partner animals was simultaneously recorded on an electronic machine designed for that purpose, as employed previously (Donald, Cutler and Moore, 1987). As in preceding studies, data have been recorded as the means for each group and probability values for the significance of the differences between control and drug-treated groups determined by the nonparametric Mann-Whitney U-test and the KruskalWallis one-way analysis of variance (ANOVA). Changes having a two-tailed probability of 0.05 or less were deemed to be statistically significant. RESULTS Behaviour of the female mice treated with yohimbine Table l shows that yohimbine induced dose-related increases in the frequency and duration of immobility of the mice and that this was associated with doserelated reductions in the frequency of social investigation and in the frequency and duration of non-social activity. Yohimbine had no significant
Yohimbine and clonidine: their effects on behaviour of female mice
413
Table 1. Changes to catgories of behaviour induced by yohimbine in female mice when encountering male partners Yohimbine Group n
Controls 18
2 mg/kg 15
5 mg/kg 15
Kruskal-Wallis H-test
Non-social activity Immobility Social investigation Aggression Aggressive defence Flight
Behavioural categories Mean duration, sec ( 4- SEM)/observation 229.1 4- 9.1 216.0 4- 6.3 187.0 + 7.6** I.I + 0.6 13.7 4- 4.3** 31.4 4- 7.3** 56.3 4- 4.9 58.2 4- 3.5 66.3 + 4.5 0.2 4- 0.2 0.4 4- 0.3 0.1 + 0.1 4.2 4- 1.6 2.2 4- 0.8 2.3 4- 1.0 10.1 4-4.4 10.24-3.4 13.5+6.0
14.8tt 25.7tt 5.4 0.6 1.0 7.3
Non-social activity Immobility Social investigation Aggression Aggressive defence Flight
Mean frequency ( 4- SEM)/observation 86.4 4- 6.0 55.5 4- 5.4** 34.5 + 3.4** 0.7 4- 0.3 5.7 + 1.3"* 9.2 + 1.3"* 31.3 + 2.1 24.5 4- 1.2" 21.1 + 1.8"* 0.2 4- 0. I 0.2 + 0. I 0.1 4- 0.1 2.4 4- 0.9 1.5 4- 0.6 1.5 + 0.6 4.8 + 2.0 3.9 4- 1.4 4.0 4- 1.4
29.1$t 26.9tt I I.Itt 2.8 0.3 0.3
*P < 0.05, **P < 0.01, between drug-treated and control mice by the Mann-Whitney U-test. ~ ' P < 0.01, between drug-treated and control groups by the KruskaI-Wallis test.
effects on the occurrence of aggression, aggressive defence or flight. The significant effects of yohimbine on the occurrence of individual elements within each of the categories of behaviour can be seen from Table 2. This shows dose-related increases in the frequency and duration of the immobile acts, "sit", "pause" and "social crouch" after yohimbine administration. Yohimbine also significantly increased the bout lengths of "sit" (mean + SEM; controls: 0.3 + 0.2; yohimbine: 2 mg/kg, 1.3 ___0.5; 5 mg/kg, 3.8 ___0.6; P < 0.01) and of "social crouch" (mean + SEM; controls: 1.1 ___0.5; yohimbine: 2 mg/kg, 0.8 ___0.8; 5 mg/kg, 3.0 + 0.5; P < 0.05).
