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Pharmacological evidence for a role of D2 dopamine receptors in the defensive behavior of the mouse
BEHAVIORAL AND NEURAL BIOLOGY 5 0 , 98--111
(1988)
Pharmacological Evidence for a Role of D2 Dopamine Receptors in the Defensive Behavior of the Mouse STEFANO PUGLISI-ALLEGRA AND SIMONA CABIB 1
Istituto di Psicobiologia e Psicofarmacologia (C.N.R.), via Reno 1, 00198 Roma, Italy In this study the role of the DA system in the expression of defensive behavior of the mouse was investigated. C57BL/6 mice subjected to three daily defeat experiences (24 h apart) exhibited an increase of defensive behaviors (upright and sideways postures and escape) as well as a decrease of activity and a decrease of social investigation compared with undefeated mice (controls) when confronted with nonaggressive Swiss mice 24 h after the last aggressive confrontation. The selective D2 DA receptor antagonist (-)-sulpiride administered before confrontation with nonaggressive opponents (fourth day) dramatically decreased defensive behaviors and produced an increase of social investigation. The selective D1 DA receptor antagonist SCH 23390 did not affect either defence or social investigation. In further experiments the behavioral effects of the selective D1 agonist SKF 38393 and of the selective D2 agonist LY171555 on naive C57BL/6 mice interacting with nonaggressive opponents of the same strain were assessed. SKF 38393 in doses up to 30 mg/kg did not produce any significant behavioral changes while LY171555 produced a clear-cut dose-dependent increase of defensive behavior as well as a decrease of social investigation and activity and an increase of immobility. The behavioral profile produced by the D2 agonist did not differ from that produced by defeat experiences. These results indicate that D2 receptors play a major role in the expression of defensive behavior in the mouse. The hypothesis that alteration in D2 receptor functioning may produce hyperdefensiveness possibly due to altered perceptive processes is discussed. © 1988Academic Press, Inc.
Animals exposed to an aggressive conspecific exhibit different defensive behaviors such as escape and submissive postures. The attack-experienced animals display defensive behaviors also in subsequent nonaggressive confrontations, thus showing that defensive behavioral patterns may be learned and included in the individual behavioral repertoire (Frischknecht, Siegfried, & Waser, 1982; Roche & Leshner, 1979; Siegfried, Frischknecht & Waser, 1982; Siegfried, Frischknecht & Waser, 1984a; Scott, 1946). t The authors thank the Eli Lilly & Co., The Schering Corporation, and Ravizza for supplying with LY171555, SCH 23390, and (-)-sulpiride. Thanks are also given to Mr. Mario Battaglia for the preparation of figures. Correspondence should be addressed to S. Puglisi-Allegra at Istituto di Psicobiologia e Psicofarmacologia, via Reno 1, 00198 Roma, Italy. 98 0163-1047/88 $3.00 Copyright © 1988 by Academic Press, Inc. All fights of reproduction in any form reserved.
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Moreover, a number of studies have shown that the attacked animal may show a decrease of pain sensitivity (analgesia) (Miczek & Thompson, 1984; Miczek, Thompson, & Shuster, 1982; Siegfried, Frischknecht & Waser, 1984b; Rodgers & Hendrie, 1983). Thus, the effects of being attacked and defeated have been related to the activaction of the endogenous opioid system produced by this stressful experience (defeat) (Miczek & Thompson, 1984; Miczek, Thompson, & Shuster, 1985; Miczek & Winslow, 1987; Siegfried et al., 1984b). Evidence exists that stressful (adversive unescapable) experiences affect brain dopamine (DA) systems in the mouse and that such effects may be modulated by endogenous opioids (Cabib, Puglisi-Allegra & Oliverio, 1984; Cabib, Puglisi-Allegra, & Oliverio, 1985; Cabib, Kempf, Schleef, Oliverio, & Puglisi-Allegra, 1988; Nabeshima, Katoh, Hiramatsu, & Kameyama, 1986). Some studies have also indicated that the DA system is involved in the expression of defensive and submissive behaviors of laboratory animals interacting with aggressive and nonaggressive conspecifics. Upchurch and Schallert (1982) showed that gerbils which were more sensitive to the cataleptic and akinetic effects of haloperidol exhibit more subordinate behavior than the haloperidol-resistant animals in nondrugged tests. Rats interacting with an aggressive male conspecific exhibit the greatest increase of DA activity in nucleus accumbens when compared with animals interacting with a nonaggressive male or with a female conspecific (Louilot, Le Moal, & Simon, 1986). Although these effects on nucleus accumbens may be a consequence of motor changes produced by different social situations, these results point to an involvement of the mesolimbic DA system in the effects of defeat experience. Moreover, both attack and social stress have been shown to increase dihydroxyphenylacetic acid and homovanillic acid in rat olfactory tubercle (Mos and Van Valkenburg, 1979). Psychostimulants, d-amphetamine and cocaine, were shown to decrease dose-dependently social behavior and dominancerelated behaviors in squirrel monkeys (Miczek & Gold, 1983; Miczek & Yoshimura, 1982) and to induce defensive behavior in mice confronted with isolated resident consepecifics (Miczek & O'Donnell, 1978). In the present study we investigated the role of the DA system in the expression of defensive behavior exhibited during a nonaggressive confrontation by mice previously exposed to three daily aggressive encounters. To this end defeated mice were treated before nonaggressive confrontation with selective D1 and D2 antagonists SCH 23390 and (-)-sulpiride (Stoof & Kebabian, 1984). In a further experiment the behavior of naive mice injected with the selective D2 agonist LY171555 and the selective D1 agonist SKF 38393 before confrontation with nonaggressive opponents was assessed. C57BL/6 mice were used in this study since they were shown to be characterized by reliable defeat responses (Siegfried et al., 1984b).
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EXPERIMENT 1 Methods
Male C57BL/6 (C57) (Charles River Labs., Calco, Italy) and SwissWebster (Swiss) (Plaisant Labs., Rome, Italy) mice were used. Upon arrival C57 mice were housed in groups of eight in standard breeding cages (27 x 21x 13.5 cm). Swiss mice were housed either with female conspecifics (aggressive opponents) or in groups of 15 (nonaggressive opponents). Four to five weeks after being housed with a female, each resident mouse was tested for attack behavior by introducing a grouphoused mouse into its home cage. During this and all subsequent tests, the female and pups were removed from the cage before introducing the intruder into the resident's cage. The mice were kept in a 12-h light/12-h dark cycle, at a temperature of 22 ___ I°C, given water and food ad lib, and tested during the second half of the light period in a sound-insulated room lighted by a 60 W lamp placed 2.4 m above the floor of the room. Test mice were exposed to three daily aggressive confrontations 24 h apart and subjected to nonaggressive confrontation (testing) 24 h after the last aggressive encounter (Kulling, Frischknecht, Pasi, Waser, & Siegfried, 1987; Siegfried, Frischknecht, Kulling, & Waser, 1986). Aggressive confrontations were carried out by introducing a C57 mouse to a Swiss mouse housed with a female. Only Swiss mice which consistently showed aggression on each testing day were used. The test animal (C57) was attacked immediately after being placed in the resident's cage. Following 30 s of attack, during which it received 50 + 5 bites, the intruder was gently removed from the resident's cage. Control animals were paired with a nonaggressive (group-housed) Swiss mouse habituated for 10 min to the text box (transparent breeding cage with fresh hardwood sawdust on the floor) during three daily 3-min sessions. On Day 4 the C57 mouse was introduced into the test box and confronted with a nonaggressive (group-housed) Swiss mouse during a 2-min testing session. The duration (s) of the following behavioral items (Cutler, Mackintosh, & Chance, 1975; Grant and Mackintosh, 1963; Mele, Cabib, Oliverio, Melchiorri, & Puglisi-Allegra, 1987; Puglisi-Allegra, Oliverio, & Mandel, 1982) shown by the test animal were recorded: upright and sideways postures, escape and crouch, social investigation (sniff-body, sniff-nose, and following), activity (horizontal and vertical), and immobility. Moreover, the behavioral items upright and sideways postures, escape and crouch were summarized as defence category. In an additional recording, the behaviors of opponents interacting with test animals was also observed. Each test was videotaped and, later, two experienced observers unaware
D2 DOPAMINE RECEPTORS AND DEFENSIVE BEHAVIOR
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TABLE 1 Mean (___SE) Duration (s) of Behavioral Categories and Defensive Items Observed in Mice during a Nonaggressive Confrontation 24 h after Repeated (1 × 3 Days) Aggressive (Defeated) or Nonaggressive (Undefeated) Confrontations Undefeated mice Category Activity Immobility Defence Soc. inv. Def. item Sideways Crouch Escape Upright
Defeated mice
71.6 16.6 5.5 27.3
_+ 8.4 -+ 4.9 _+ 2.1 - 8.3
42.0 31.6 37.2 6.9
+- 7.1a _+ 7.0 _+ 4.0c - 4.1b
1.4 0.6 0.6 2.4
--- 0.6 -+ 0.6 --- 0.2 +- 1.8
11.8 2.5 5.6 17.3
--- 2.0~ -+ 0.7 -+ 1.1d -4- 5.1°
a p < .05; bp < .02; ~p < .01; dp< .005; ~p < .001 in comparison with undefeated mice (t test).