Yohimbine had significant effects on the occurrence of four acts of non-social activity. The duration and frequency of "scan" and "dig" were dose-dependently decreased by yohimbine. However, the bout length of "scan" was increased by yohimbine (mean + SEM; controls: 2.5 + 0.1; yohimbine: 2mg/kg, 3.1+0.2, P<0.05; 5mg/kg, 3.7+0.2, P <0.01), while that of "dig" was reduced (mean + SEM; controls 2.2 + 0.4; yohimbine: 2 mg/kg, 2.2 + 0.5; 5 mg/kg, 0.3 + 0.2; P < 0.01). The frequency of "explore" also was decreased in a dose-dependent manner by yohimbine, yet the bout length of "explore" was dose-dependently raised (mean + SEM; controls: 3.1 +0.3; yohimbine:
Table 2. Significant effects of yohimbine on the elements of behaviour shown by female mice when encountering male partners Yohimbine Group n
ControLs 18
2 mg/kg 15
5 mg/kg 15
KruskaI-Wanis H-test
Behavioural elements Mean duration, set' ( 4- SEM)/obsert, ation Non-social activity Explore Scan Dig Wash Immobility Sit Pause Social investigation Attend Investigate Social crouch
119.74-6.8 86.1 4- 7.3 11.3 + 2.7 2.0 4- 0.7
143.5+_5.3" 53.7 4- 5.1"* 5.6 + 1.7 2.5 4- 0.8
135.24-7.3 34.9 4- 5.6** 0.7 4- 0.5** 7.4 +_ 1.6"
9.5t 29.6tt 16.7tt 7.1t
0.3 4- 0.2 0.8 4- 0.5
8.9 4- 4.3 4.8 4- 1.1"*
26.8 + 7.4** 4.6 4- 0.6**
22.3tt 26.4t
8.9 _+ I.I 23.3 4- 2.7 8.6 + 2.3**
5.6 0.2 13.2t~
8.3 4- 0.9 25.2 4- 2.6 1.3 4- 0.6
6.2 _+ 0.6 25.2 + 2.3 4.3 +_ 3.0
Mean ]?equency ( 4- SEM)/obsert, ation Non-social activity Explore Scan Dig Immobility Sit Pause Social investigation Attend Investigate Social crouch
41.9 + 2.7 36.2 + 2.9 4.1 4- 0.9
28.5 + 1.4"* 18.3 4- 1.9"* 1.8 + 0.4
20.9 + 1.6"* 9.7 + 1.8"* 0.3 4- 0.2**
28.4tf 35.1tf 16.8tq
0.2 4- 0.1 0.6 + 0.3
2.4 4- I.I 3.5 + 0.7**
5.8 _+ 1.4"* 3.5 +_ 0.5**
22.0t+ 26.8t~"
8.5 4- 0.8 I 1.2 4- 1.1 0.3 4- 0.1
5.7 4- 0.4* 7.7 4- 0.6* 1.3 _4-0.7
5.3 4- 0.5* 5.7 __.0.5** 2.2 +_ 0.5**
13.7t 19.0tt 12.2t
*P < 0.05, **P < 0.01, between drug-treated and control mice by the Mann-Whitney U-test. t t P < 0.01, t P < 0.05, between drug-treated and control groups by the Kruskal-Wallis test.
414
MARGARET G . CUTLER Table 3. Changes to catgories of behaviour induced by clonidine in female mice when encountering male partners Clonidine Group n
Controls 18
0.01 mg/kg 15
0.05 mg/kg 15
Kruskal-Wallis H-test
Non-social activity Immobility Social investigation Aggression Aggressive defence Flight
Behavioural categories Mean duration, sec ( + SEM)/observation 229.1 _+ 9.1 205.7 _+ 13.8 195.0 _+ 18.5 I.I _+ 0.6 24.9 _+ 11.2"* 36,4 _+ 14.6"* 56.3 _+ 4.9 50.7 _+ 5.5 59.3 _+ 6.1 0.2 _+ 0.2 0.4 _+ 0.2 0,1 + 0.1 4.2 _+ 1.6 4.5 _+ 2.2 1.4 + 0.9 12.1 -+ 4.4 14.0 _+ 6.3 8.3 _+ 4.9
8.3 13.4t't 3.8 0.7 1.5 0.9
Non-social activity Immobility Social investigation Aggression Aggressive defence Flight
Mean frequency ( ± SEM)/observation 86.4 _+ 6.0 66.7 _+ 8.4 60.1 _+ 8.1" 0.7 _+ 0.3 3.3 _+ 0.9** 4.6 _+ 0.9** 31.3 _+ 2.1 23.9 + 2.3* 23.9 + 2.4* 0.2 + 0.1 0.1 + 0.1 0.1 -+ 0.1 2.4 -+ 0.9 1.4 _+ 0.7 1.8 _+ 0.5 4.8 _+ 2.0 3.7 _+ 1.4 2.7 _+ 1.4
6.5"t 14.8'I"'I" 5.3"I" 0.5 0.9 1.9
*P < 0.05, **P < 0.01, between drug-treated and control mice by the Mann-Whitney U-test. t t P < 0.01, t P < 0.05, between drug-treated and control groups by the Kruskal-Wallis test.
2 mg/kg, 5.3 _ 0.4; 5 mg/kg, 6.9 + 0.6; P < 0.01). As a result of these contrasting influences, the duration of exploration was slightly increased in the mice given yohimbine at 5 mg/kg and significantly raised in the group given yohimbine at 2 mg/kg. Duration of the act, "wash", although not its frequency, was raised by yohimbine at the higher dose of 5 mg/kg. Significant effects of yohimbine on elements of social investigation, included a dose-related decrease in the frequency of "attend" and "investigate" (Table 2). There were, however, no significant changes to the duration of these elements. The bout length of "investigate" was increased by yohimbine at both dose levels (mean + SEM; controls: 2.3 + 0.2; yohimbine: 2 mg/kg, 3.4_+ 0.3, P < 0.05; 5 mg/kg, 4.2 _ 0.4, P < 0.01), and that of "attend" was raised by yohimbine at 5 mg/kg (mean + SEM; controls: i.0 __.0.1 ; yohimbine: 2 mg/kg, 1.1 ___0.1 ; 5 mg/kg 1.7 ___0.2, P < 0.05).