of the treatment condition of each animal independently recorded behavioral items using a k e y b o a r d system connected to an Apple computer. The interobserver reliability of the recorded items was determined on the basis of recordings of social interactions observed in 40 mice during 5 min testing sessions. The r values ranged from 0.74 to 0.95. Test (defeated) mice from different breeding cages were randomly assigned to the following drug treatment: ( - ) - s u l p i r i d e (12.5, 25, and 50 mg/kg), S C H 23390 (0.03, 0.1, and 0.25 mg/kg) and their vehicles. Each experimental group included 6-12 animals. S C H 23390 ((R)-( + )-8-chloro2,3,4,5-tetrahydro-3-methyl-5-phenyl- 1H-3-benzazepine-7-ol maleate; Schering Corp.) was dissolved in distilled water (H20). ( - ) - S u l p i r i d e (Ravizza, Italy) was dissolved in HC1 and diluted in H20, and the pH was adjusted to 7.4 with N a O H . All drugs were injected intraperitoneally (ip) in a volume of 10 m l / k g 20 min before testing. The test doses were chosen on the basis of preliminary experiments. Results were analyzed statistically by one-way analysis of variance (ANOVA). Further analyses for individual between-group comparisons were carried out with post hoc tests (Duncan multiple range test). Student's t test (two-tailed) was also used.
Results and Discussion When c o m p a r e d with undefeated mice (controls), C57 mice subjected to repeated defeat experiences exhibited a decrease of activity, a decrease of social investigation, and an increase of defence when confronted with nonaggressive mice (Table 1). A trend toward increased immobility was
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TABLE 2 Effects of Different Doses of ( -)-Sulpiride (Sulp mg/kg) on Mean (-+ SE) Duration (s) of Behavioral Categories and Defensive Items Observed in Defeated Mice Interacting with a Nonaggressive Opponent Vehicle
Sulp 12
Category Activity Immobility Soc. inv. Defence
47.0 24.6 1.3 40.7
_+ _ + -
7.1 6.9 0.8 3.0
41.0 27.7 10.5 28.7
-+ 4.1 _+ 6.7 +_ 6.1 _+ 8.5
Def. item Sideways Crouch Escape Upright
12.3 4.8 4.8 18.8
_+ _+ _+ _+
2.5 1.4 1.0 4.5
6.3 3.5 2.2 16.5
-+ _+ + -4-
p < .05;
bp <
2.4 a 1.5 0.7 b 7.9
Sulp 25
Sulp 50
57.0 24.6 15.5 6.5
+ _+ _+ _+
9.7 4.7 3.& 5.5 b
27.3 54.7 28.0 20.8
_+ -------
2.6 12.6 a 5.6 b 5.7 ~
2.1 0.3 0.8 3.1
_+ --+ -
1.4 b 0.3 0.6 b 1.9 b
2.0 12.6 0.4 5.8
--_+ _+ -+
0.8 b 4.46 0.2 b 1.3 a
.01, in comparison with vehicle-treated mice (Duncan test).
also found. All the items in the defensive category increased significantly, except crouch (Table 1). ANOVA showed a significant effect of (-)-sulpiride on defence (F(3, 20) = 5.62, p < .01) and on social investigation (F(3, 20) = 9.17, p < .002). Individual between-group comparisons showed dose-related decrease of defence and increase of social investigation (Table 2). ANOVA also revealed a significant effect on immobility (F(3, 20) = 3.25, p < .05) and post hoc test showed that only the 50 mg/kg dose increased this behavior (Table 2) while no significant effects on activity were revealed, although a tendency to a decrease of this behavior appeared at 50 mg/kg of (-)-sulpiride (Table 2). As regards defensive items the D2 antagonist affected upright (F(3, 20) = 5.01, p < .02) and sideways (F(3, 20) = 5.99, p < .01) postures, escape (F(3, 20) = 6.68, p < .005), and also crouch (F(3, 20) = 5.64, p < .01). As shown by individual betweengroup comparisons (-)-sulpiride dose-dependently antagonized upright and sideways postures as well as escape (Table 2). Although crouch decreased without statistical significance at the doses of 12.5 and 25 mg/kg, a significant increase at a dose of 50 mg/kg was evident (Table 2). It is worth noting that crouch was not significantly affected by defeat in our experimental conditions. SCH 23390 did not produce any significant effect either on defence or on social investigation while ANOVA revealed significant effects on activity (F(3, 20) = 4.27, p < .02) and immobility (F(3, 20) = 6.69, p < .005). Individual between-group comparisons showed that the doses of 0.1 and 0.25 mg/kg produced a significant decrease of activity and a increase of immobility while the dose of 0.03 mg/kg was not effective
D2 D O P A M I N E R E C E P T O R S A N D D E F E N S I V E B E H A V I O R
103
TABLE 3 Effects of Different Doses of SCH 23390 (SCH mg/kg) on Mean ( ± SE) Duration (s) of Behavioral Categories and Defensive Items Observed in Defeated Mice Interacting with a Nonaggressive O p p o n e n t Vehicle
SCH 0.03
SCH 0.1
SCH 0.25
Category Activity Immobility Soc. inv. Defence
42.1 23.0 1.3 39.1
± ++
7.0 5.9 0.8 4.3
43.5 30.5 4.1 38.6
--± -+ -
7.8 3.1 1.9 9.6
24.3 41.7 1.4 52.7
+ 3.3 ° --- 8.6" ± 1.1 ± 15.0
17.1 59.0 1.6 41.6
± --± +
5.1" 6.0 b 0.8 11.7
Def. item Sideways Crouch Escape Upright
10.0 4.8 7.3 17.0
--- 3.3 -+-+ 1.4 --- 0.6 ± 2.6
13.3 1.5 8.8 15.8
--± -+ --+
3.0 1.3 2.9 4.1
10.1 20.2 2.4 20.0
--- 4.9 ± 4.C --- 1.0 +- 6.0
6.1 18.0 0.3 17.2
--± --±
2.5 6.9 ° 0.2" 3.9
" p < .05; b p < .01 in comparison with vehicle-treated mice (Duncan test).
on both behaviors (Table 3). When single defensive items are considered SCH 23390 did not significantly affect either upright or sideways postures while significant effects were evident on escape (F(3, 20) = 6.02, p < .005) and crouch (F(3, 20) = 6.28, p < .005). In particular while only the dose of 0.25 mg/kg decreased escape significantly, both 0.1 and 0.25 mg/kg increased crouch (Table 3). To summarize, defensive behavior exhibited by defeated mice interacting with nonaggressive opponents was dose-dependently reduced by the D2 selective antagonist (-)-sulpiride but unaffected by the D1 antagonist SCH 23390 at the doses used in these experiments. Also social investigation was unaffected by SCH while a dose-dependent increase of this behavior was evident in (-)-sulpiride-treated mice. As far as the behavioral items in the defence category are concerned, sideways and upright postures decreased in (-)-sulpiride-treated mice but not in SCH 23390-treated mice while escape was reduced by all doses of (-)-sulpiride and by only the highest dose of SCH 23390. Crouch was the only item to be increased both by the high dose of ( - )-sulpiride and by 0.1 and 0.25 mg/kg of SCH 23390. It is worth noting that crouch is not significantly affected by previous defeat in our experimental conditions (Table 1). At the same time, modulation of crouch by the two DA antagonists seems to be strictly related to modulation of overall activity. In fact, at the dose of 50 mg/kg of (-)-sulpiride (the only dose that affects crouch) a nonsignificant decrease of activity and a significant increase of immobility were evident while 0.1 and 0.25 mg/kg of SCH 23390 both significantly reduced activity and increased immobility. Moreover, as we observed in preliminary experiments, mice tested individually
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in an activity meter (for the method see Cabib & Puglisi-Allegra, 1985) following administration of ( -)-sulpiride and SCH 23390 showed a dramatic decrease of locomotor activity ((-)-sulpiride 50 mg/kg = - 6 0 % ; SCH 23390 0.1 mg/kg = - 6 5 % and 0.25 mg/kg = - 8 7 % compared with vehicle injected animals). Since akinetic animals have been shown to be treated by normal animals as subordinates (Thiessen and Upchurch, 1981), the behavior of the opponent interacting with an akinetic mouse might therefore increase crouch in testing mice. Further analysis possibly involving also ultrasound recordings could elucidate this point. It is worth noting that no overt offensive behavior was observed either in test mice or in opponents throughout the experiments. These results suggest that defensive behavior exhibited by defeatexperienced mice when confronted with nonaggressive opponents is mediated through the D2 receptor. In order to check this hypothesis we investigated the behavioral effects of the selective D2 agonist LY171555 (Itoh, Goldman & Kebabian, 1985) and of the selective D1 agonist SKF 38393 (Setler, Sarau, Zirkle, & Saunders, 1978) on naive C57 mice interacting with a nonaggressive opponent of the same strain. EXPERIMENT 2
Methods Group-housed male C57 mice aged 11-12 weeks and weighing 23-24 g were used in this experiment. Each test mouse was placed in the testing box (see Expt 1) 5 min before a C57 mouse from a different breeding cage was introduced into the box. Resident and intruder mice did not differ by more than 1 g in weight. Each animal was tested only once. Behavioral recordings were carried out as described in Expt 1 during a 5-rain testing session. No attack on the intruder or the resident was observed in these experimental conditions. Test mice were randomly assigned to different drug treatment (vehicles; SKF 38393 3, 10, 20, 30 mg/kg; LY171555 0.1, 0.5, 2, 5 mg/kg). Each experimental group comprised six animals. SKF 38393 (l-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diolhydrochloride; Smith, Kline & French Laboratories) and LY171555 (trans(-)-4aR, 4a, 5, 6, 7, 8, 8a, 9-octahydro-5-propyl-lH(or 2H)-pyrazolo (3,4,-g) quinoline monohydrochloride; Eli Lilly & Co.) were dissolved in distilled water and injected subcutaneously (sc) 25 min before testing. Results were analyzed statistically as described in Expt 1. Results and Discussion Since the test used in the second experiment was different from that used in the first one, the percentage time of behavioral categories exhibited by naive, untreated mice in the two tests was compared (Fig. 1). t test
D2 D O P A M I N E R E C E P T O R S A N D D E F E N S I V E B E H A V I O R TEST I
[] ACTIVITY [] IMMOBILITY
105
TEST 2
"
:
/
,
/
Fir. I. T i m e spent in behavioral categories by undefeated C57 mice interacting with a nonaggressive o p p o n e n t in the two testing conditions used in this study. Results are e x p r e s s e d in percentage ( m e a n values of 12 mice per group) of recorded behaviors. Note: activity = 57 ± 7.3 (Test 1), 63 ± 5.3 (Test 2); immobility = 15 ± 4.1 (Test 1), 20 --+3.4 (Test 2); defence = 3 ± 1.! (Test 1), 5 - 1.5 (Test 2); social investigation = 23 ± 6.5 (Test 1), 13 ± 3.8 (Test 2). N o statistical difference was evident b e t w e e n testing conditions (t test).
did not show any significant difference between the two tests, namely social interaction with a nonaggressive Swiss or C57 opponent. SKF 38393 did not produce any significant effect on any of the behavioral categories observed in this test situation or on single defensive items (Table 4). The results obtained with LY171555 are presented in Fig. 2. ANOVA TABLE 4 Effects o f Different D o s e s of S K F 38393 ( S K F m g / k g ) on M e a n ( ± SE) Duration (s) o f Behavioral Categories and Defensive Items O b s e r v e d in Naive Mice Interacting with a Nonaggressive Opponent Vehicle Category Activity Immobility Soc. inv. Defensive Def. item Sideways Crouch Escape Upright
143.7 80.5 38.8 10.4 2.4 3.0 2.4 2.7
SKF 3
___ 25.8 --- 20.4 ± 10.7 ± 3.7 ± ± ± ±
0.8 1.5 0.7 1.2
145.0 65.1 34.2 6.8 2.5 1.3 2.0 1.0
_+ --± ± ± ± ±
S K F 10
17.6 133.7 ± 17.6 89.2 ± 6.2 32.2 _+ 2.7 6.5 _+ 0.6 0.8 0.8 0.3
1.5 1.5 2.0 1.5
_ ± _ _+_
S K F 20
20.9 128.0 ± 21.9 82.8 8.5 25.1 ± 1.9 5.0 --0.8 0.3 0.5 0.5
1.6 1.1 1.1 2.2
S K F 30
15.1 125.0 _+ 17.7 19.8 87.0 ± 7.7 7.0 20.7 _ 6.9 2.7 8.1 -+ 1.3
± 0.6 --- 0.6 ± 0.6 ± 1.2
2.7 1.5 2.5 2.0
± ± ±
2.1 0.5 1.0 0.5
Note. A N O V A s h o w e d no significant effects for a n y behavioral category and defensive item.
106
P U G L I S I - A L L E G R A A N D CABIB m RCTIVITY r ~ IMMOBILITY gr/A DEFENCE k ~ BOC. INV. 200
*
150
*
100
*
g m
50
,
o VEHICLE
0. I
0.5 LTITIBS5 {mg/Kg)
2
B
Fro. 2. Effects of LY 171555 on mean ( - SE) duration (s) of behavioral categories exhibited by naive mice interacting with a nonaggressive opponent. *p < .01 in comparison with vehicle-treated mice (Duncan test).
showed a significant decrease of activity (F(4, 25) = 11.04, p < .001), a significant increase of immobility (F(4, 25) = 8.13, P < .001), a significant increase of defence (F(4, 25) = 22.72, p < .001), and a significant decrease of social investigation (F(4, 25) = 43.46, p < .001). The Duncan test indicated that the dose of 0.1 mg/kg of LY171555 was already effective in reducing activity and in increasing immobility but still ineffective in altering defensive and social behavior. The 5 mg/kg dose produced the greatest effects on defence and social investigation. The results related to defensive behaviors are presented in Table 5. ANOVA showed a significant effect of LY171555 in increasing sideways (F(4, 25) = 18.9, p < .001) and upright postures (/7(4, 25) = 20.61, p < .001) and escape (F(4, 25) = 13.46, p < .001) but no apparent effect on crouch. The Duncan test revealed that the 0.1 mg/kg dose of LY171555 was effective only in increasing sideways postures. It is worth noting that neither stereotypic nor defensive behavior was observed in animals treated with LY171555 during the 5-min pretest TABLE 5 Effects of LY171555 (LY mg/kg) on Mean ( ± S E ) Duration (s) of Defensive Behavioral Items Observed in Naive Mice Interacting with a Nonaggressive Opponent Def. item Sideways Crouch Escape Upright
Vehicle 1.3 1.1 1.1 4.0
_ _+ ±
0.4 0.6 0.6 1.3
LY 0.1 19.8 9.0 6.7 6.1
± ± ±
2.9" 3.9 2.9 1.7
LY 0.5 24.6 3.5 26.3 21.6
± ± ± ±
2.3 h 1.7 4.9" 1.9"
LY 2 22.6 7.3 40.0 34.8
± 5.1 ~ - 2.9 ± 10.1b ± 6.5"
LY 5 62.5 5.1 33.1 101.0
± -+_+ ±
a p < .05 and b p < .01 in comparison with vehicle-treated mice (Duncan test).