Behaviour of the female mice treated with clonidine Table 3 shows that clonidine induced a doserelated increase in the frequency and duration of immobility. Although clonidine had no significant effects on the duration of other categories of behaviour, it reduced the frequency of social investigation at 0.01 and 0.05mg/kg and decreased the frequency of non-social activity at 0.05mg/kg. Clonidine did not alter the frequency of aggressive behaviour, flight or aggressive defence. Significant effects of cionidine on the occurrence of individual elements within the behavioural categories are shown in Table 4. Clonidine increased frequency and duration of the element, "sit". Within the category of social investigation, "investigate" was significantly reduced in frequency and duration by clonidine at 0.01 and 0.5 mg/kg. Cionidine had significant effects on two elements of non-social activity.
Table 4. Significant effects of clonidine on the elements of behaviour shown by female mice when encountering male partners Clonidine Group n
Controls 18
0.01 mg/kg 15
0.05 mg/kg 15
Kruskal-Wallis H-test
Oehavioural elements Mean duration, sec ( +_SEM)/observation Non-social activity Explore Suhstrate sniff Immobility Sit Social investigation Investigate
119.7_+6.8 9.4 +_ 2.2
107.9_+6.3 5.0 -+ 1.3
104.9_+9.8 1.5 +_ 1.0"*
2.6 13.8"t't
0.3_+_0.2
22.5-+ I1.1"
35.3_+ 14.7"*
I 1.4"tt
25.2 _+ 2.6
17.9 _+ 2.0*
15.8 _+ 3.7**
I 1.5"i"l"
Mean [requeno" ( ± SEM)/obserration Non-social activity Explore Substrate sniff Immobility Sit Social investigation Investigate
41.9 ± 2.7 2.9 _+ 0.5
32.2 _+ 4.3 1.8 _+ 0.4
30.5 _+ 3.9* 0.7 _+ 0.3**
6.6 18.3tt
0.2 _+ 0.1
2. I _+ 0.8*
3.7 +_ 1.0"*
14.9~t
11.2 ± 1.1
6.6 ± 0.9**
6.6 _+ 1.0"
12.5tt
*P < 0.05, **P < 0.01, between drug-treated and control mice by the Mann-Whitney U-test. t'['P < 0.01, between drug-treated and control groups by the Kruskal-Wallis test.
Yohimbine and clonidine: their effects on behaviour of female mice It reduced the frequency and duration of substrate sniffing at 0.05mg/kg, and decreased frequency although not duration of the act, "explore" at 0.5 mg/kg. Clonidine increased the bout length of "explore" at 0.05 mg/kg (mean + SEM; controls: 3.1 +__0.31; clonidine; 0.01 mg/kg, 3.9 _ 0.41; 0.05 mg/kg, 4.0 +__0.1, P <0.05). Behaviour o f the male partners
There were no significant differences in the duration of non-social behaviour, social investigation or aggression between the groups of partner males. None of the partner males showed flight or aggressive defence during the social encounters. DISCUSSION
The anxiogenic effects of yohimbine in rodents at pharmacological dose levels involve an increase of arousal (Sanger, 1988) coupled with inhibition of locomotor activity (Lapin, 1990; Merlo Pich and Samanin, 1989; Papeschi et al., 1971; Sanghvi and Gershon, 1974). Gray (1987) interpreted these effects as activation of the behavioural inhibition system in the locus coeruleus and dorsal noradrenergic bundle. It appears that the antagonist effects of yohimbine on presynaptic ct2-adrenoceptors in the locus coeruleus reduce negative feedback, increase the firing rate of coeruleus neurons and thus enhance central noradrenergic activity (Goldberg and Robertson, 1983; Starke, Borowski and Endo, 1975). The present ethological study has shown that yohimbine induced dose-related increases in the frequency, duration and bout length of the immobile postures, "sit" and "social crouch" in female mice when engaged in encounters with male partners. Yohimbine also reduced the frequency of social investigation and induced a dose-related decrease in the frequency and duration of non-social activity. There appeared to be a decreased rate of"switching" from one behavioural activity to another since the bout lengths of the behavioural acts, "explore", "scan", "attend" and "investigate" were significantly increased by yohimbine and "pausing" between each of the behavioural activities was enhanced. Interestingly, yohimbine was found to reduce bout lengths of the strenuous activity of sawdust digging. Yohimbine had no significant effects on aggression or flight during the present test, but this may arise from the fact that urine excreted by female mice contains an olfactory principle with aggressioninhibiting properties (Dixon and Mackintosh, 1971), so that females are not subjected to attack from male partners. In male mice, it has been shown that yohimbine inhibits isolation-induced attack (Kemble et al., 1991) and acts to reduce social investigation and increase social distances (Aitken and Cutler, 1990; Kemble et al., 1991). Thus, in male mice, the behavioural actions of yohimbine bear some resem-