9.5 b 1.9 4.W 14.1b
D2 DOPAMINE RECEPTORS AND DEFENSIVE BEHAVIOR
107
habituation period, the only difference with control animals being a depression of spontaneous activity. Furthermore, no overt offensive behavior was observed either in test mice or in opponents of control and drug-treated groups. As shown by these results, the effects of LY171555 on activity, immobility, and defence seem unrelated to each other. In fact, the 2 mg/kg dose did not reduce activity compared with vehicle, while defensive behavior was significantly increased by this same dose. At the dose of 5 mg/kg defence reached its greatest values, activity was lower than in control animals, while immobility was unchanged. Finally, the dosedependent increase of defensive behavior over the range of doses commonly considered to affect postsynaptic receptors (Braun & Chase, 1986; Koller, Herbster, Anderson, Wack, & Gordon, 1987) suggests their involvement in this behavioral effect of LY171555. The profile of the effects of LY171555 on defensive behaviors, social investigation, activity, and immobility of C57 mice interacting with a nonaggressive conspecific does not differ from that of the effects of defeat experiences on the behavior of animals confronted with nonaggressive opponents as shown in the Expt 1. Although animals pretreated with LY171555 did not experience defeat in the way the animals tested in Expt 1 did, they exhibited defensive upright and sideways postures which paralleled those exhibited by defeatexperienced mice. The submissive behavior of these mice suggests that they perceive the opponent as a threatening stimulus toward which they exhibit a behavioral pattern capable of inhibiting aggression. Moreover the high escape exhibited by the defeat-experienced animals and those treated with LY171555 shows that mice in both experimental conditions avoid contact with the opponent. This behavioral output may depend on a fear-motivated response possibly produced by social experience (defeat) in one case and by pharmacological treatment in the other. Thus the D2 agonist seems to affect the same neural mechanisms as those involved in the expression of behavioral patterns exhibited by defeat-experienced mice. These results support the conclusions of Expt 1, namely that D2 receptors play a major role in the expression of defensive submissive behaviors.
GENERAL DISCUSSION A number of studies have pointed to the involvement of DA systems in the expression of submissive and defensive behaviors in laboratory animals like rodents and monkeys. Gerbils charactized by high sensitivity to the cataleptic and akinetic effects of haloperidol were shown to exhibit more subordinate behavior than the haloperidol-resistant animals in nondrugged tests (Upchurch & Schallert, 1982). d-Amphetamine and cocaine dose-dependently decrease social behavior and dominance-related be-
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PUGLISI-ALLEGRA AND CABIB
haviors in squirrel monkeys (Miczek & Gold, 1983; Miczek & Yoshimura, 1982) and induce submissive and defensive (escape) behavior in mice confronted with isolated resident conspecifics (Miczek & O'Donnell, 1978). Louilot et al. (1986) observed that rats interacting with an aggressive male conspecific showed higher increase of DA activity in nucleus accumbens when compared with animals interacting with a nonaggressive male or with female conspecifics. These results point to a role of the mesolimbic DA system in the effects of defeat experience. Moreover nucleus accumbens has been shown to be under inhibitory control of dopaminergic transmission in the amygdala (Louilot, Simon, Taghzouti, & Le Moal, 1985), a brain area involved in social learning processes related to defeat (Luiten, Koolhaas, de Boer, & Koopmans, 1985). Our present results indicate that defensive behaviors exhibited by defeatexperienced mice when confronted with nonaggressive opponents involve D2 receptor activation. In fact, the selective D2 receptor antagonist ( - )sulpiride, but not the selective D1 receptor antagonist SCH 23390, antagonizes the effects of defeat on defensive behaviors. It may be that during the stressful experience of being defeated D2 receptors are activated, producing sustained defensive response in the mouse. Subsequent confrontations with nonaggressive opponents could then induce in defeated mice a conditioned activation of the same receptor mechanisms and the expression of defensive behavioral patterns. The fact that the selective D2 agonist LY171555 but not the selective D1 agonist SKF 38393 produced, in naive mice interacting with nonaggressive conspecifics, a behavioral profile paralleling that of defeatexperienced animals further points to the major role of D2 receptor in modulating defensive behavior in the mouse. On the basis of these results it can be hypothesized that submissive animals are characterized by an imbalance between D1 and D2 receptors compared with dominant animals. In the light of this hypothesis the different behavioral effects of amphetamine on dominant and submissive monkeys (Haber, Barchas, & Barchas, 1977; Miczek & Gold, 1983) may be interpreted in terms of different balance of DA receptor types in relation to social status. Furthermore, it can be argued that alteration of D1/D2 receptor balance may lead to pathological hyperdefensiveness. This could be also viewed as a distorted perception of environmental stimuli causing the individual to perceive danger and/or threat where absent. Such a perceptive malfunctioning is reminiscent of paranoia. In fact, a paranoid person seems to perceive serious threats to himself and coping with such a threat dominates his entire life (Sullivan, 1965). The survival of man as well as animals depends on effective recognition of actual danger and the ability to perceive real danger or threat is not peculiar to the human
D2 DOPAMINE RECEPTORS AND DEFENSIVE BEHAVIOR
109
species. The view of paranoia as an alteration of this ability may thus provide a fruitful way to develop animal models of such pathology. The results of our study provide evidence on the role of D2 receptors in the expression of defensive behavior in the mouse and suggest that alterations in D2 receptor functioning may produce hyperdefensiveness, possibly as a result of altered perceptive processes. Further experiments will elucidate this point and clarify which brian areas and DA systems are involved in the behavioral effects observed in the present study.
REFERENCES Braun, A. R., & Chase, T. N. (1986). Obligatory D-I/D-2 receptor interaction in the generation of dopamine agonist related behaviors. European Journal of Pharmacology, 131, 301-306. Cabib, S., Kempf, E., Schleef, C., Oliverio, A., & Puglisi-Allegra, S. (1988). Effects of immobilization stress on dopamine and its metabolites in different brain areas of the mouse: Role of genotype and stress duration. Brain Research, 441, 153-160. Cabib, S., & Puglisi-Allegra, S. (1985). Different effects of apomorphine on climbing behavior and locomotor activity in three strains of mice. Pharmacology Biochemistry and Behavior, 23, 555-557. Cabib, S., Puglisi-Allegra, S., & Oliverio, A. (1984). Chronic stress enhances apomorphineinduced stereotyped behavior in mice: Involvement of endogenous opioids. Brain Research, 298, 138-140. Cabib, S., Puglisi-Allegra, S., & Oliverio, A. (1985). A genetic analysis of sterotypy in the mouse: Dopaminergic plasticity following chronic stress. Behavioral and Neural Biology, 44, 239-248. Cutler, M. G., Mackintosh, J. H., & Chance, M. R. A. (1975). Effects of cannabis resins on social behavior in the laboratory mouse. Psychopharmacologia, 41, 271-276. Frischknecht, H. R., Siegfried, B., & Waser, P. G., (1982). Learning of submissive behavior in mice: A new model. Behavioral Processes, 7, 235-245. Grant, E. C., & Mackintosh, J. H. (1963). A comparisons of the social postures of some laboratory rodents. Behaviour, 21, 246-257. Haber, S., Barchas, P. R., & Barchas, J. D. (1977). Effects of amphetamine on social behaviors of rhesus macaques: An animal model of paranoia. In I. Hanin, & E. Usdin, (Eds.), Animal models in psychiatry and neurology (pp. 107-115). Oxford: Pergamon. Koller, W., Herbster, G., Anderson, D., Wack, R., & Gordon, J. (1987). Quinpirole hydrochloride, a potential anti-parkinsonism drug. Neuropharmacology, 26, 10311036. Kulling, P., Frischknecht, H. R., Pasi, A., Waser, P. G., & Siegfried, B. (1987). Effects of repeated as compared to single aggressive confrontation on nociception and defensive behavior in C57BL/6 and DBA/2 mice. Physiology and Behavior 39, 599-605. Itoh, Y., Goldman, M. E., & Kebabian, J. W. (1985). T1 333, a benzhydro(g)quinoline, stimulates both D-1 and D-2 dopamine receptors: Implications for the selectivity of LY 141865 toward the D-2 receptors. European Journal of Pharmacology, 108, 1985. Louilot, A., Le Moal, M., & Simon, H. (1986). Differential reactivity of dopaminergic neurons in the nucleus accumbens in response to different behavioral situations. An in vivo voltametric study in free moving rats. Brain Research, 397, 395-400. Louilot, A., Simon, H., Taghzouti, K., & Le Moal, M. (1985). Modulation of dopaminergic activity in the nucleus accumbens following facilitation or blockade of the dopaminergic transmission in the amygdala: A study by in vivo differential pulse voltametry. Brain Research, 346, 141-145. Luiten, P. G. M., Koolhaas, J. M., de Boer, S., & Koopmans, S. J. (1985). The cortico-
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medial amygdala in the central nervous system organization of agonistic behavior. Brain Research, 332, 283-297. Mele, A., Cabib, S., Oliverio, A., Melchiorri, P., & Puglisi-Allegra, S. (1987). Effects of corticotropin releasing factor and sauvagine on social behavior of isolated mice. Peptides, 8, 935-938. Miczek, K. A., & Gold, L. H. (1983). d-Amphetamine in squirrel monkeys of different social status: Effects on social and agonistic behavior, locomotion and stereotypies. Psychopharmacology, 81, 183-190. Miczek, K. A., & O'Donnell, J. M. (1978). Intruder-evoked aggression in isolated and nonisolated mice: Effects of psychomotor stimulants and 1-Dopa. Psychopharmachology, 57, 47-55. Miczek, K. A., & Thompson, M. L. (1984). Analagesia resulting from defeat in a social confrontation: the role of endogenous opioids in brain. In R. Bandler (Ed.), Modulation of sensorimotor activity during alteration in behavioral states (pp. 431-456). New York: A. R. Liss. Miczek, K. A., Thompson, M. L., & Shuster, L. (1982). Opioid-like analgesia in defeated mice. Science, 215, 1520-1522. Miczek, K. A., Thompson, M. L., & Shuster, L. (1985). Naloxone injections into the peraqueductal grey area and arcuate nucleus block analgesia in defeated mice. Psychopharmacology, 87, 39-42. Miczek, K. A., & Winslow, J. T. (1987). Analgesia and decrement in operant performance in socially defeated mice: Selective cross-tolerance to morphine and antagonism by naltrexone. Psychopharmacology, 92, 444-451. Miczek, K. A., & Yoshimura, H. (1982). Disruption of primate behavior by d-amphetamine and cocaine: Differential antagonism by antipsychotics. Psychopharmacology, 76, 163171. Mos, J., & Van Valkenburg, C. F. M. (1979). Specific effect of social stress and aggression on regional dopamine metabolism in rat brain. Neuroscience Letters, 15, 325-327. Nabeshima, T., Katoh, A., Hiramatsu, M., & Kameyama, T. (1986). A role played by dopamine and opioid neuronal systems in stress-induced motor suppression (conditioned suppression of motility) in mice. Brain Research, 398, 354-360. Puglisi-Allegra, S. Oliverio, A., & Mandel, P. (1982). Effects of opiate antagonists on social and aggressive behavior of isolated mice. Pharmacology Biochemistry and Behavior, 17, 691-694. Roche, K. E., & Leshner, A. I. (1979). ACTH and vasopressin treatments immediately after a defeat increase future submissiveness in male mice. Science, 204, 1343-1344. Rodgers, R. J., & Hendrie, C. A. (1983). Social conflict activates status-dependent endogenous analgesic or hyperalgesic mechanisms in male mice: Effects of naloxone on nociception and behaviour. Physiology and Behavior, 30, 775-780. Scott, J. P. (1946). Incomplete adjustment caused by frustration of untrained fighting mice. Journal of Comparative Psychology, 39, 379-390. Setler, P., Sarau, H. M., Zirkle, C. L., & Saunders, H. L. (1978). The central effect of a novel dopamine agonist. European Journal of Pharmacology, 50, 419-430. Siegfried, B., Frischknecht, H. R., Kulling, P., & Waser, P. G. (1986). Defeat-induced analgesia and the conditioned display of submissive postures and escapes in strains of mice. In H. Matthies, (Ed.), Advances in biosciences. Learning and memory: Mechanisms of information storage in the nervous system (Vol. 59, pp. 295-298). Oxford: Pergamon. Siegfried, B., Frischknecht, H. R., & Waser, P. G. (1982). A new learning model for submissive behavior in mice: Effects of naloxone. Aggressive Behavior, 8, 112-115. Siegfried, B., Frischknecht, H. R., & Waser, P. G. (1984a). Vasopressin impairs or enhances
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retention of learned behavior in mice depending on the time of application. Behavioral Brain Research, 11, 259-269. Siegfried, B., Frischknecht, H. R., & Waser, P. G. (1984b). Defeat, learned submissiveness and analgesia in mice: Effect of genotype. Behavioral and Neural Biology, 42, 9197. Stoof, J. C., & Kebabian, J. W. (1984). Two dopamine receptors: Biochemistry, physiology and pharamcology. Life Sciences, 35, 2281-2296. Sullivan, H. S. (1965). Clinical studies in psychiatry. In H. S. Perry, M. L. Gawel, & Gibbon (Eds.), The collected works of Harry Stack Sullivan, (Vol. 2). New York. Norton. Thiessen, D. D., & Upchurch, M. (1981). Haloperidol and clonidine increase, and apomorphine decreases ultrasonic vocalizations by gerbils. Psychopharmacology, 75, 287-290. Upchurch, M., & Schallert, T. (1982). Neuroleptic-sensitive posture and movement related to subordinate social status in mongolian gerbils (Meriones unguiculatus). Behavioral and neural Biology, 35, 308-314.
(1988)
Pharmacological Evidence for a Role of D2 Dopamine Receptors in the Defensive Behavior of the Mouse STEFANO PUGLISI-ALLEGRA AND SIMONA CABIB 1
Istituto di Psicobiologia e Psicofarmacologia (C.N.R.), via Reno 1, 00198 Roma, Italy In this study the role of the DA system in the expression of defensive behavior of the mouse was investigated. C57BL/6 mice subjected to three daily defeat experiences (24 h apart) exhibited an increase of defensive behaviors (upright and sideways postures and escape) as well as a decrease of activity and a decrease of social investigation compared with undefeated mice (controls) when confronted with nonaggressive Swiss mice 24 h after the last aggressive confrontation. The selective D2 DA receptor antagonist (-)-sulpiride administered before confrontation with nonaggressive opponents (fourth day) dramatically decreased defensive behaviors and produced an increase of social investigation. The selective D1 DA receptor antagonist SCH 23390 did not affect either defence or social investigation. In further experiments the behavioral effects of the selective D1 agonist SKF 38393 and of the selective D2 agonist LY171555 on naive C57BL/6 mice interacting with nonaggressive opponents of the same strain were assessed. SKF 38393 in doses up to 30 mg/kg did not produce any significant behavioral changes while LY171555 produced a clear-cut dose-dependent increase of defensive behavior as well as a decrease of social investigation and activity and an increase of immobility. The behavioral profile produced by the D2 agonist did not differ from that produced by defeat experiences. These results indicate that D2 receptors play a major role in the expression of defensive behavior in the mouse. The hypothesis that alteration in D2 receptor functioning may produce hyperdefensiveness possibly due to altered perceptive processes is discussed. © 1988Academic Press, Inc.
Animals exposed to an aggressive conspecific exhibit different defensive behaviors such as escape and submissive postures. The attack-experienced animals display defensive behaviors also in subsequent nonaggressive confrontations, thus showing that defensive behavioral patterns may be learned and included in the individual behavioral repertoire (Frischknecht, Siegfried, & Waser, 1982; Roche & Leshner, 1979; Siegfried, Frischknecht & Waser, 1982; Siegfried, Frischknecht & Waser, 1984a; Scott, 1946). t The authors thank the Eli Lilly & Co., The Schering Corporation, and Ravizza for supplying with LY171555, SCH 23390, and (-)-sulpiride. Thanks are also given to Mr. Mario Battaglia for the preparation of figures. Correspondence should be addressed to S. Puglisi-Allegra at Istituto di Psicobiologia e Psicofarmacologia, via Reno 1, 00198 Roma, Italy. 98 0163-1047/88 $3.00 Copyright © 1988 by Academic Press, Inc. All fights of reproduction in any form reserved.
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Moreover, a number of studies have shown that the attacked animal may show a decrease of pain sensitivity (analgesia) (Miczek & Thompson, 1984; Miczek, Thompson, & Shuster, 1982; Siegfried, Frischknecht & Waser, 1984b; Rodgers & Hendrie, 1983). Thus, the effects of being attacked and defeated have been related to the activaction of the endogenous opioid system produced by this stressful experience (defeat) (Miczek & Thompson, 1984; Miczek, Thompson, & Shuster, 1985; Miczek & Winslow, 1987; Siegfried et al., 1984b). Evidence exists that stressful (adversive unescapable) experiences affect brain dopamine (DA) systems in the mouse and that such effects may be modulated by endogenous opioids (Cabib, Puglisi-Allegra & Oliverio, 1984; Cabib, Puglisi-Allegra, & Oliverio, 1985; Cabib, Kempf, Schleef, Oliverio, & Puglisi-Allegra, 1988; Nabeshima, Katoh, Hiramatsu, & Kameyama, 1986). Some studies have also indicated that the DA system is involved in the expression of defensive and submissive behaviors of laboratory animals interacting with aggressive and nonaggressive conspecifics. Upchurch and Schallert (1982) showed that gerbils which were more sensitive to the cataleptic and akinetic effects of haloperidol exhibit more subordinate behavior than the haloperidol-resistant animals in nondrugged tests. Rats interacting with an aggressive male conspecific exhibit the greatest increase of DA activity in nucleus accumbens when compared with animals interacting with a nonaggressive male or with a female conspecific (Louilot, Le Moal, & Simon, 1986). Although these effects on nucleus accumbens may be a consequence of motor changes produced by different social situations, these results point to an involvement of the mesolimbic DA system in the effects of defeat experience. Moreover, both attack and social stress have been shown to increase dihydroxyphenylacetic acid and homovanillic acid in rat olfactory tubercle (Mos and Van Valkenburg, 1979). Psychostimulants, d-amphetamine and cocaine, were shown to decrease dose-dependently social behavior and dominancerelated behaviors in squirrel monkeys (Miczek & Gold, 1983; Miczek & Yoshimura, 1982) and to induce defensive behavior in mice confronted with isolated resident consepecifics (Miczek & O'Donnell, 1978). In the present study we investigated the role of the DA system in the expression of defensive behavior exhibited during a nonaggressive confrontation by mice previously exposed to three daily aggressive encounters. To this end defeated mice were treated before nonaggressive confrontation with selective D1 and D2 antagonists SCH 23390 and (-)-sulpiride (Stoof & Kebabian, 1984). In a further experiment the behavior of naive mice injected with the selective D2 agonist LY171555 and the selective D1 agonist SKF 38393 before confrontation with nonaggressive opponents was assessed. C57BL/6 mice were used in this study since they were shown to be characterized by reliable defeat responses (Siegfried et al., 1984b).