415
blance to those of anxiogenic benzodiazepine inverse agonists and partial inverse agonists, which reduce the aggression of dominant animals and, in some tests, decrease social investigation (Beck and Cooper, 1986; Cutler and Aitken, 1991; Piret, Depaulis and Vergnes, 1991; Sulcova, Krsiak and Donat, 1992). Yohimbine appears to differ from the benzodiazepine inverse agonists in its marked enhancement of immobility and lack of significant effect on defensive and submissive behaviour. It is possible that the immobility and reduction of motor activity induced by yohimbine may in part be related to its interaction at other receptor sites. Although the behavioural and biochemical responses to yohimbine are consistent with presynaptic ~t2adrenoceptor blockade, yohimbine does bind with lower affinity to other receptors, including the ~ttadrenoceptors (Doxey, Lane, Roach and Virdee, 1984), serotonin (Dwoskin, Neal and Sparber, 1988) and dopamine D2 (van Oene, de Vries and Horn, 1984) receptors. The experiments of Johnston and File (1989) indicate that yohimbine may be producing its anxiogenic responses by activity on both the noradrenergic and dopaminergic systems. Dopamine is well known to play a vital role in the initiation of movement (Waddington, 1989), and D2 dopamine agonists, such as quinpirole, reduce motor activity and induce immobility of mice when given at small doses (Gao and Cutler, 1993). This most probably arises from activation of autoreceptors in the nigros-" triatal and mesocorticolimbic dopamine systems, so that neuronal firing rate is suppressed. Clonidine, despite its antagonism of the anxiogenic actions of yohimbine (Goldberg and Robertson, 1983), shows a profile of behavioural effects in rodents which differ from those of many anxiolytic agents due to its motor-depressant, sedative, hypotensive and analgesic actions (Drew et al., 1979; Durcan et al., 1985; Goldberg and Robertson, 1983). Its agonist activity at ct2-adrenoceptors results in a decrease of central sympathetic activity (Charney et al., 1983), and synchronization of EEG rhythm that is associated with the sedative action (Parale, Nayar and Kulkarni, 1986). In the present ethopharmacological experiments, clonidine was found to dose-dependently reduce non-social activity and increase "sitting". At the highest dose tested, clonidine reduced the frequency of exploration but increased its bout length, indicating that reduction of motor activity was a major behavioural effect of clonidine in this paradigm. An additional behavioural action of clonidine was to dose-dependently reduce sniffing of the sawdust substrate, which may have arisen from reduced attention to olfactory stimuli in the cage. Furthermore, clonidine reduced the frequency and duration of the specific social act, "investigate", although it did not significantly change the time spent by the animals in overall social investigation. The profile of effects of clonidine in this test differed markedly from those noted previously in female mice
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MARGARETG. CUTLER
treated with the anxiolytic agents, b u s p i r o n e a n d granisetron, b o t h o f which increased social investigation a n d reduced defensive b e h a v i o u r (Cutler, 1991). In conclusion, the present results indicate t h a t the sedative a n d o t h e r p s y c h o p h a r m a c o l o g i c a l actions o f clonidine m a y complicate its use as a n anxiolytic agent. Such a conclusion supports the findings of several clinical studies ( R e d m o n d , 1982; U h d e et al., 1989). The fact t h a t clonidine impairs a t t e n t i o n a n d p e r f o r m a n c e provides a further limit to its clinical usefulness (Clark, Geffen a n d Geffen, 1986). Y o h i m b i n e , in contrast, provides a useful preclinical model for studying " a l t e r n a t i v e " m o d e s o f anxiogenic action from those o f the benzodiazepine inverse agonists. Acknowledgements--I wish to express my thanks to Mrs V. Graham and Mr B. Leiper for technical assistance. REFERENCES
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