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EXPERIMENT 1 Methods
Male C57BL/6 (C57) (Charles River Labs., Calco, Italy) and SwissWebster (Swiss) (Plaisant Labs., Rome, Italy) mice were used. Upon arrival C57 mice were housed in groups of eight in standard breeding cages (27 x 21x 13.5 cm). Swiss mice were housed either with female conspecifics (aggressive opponents) or in groups of 15 (nonaggressive opponents). Four to five weeks after being housed with a female, each resident mouse was tested for attack behavior by introducing a grouphoused mouse into its home cage. During this and all subsequent tests, the female and pups were removed from the cage before introducing the intruder into the resident's cage. The mice were kept in a 12-h light/12-h dark cycle, at a temperature of 22 ___ I°C, given water and food ad lib, and tested during the second half of the light period in a sound-insulated room lighted by a 60 W lamp placed 2.4 m above the floor of the room. Test mice were exposed to three daily aggressive confrontations 24 h apart and subjected to nonaggressive confrontation (testing) 24 h after the last aggressive encounter (Kulling, Frischknecht, Pasi, Waser, & Siegfried, 1987; Siegfried, Frischknecht, Kulling, & Waser, 1986). Aggressive confrontations were carried out by introducing a C57 mouse to a Swiss mouse housed with a female. Only Swiss mice which consistently showed aggression on each testing day were used. The test animal (C57) was attacked immediately after being placed in the resident's cage. Following 30 s of attack, during which it received 50 + 5 bites, the intruder was gently removed from the resident's cage. Control animals were paired with a nonaggressive (group-housed) Swiss mouse habituated for 10 min to the text box (transparent breeding cage with fresh hardwood sawdust on the floor) during three daily 3-min sessions. On Day 4 the C57 mouse was introduced into the test box and confronted with a nonaggressive (group-housed) Swiss mouse during a 2-min testing session. The duration (s) of the following behavioral items (Cutler, Mackintosh, & Chance, 1975; Grant and Mackintosh, 1963; Mele, Cabib, Oliverio, Melchiorri, & Puglisi-Allegra, 1987; Puglisi-Allegra, Oliverio, & Mandel, 1982) shown by the test animal were recorded: upright and sideways postures, escape and crouch, social investigation (sniff-body, sniff-nose, and following), activity (horizontal and vertical), and immobility. Moreover, the behavioral items upright and sideways postures, escape and crouch were summarized as defence category. In an additional recording, the behaviors of opponents interacting with test animals was also observed. Each test was videotaped and, later, two experienced observers unaware
D2 DOPAMINE RECEPTORS AND DEFENSIVE BEHAVIOR
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TABLE 1 Mean (___SE) Duration (s) of Behavioral Categories and Defensive Items Observed in Mice during a Nonaggressive Confrontation 24 h after Repeated (1 × 3 Days) Aggressive (Defeated) or Nonaggressive (Undefeated) Confrontations Undefeated mice Category Activity Immobility Defence Soc. inv. Def. item Sideways Crouch Escape Upright
Defeated mice
71.6 16.6 5.5 27.3
_+ 8.4 -+ 4.9 _+ 2.1 - 8.3
42.0 31.6 37.2 6.9
+- 7.1a _+ 7.0 _+ 4.0c - 4.1b
1.4 0.6 0.6 2.4
--- 0.6 -+ 0.6 --- 0.2 +- 1.8
11.8 2.5 5.6 17.3
--- 2.0~ -+ 0.7 -+ 1.1d -4- 5.1°
a p < .05; bp < .02; ~p < .01; dp< .005; ~p < .001 in comparison with undefeated mice (t test).
of the treatment condition of each animal independently recorded behavioral items using a k e y b o a r d system connected to an Apple computer. The interobserver reliability of the recorded items was determined on the basis of recordings of social interactions observed in 40 mice during 5 min testing sessions. The r values ranged from 0.74 to 0.95. Test (defeated) mice from different breeding cages were randomly assigned to the following drug treatment: ( - ) - s u l p i r i d e (12.5, 25, and 50 mg/kg), S C H 23390 (0.03, 0.1, and 0.25 mg/kg) and their vehicles. Each experimental group included 6-12 animals. S C H 23390 ((R)-( + )-8-chloro2,3,4,5-tetrahydro-3-methyl-5-phenyl- 1H-3-benzazepine-7-ol maleate; Schering Corp.) was dissolved in distilled water (H20). ( - ) - S u l p i r i d e (Ravizza, Italy) was dissolved in HC1 and diluted in H20, and the pH was adjusted to 7.4 with N a O H . All drugs were injected intraperitoneally (ip) in a volume of 10 m l / k g 20 min before testing. The test doses were chosen on the basis of preliminary experiments. Results were analyzed statistically by one-way analysis of variance (ANOVA). Further analyses for individual between-group comparisons were carried out with post hoc tests (Duncan multiple range test). Student's t test (two-tailed) was also used.
Results and Discussion When c o m p a r e d with undefeated mice (controls), C57 mice subjected to repeated defeat experiences exhibited a decrease of activity, a decrease of social investigation, and an increase of defence when confronted with nonaggressive mice (Table 1). A trend toward increased immobility was
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TABLE 2 Effects of Different Doses of ( -)-Sulpiride (Sulp mg/kg) on Mean (-+ SE) Duration (s) of Behavioral Categories and Defensive Items Observed in Defeated Mice Interacting with a Nonaggressive Opponent Vehicle
Sulp 12
Category Activity Immobility Soc. inv. Defence
47.0 24.6 1.3 40.7
_+ _ + -
7.1 6.9 0.8 3.0
41.0 27.7 10.5 28.7
-+ 4.1 _+ 6.7 +_ 6.1 _+ 8.5
Def. item Sideways Crouch Escape Upright
12.3 4.8 4.8 18.8
_+ _+ _+ _+
2.5 1.4 1.0 4.5
6.3 3.5 2.2 16.5
-+ _+ + -4-
p < .05;
bp <
2.4 a 1.5 0.7 b 7.9
Sulp 25
Sulp 50
57.0 24.6 15.5 6.5
+ _+ _+ _+
9.7 4.7 3.& 5.5 b
27.3 54.7 28.0 20.8
_+ -------
2.6 12.6 a 5.6 b 5.7 ~
2.1 0.3 0.8 3.1
_+ --+ -
1.4 b 0.3 0.6 b 1.9 b
2.0 12.6 0.4 5.8
--_+ _+ -+
0.8 b 4.46 0.2 b 1.3 a
.01, in comparison with vehicle-treated mice (Duncan test).
also found. All the items in the defensive category increased significantly, except crouch (Table 1). ANOVA showed a significant effect of (-)-sulpiride on defence (F(3, 20) = 5.62, p < .01) and on social investigation (F(3, 20) = 9.17, p < .002). Individual between-group comparisons showed dose-related decrease of defence and increase of social investigation (Table 2). ANOVA also revealed a significant effect on immobility (F(3, 20) = 3.25, p < .05) and post hoc test showed that only the 50 mg/kg dose increased this behavior (Table 2) while no significant effects on activity were revealed, although a tendency to a decrease of this behavior appeared at 50 mg/kg of (-)-sulpiride (Table 2). As regards defensive items the D2 antagonist affected upright (F(3, 20) = 5.01, p < .02) and sideways (F(3, 20) = 5.99, p < .01) postures, escape (F(3, 20) = 6.68, p < .005), and also crouch (F(3, 20) = 5.64, p < .01). As shown by individual betweengroup comparisons (-)-sulpiride dose-dependently antagonized upright and sideways postures as well as escape (Table 2). Although crouch decreased without statistical significance at the doses of 12.5 and 25 mg/kg, a significant increase at a dose of 50 mg/kg was evident (Table 2). It is worth noting that crouch was not significantly affected by defeat in our experimental conditions. SCH 23390 did not produce any significant effect either on defence or on social investigation while ANOVA revealed significant effects on activity (F(3, 20) = 4.27, p < .02) and immobility (F(3, 20) = 6.69, p < .005). Individual between-group comparisons showed that the doses of 0.1 and 0.25 mg/kg produced a significant decrease of activity and a increase of immobility while the dose of 0.03 mg/kg was not effective
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TABLE 3 Effects of Different Doses of SCH 23390 (SCH mg/kg) on Mean ( ± SE) Duration (s) of Behavioral Categories and Defensive Items Observed in Defeated Mice Interacting with a Nonaggressive O p p o n e n t Vehicle
SCH 0.03
SCH 0.1
SCH 0.25
Category Activity Immobility Soc. inv. Defence
42.1 23.0 1.3 39.1
± ++
7.0 5.9 0.8 4.3
43.5 30.5 4.1 38.6
--± -+ -
7.8 3.1 1.9 9.6
24.3 41.7 1.4 52.7
+ 3.3 ° --- 8.6" ± 1.1 ± 15.0
17.1 59.0 1.6 41.6
± --± +
5.1" 6.0 b 0.8 11.7
Def. item Sideways Crouch Escape Upright
10.0 4.8 7.3 17.0
--- 3.3 -+-+ 1.4 --- 0.6 ± 2.6
13.3 1.5 8.8 15.8
--± -+ --+
3.0 1.3 2.9 4.1
10.1 20.2 2.4 20.0
--- 4.9 ± 4.C --- 1.0 +- 6.0
6.1 18.0 0.3 17.2
--± --±
2.5 6.9 ° 0.2" 3.9
" p < .05; b p < .01 in comparison with vehicle-treated mice (Duncan test).
on both behaviors (Table 3). When single defensive items are considered SCH 23390 did not significantly affect either upright or sideways postures while significant effects were evident on escape (F(3, 20) = 6.02, p < .005) and crouch (F(3, 20) = 6.28, p < .005). In particular while only the dose of 0.25 mg/kg decreased escape significantly, both 0.1 and 0.25 mg/kg increased crouch (Table 3). To summarize, defensive behavior exhibited by defeated mice interacting with nonaggressive opponents was dose-dependently reduced by the D2 selective antagonist (-)-sulpiride but unaffected by the D1 antagonist SCH 23390 at the doses used in these experiments. Also social investigation was unaffected by SCH while a dose-dependent increase of this behavior was evident in (-)-sulpiride-treated mice. As far as the behavioral items in the defence category are concerned, sideways and upright postures decreased in (-)-sulpiride-treated mice but not in SCH 23390-treated mice while escape was reduced by all doses of (-)-sulpiride and by only the highest dose of SCH 23390. Crouch was the only item to be increased both by the high dose of ( - )-sulpiride and by 0.1 and 0.25 mg/kg of SCH 23390. It is worth noting that crouch is not significantly affected by previous defeat in our experimental conditions (Table 1). At the same time, modulation of crouch by the two DA antagonists seems to be strictly related to modulation of overall activity. In fact, at the dose of 50 mg/kg of (-)-sulpiride (the only dose that affects crouch) a nonsignificant decrease of activity and a significant increase of immobility were evident while 0.1 and 0.25 mg/kg of SCH 23390 both significantly reduced activity and increased immobility. Moreover, as we observed in preliminary experiments, mice tested individually
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in an activity meter (for the method see Cabib & Puglisi-Allegra, 1985) following administration of ( -)-sulpiride and SCH 23390 showed a dramatic decrease of locomotor activity ((-)-sulpiride 50 mg/kg = - 6 0 % ; SCH 23390 0.1 mg/kg = - 6 5 % and 0.25 mg/kg = - 8 7 % compared with vehicle injected animals). Since akinetic animals have been shown to be treated by normal animals as subordinates (Thiessen and Upchurch, 1981), the behavior of the opponent interacting with an akinetic mouse might therefore increase crouch in testing mice. Further analysis possibly involving also ultrasound recordings could elucidate this point. It is worth noting that no overt offensive behavior was observed either in test mice or in opponents throughout the experiments. These results suggest that defensive behavior exhibited by defeatexperienced mice when confronted with nonaggressive opponents is mediated through the D2 receptor. In order to check this hypothesis we investigated the behavioral effects of the selective D2 agonist LY171555 (Itoh, Goldman & Kebabian, 1985) and of the selective D1 agonist SKF 38393 (Setler, Sarau, Zirkle, & Saunders, 1978) on naive C57 mice interacting with a nonaggressive opponent of the same strain. EXPERIMENT 2
Methods Group-housed male C57 mice aged 11-12 weeks and weighing 23-24 g were used in this experiment. Each test mouse was placed in the testing box (see Expt 1) 5 min before a C57 mouse from a different breeding cage was introduced into the box. Resident and intruder mice did not differ by more than 1 g in weight. Each animal was tested only once. Behavioral recordings were carried out as described in Expt 1 during a 5-rain testing session. No attack on the intruder or the resident was observed in these experimental conditions. Test mice were randomly assigned to different drug treatment (vehicles; SKF 38393 3, 10, 20, 30 mg/kg; LY171555 0.1, 0.5, 2, 5 mg/kg). Each experimental group comprised six animals. SKF 38393 (l-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diolhydrochloride; Smith, Kline & French Laboratories) and LY171555 (trans(-)-4aR, 4a, 5, 6, 7, 8, 8a, 9-octahydro-5-propyl-lH(or 2H)-pyrazolo (3,4,-g) quinoline monohydrochloride; Eli Lilly & Co.) were dissolved in distilled water and injected subcutaneously (sc) 25 min before testing. Results were analyzed statistically as described in Expt 1. Results and Discussion Since the test used in the second experiment was different from that used in the first one, the percentage time of behavioral categories exhibited by naive, untreated mice in the two tests was compared (Fig. 1). t test
D2 D O P A M I N E R E C E P T O R S A N D D E F E N S I V E B E H A V I O R TEST I
[] ACTIVITY [] IMMOBILITY
105
TEST 2
"
:
/
,
/
Fir. I. T i m e spent in behavioral categories by undefeated C57 mice interacting with a nonaggressive o p p o n e n t in the two testing conditions used in this study. Results are e x p r e s s e d in percentage ( m e a n values of 12 mice per group) of recorded behaviors. Note: activity = 57 ± 7.3 (Test 1), 63 ± 5.3 (Test 2); immobility = 15 ± 4.1 (Test 1), 20 --+3.4 (Test 2); defence = 3 ± 1.! (Test 1), 5 - 1.5 (Test 2); social investigation = 23 ± 6.5 (Test 1), 13 ± 3.8 (Test 2). N o statistical difference was evident b e t w e e n testing conditions (t test).
did not show any significant difference between the two tests, namely social interaction with a nonaggressive Swiss or C57 opponent. SKF 38393 did not produce any significant effect on any of the behavioral categories observed in this test situation or on single defensive items (Table 4). The results obtained with LY171555 are presented in Fig. 2. ANOVA TABLE 4 Effects o f Different D o s e s of S K F 38393 ( S K F m g / k g ) on M e a n ( ± SE) Duration (s) o f Behavioral Categories and Defensive Items O b s e r v e d in Naive Mice Interacting with a Nonaggressive Opponent Vehicle Category Activity Immobility Soc. inv. Defensive Def. item Sideways Crouch Escape Upright
143.7 80.5 38.8 10.4 2.4 3.0 2.4 2.7
SKF 3
___ 25.8 --- 20.4 ± 10.7 ± 3.7 ± ± ± ±
0.8 1.5 0.7 1.2
145.0 65.1 34.2 6.8 2.5 1.3 2.0 1.0
_+ --± ± ± ± ±
S K F 10
17.6 133.7 ± 17.6 89.2 ± 6.2 32.2 _+ 2.7 6.5 _+ 0.6 0.8 0.8 0.3
1.5 1.5 2.0 1.5
_ ± _ _+_
S K F 20
20.9 128.0 ± 21.9 82.8 8.5 25.1 ± 1.9 5.0 --0.8 0.3 0.5 0.5
1.6 1.1 1.1 2.2
S K F 30
15.1 125.0 _+ 17.7 19.8 87.0 ± 7.7 7.0 20.7 _ 6.9 2.7 8.1 -+ 1.3
± 0.6 --- 0.6 ± 0.6 ± 1.2
2.7 1.5 2.5 2.0
± ± ±
2.1 0.5 1.0 0.5
Note. A N O V A s h o w e d no significant effects for a n y behavioral category and defensive item.
106
P U G L I S I - A L L E G R A A N D CABIB m RCTIVITY r ~ IMMOBILITY gr/A DEFENCE k ~ BOC. INV. 200
*
150
*
100
*
g m
50
,
o VEHICLE
0. I
0.5 LTITIBS5 {mg/Kg)
2
B
Fro. 2. Effects of LY 171555 on mean ( - SE) duration (s) of behavioral categories exhibited by naive mice interacting with a nonaggressive opponent. *p < .01 in comparison with vehicle-treated mice (Duncan test).
showed a significant decrease of activity (F(4, 25) = 11.04, p < .001), a significant increase of immobility (F(4, 25) = 8.13, P < .001), a significant increase of defence (F(4, 25) = 22.72, p < .001), and a significant decrease of social investigation (F(4, 25) = 43.46, p < .001). The Duncan test indicated that the dose of 0.1 mg/kg of LY171555 was already effective in reducing activity and in increasing immobility but still ineffective in altering defensive and social behavior. The 5 mg/kg dose produced the greatest effects on defence and social investigation. The results related to defensive behaviors are presented in Table 5. ANOVA showed a significant effect of LY171555 in increasing sideways (F(4, 25) = 18.9, p < .001) and upright postures (/7(4, 25) = 20.61, p < .001) and escape (F(4, 25) = 13.46, p < .001) but no apparent effect on crouch. The Duncan test revealed that the 0.1 mg/kg dose of LY171555 was effective only in increasing sideways postures. It is worth noting that neither stereotypic nor defensive behavior was observed in animals treated with LY171555 during the 5-min pretest TABLE 5 Effects of LY171555 (LY mg/kg) on Mean ( ± S E ) Duration (s) of Defensive Behavioral Items Observed in Naive Mice Interacting with a Nonaggressive Opponent Def. item Sideways Crouch Escape Upright
Vehicle 1.3 1.1 1.1 4.0
_ _+ ±
0.4 0.6 0.6 1.3
LY 0.1 19.8 9.0 6.7 6.1
± ± ±
2.9" 3.9 2.9 1.7
LY 0.5 24.6 3.5 26.3 21.6
± ± ± ±
2.3 h 1.7 4.9" 1.9"
LY 2 22.6 7.3 40.0 34.8
± 5.1 ~ - 2.9 ± 10.1b ± 6.5"
LY 5 62.5 5.1 33.1 101.0
± -+_+ ±
a p < .05 and b p < .01 in comparison with vehicle-treated mice (Duncan test).
9.5 b 1.9 4.W 14.1b
D2 DOPAMINE RECEPTORS AND DEFENSIVE BEHAVIOR
107
habituation period, the only difference with control animals being a depression of spontaneous activity. Furthermore, no overt offensive behavior was observed either in test mice or in opponents of control and drug-treated groups. As shown by these results, the effects of LY171555 on activity, immobility, and defence seem unrelated to each other. In fact, the 2 mg/kg dose did not reduce activity compared with vehicle, while defensive behavior was significantly increased by this same dose. At the dose of 5 mg/kg defence reached its greatest values, activity was lower than in control animals, while immobility was unchanged. Finally, the dosedependent increase of defensive behavior over the range of doses commonly considered to affect postsynaptic receptors (Braun & Chase, 1986; Koller, Herbster, Anderson, Wack, & Gordon, 1987) suggests their involvement in this behavioral effect of LY171555. The profile of the effects of LY171555 on defensive behaviors, social investigation, activity, and immobility of C57 mice interacting with a nonaggressive conspecific does not differ from that of the effects of defeat experiences on the behavior of animals confronted with nonaggressive opponents as shown in the Expt 1. Although animals pretreated with LY171555 did not experience defeat in the way the animals tested in Expt 1 did, they exhibited defensive upright and sideways postures which paralleled those exhibited by defeatexperienced mice. The submissive behavior of these mice suggests that they perceive the opponent as a threatening stimulus toward which they exhibit a behavioral pattern capable of inhibiting aggression. Moreover the high escape exhibited by the defeat-experienced animals and those treated with LY171555 shows that mice in both experimental conditions avoid contact with the opponent. This behavioral output may depend on a fear-motivated response possibly produced by social experience (defeat) in one case and by pharmacological treatment in the other. Thus the D2 agonist seems to affect the same neural mechanisms as those involved in the expression of behavioral patterns exhibited by defeat-experienced mice. These results support the conclusions of Expt 1, namely that D2 receptors play a major role in the expression of defensive submissive behaviors.
GENERAL DISCUSSION A number of studies have pointed to the involvement of DA systems in the expression of submissive and defensive behaviors in laboratory animals like rodents and monkeys. Gerbils charactized by high sensitivity to the cataleptic and akinetic effects of haloperidol were shown to exhibit more subordinate behavior than the haloperidol-resistant animals in nondrugged tests (Upchurch & Schallert, 1982). d-Amphetamine and cocaine dose-dependently decrease social behavior and dominance-related be-
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PUGLISI-ALLEGRA AND CABIB
haviors in squirrel monkeys (Miczek & Gold, 1983; Miczek & Yoshimura, 1982) and induce submissive and defensive (escape) behavior in mice confronted with isolated resident conspecifics (Miczek & O'Donnell, 1978). Louilot et al. (1986) observed that rats interacting with an aggressive male conspecific showed higher increase of DA activity in nucleus accumbens when compared with animals interacting with a nonaggressive male or with female conspecifics. These results point to a role of the mesolimbic DA system in the effects of defeat experience. Moreover nucleus accumbens has been shown to be under inhibitory control of dopaminergic transmission in the amygdala (Louilot, Simon, Taghzouti, & Le Moal, 1985), a brain area involved in social learning processes related to defeat (Luiten, Koolhaas, de Boer, & Koopmans, 1985). Our present results indicate that defensive behaviors exhibited by defeatexperienced mice when confronted with nonaggressive opponents involve D2 receptor activation. In fact, the selective D2 receptor antagonist ( - )sulpiride, but not the selective D1 receptor antagonist SCH 23390, antagonizes the effects of defeat on defensive behaviors. It may be that during the stressful experience of being defeated D2 receptors are activated, producing sustained defensive response in the mouse. Subsequent confrontations with nonaggressive opponents could then induce in defeated mice a conditioned activation of the same receptor mechanisms and the expression of defensive behavioral patterns. The fact that the selective D2 agonist LY171555 but not the selective D1 agonist SKF 38393 produced, in naive mice interacting with nonaggressive conspecifics, a behavioral profile paralleling that of defeatexperienced animals further points to the major role of D2 receptor in modulating defensive behavior in the mouse. On the basis of these results it can be hypothesized that submissive animals are characterized by an imbalance between D1 and D2 receptors compared with dominant animals. In the light of this hypothesis the different behavioral effects of amphetamine on dominant and submissive monkeys (Haber, Barchas, & Barchas, 1977; Miczek & Gold, 1983) may be interpreted in terms of different balance of DA receptor types in relation to social status. Furthermore, it can be argued that alteration of D1/D2 receptor balance may lead to pathological hyperdefensiveness. This could be also viewed as a distorted perception of environmental stimuli causing the individual to perceive danger and/or threat where absent. Such a perceptive malfunctioning is reminiscent of paranoia. In fact, a paranoid person seems to perceive serious threats to himself and coping with such a threat dominates his entire life (Sullivan, 1965). The survival of man as well as animals depends on effective recognition of actual danger and the ability to perceive real danger or threat is not peculiar to the human
D2 DOPAMINE RECEPTORS AND DEFENSIVE BEHAVIOR
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species. The view of paranoia as an alteration of this ability may thus provide a fruitful way to develop animal models of such pathology. The results of our study provide evidence on the role of D2 receptors in the expression of defensive behavior in the mouse and suggest that alterations in D2 receptor functioning may produce hyperdefensiveness, possibly as a result of altered perceptive processes. Further experiments will elucidate this point and clarify which brian areas and DA systems are involved in the behavioral effects observed in the present study.
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