Social play alters regional brain opioid receptor binding in juvenile rats

BRAIN RESEARCH ELSEVIER

Brain Research 680 (1995) 148-156

Research report

Social play alters regional brain opioid receptor binding in juvenile rats Louk J.M.J. Vanderschuren a, *, Elliot A. Stein b, Victor M. Wiegant a, Jan M. Van Ree a a Department of Medical Pharmacology, Rudolf Magnus Institute/or Neurosciences, Faculty of Medicine, Utrecht University, Utrecht, The Netherlands b Departments of Psychiatry and Pharmacology, Medical College of Wisconsin, Milwaukee, WI, USA Accepted 7 February 1995

Abstract

An in vivo autoradiographic procedure was employed to visualize local changes in brain opioid receptor occupancy in juvenile rats. This procedure is based on the assumption that released endogenous ligand will exclude exogenously applied tracer, in this case [3H]diprenorphine, from opioid receptors. Increases in availability of opioid peptides will then result in decreased opioid receptor binding. From behavioral studies there is ample evidence that opioid systems are involved in the regulation of social play behavior in juvenile rats. In the present study, changes in regional brain opioid activity as a result of social isolation-induced social play behavior were monitored. Twenty-one-day-old rats were socially isolated for 0, 3.5 or 24 h prior to testing, and tested alone or in a dyadic encounter. After behavioral testing, [3H]diprenorphine was administered and the brain was prepared for autoradiography. Social isolation caused increases in social behavior (dyadic encounters) but not in non-social behavior (singly tested animals). Modest differences in brain opioid receptor binding due to social isolation, social play behavior, or an interaction of the two, were found in claustrum, nucleus accumbens, globus pallidus, paraventricular and arcuate nuclei of the hypothalamus, and the dorsolateral and paratenial thalamic nuclei. These results support the notion that opioid systems are involved in the regulation of social play behavior. In addition, the observation of changes in opioid binding in areas involved in reward processes, adds evidence to the hypothesis that opioid systems are involved in the regulation of the rewarding aspects of social play in juvenile rats.

Keywords: Opioid receptor; In vivo autoradiography; Social play; Social isolation; Diprenorphine

I. Introduction

In mammals, social play is the earliest form of non-mother directed social behavior. In rats, its occurrence is restricted, for the most part, to the period between weaning and sexual maturation [74]. Although its period of occurrence is rather short, its abundance in that period, its rewarding value, and the sensitivity of rats to social isolation in this period of life suggest that play behavior is of major importance. The function of play may be the development of social and cognitive skills [6,45,47,48,71]. In rats, a distinction can be made between social behaviors related to play (pinning, boxing-wrestling, and following-chasing) and social behaviors unrelated to play (social exploration, contact

* Corresponding author. Present address: Dept. of Pharmacology, Faculty of Medicine, Free University, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. Fax: (31) (20) 4448100. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00256-1

behavior), on basis of both appearance in ontogeny [2,11,31,55] a n d n e u r o b i o l o g i c a l b a c k g r o u n d s [3,5,32,59,73,77]. Opioid systems have been suggested to play a prominent role in the regulation of social play (e.g. [51,56,57]). This fits well to the finding that social play has a high rewarding value [14,33,52], since opioid systems have been implicated in reward processes [12,76,82]. Previously, it has been shown that social play, rather than social behaviors unrelated to play are regulated by opioid systems [77]. In addition, opioidergic effects on social play were shown to be mediated through /.L- and K-opioid receptor systems [78]. In an effort to elucidate the brain sites at which opioids affect social play, an in vivo autoradiographic procedure was employed to measure changes in brain opioid receptor binding after social play behavior. The rationale of the in vivo receptor binding technique is that prereleased peptide, occupying opioid

L.J.M.J. Vanderschuren et aL / Brain Research 680 (1995) 148-156

receptors, will exclude exogenously applied tracer and decrease receptor binding as assessed by autoradiography (for review see [54]). In vivo autoradiographic analyses have previously been used to assess changes in brain opioid receptor binding upon stress [63], deprivation-induced drinking [10], hypothalamic stimulationinduced feeding [69] and ventral tegmental area selfstimulation [68]. Using social interaction as a stimulus, changes in brain opioid receptor binding have been shown [56]. The present study was designed to replicate and to extend these findings, since in this early study only seven brain areas were investigated. For that purpose, 21-day-old rats were socially isolated for 0, 3.5 or 24 h. These isolation periods have been shown to produce minimal, half-maximal and maximal increases, respectively, in the amount of social play [51]. Subsequently, animals were allowed to play in a dyadic encounter for 15 min or were placed singly into the test cage for that period, after which in vivo autoradiography was performed.

2. Materials and methods

2.1. Animals and housing conditions Male Wistar rats bred from our own live stock were used. Two weeks prior to experimentation, 7-day-old rats were housed in litters of 8 with their mothers in a dimly lighted animal room (20-40 Ix, lights on 6.00 a.m., lights off 8.00 p.m.) in macrolon home cages measuring 40 x 26 × 20 cm (1 x w x h). The animal rooms were temperature controlled (22 _+ 1°C) and standard food (Hope Farms) and tap water were available ad lib.

2.2. Behavioral procedure Testing was performed in a sound attenuated inner chamber located in a dimly lighted outer room. Background noise was produced by a fan. The testing arena consisted of an acrylic plastic cage measuring 35 × 35 × 50 cm (1 x w × h) with approximately 2 cm of wood shavings covering the floor. The test cage was illuminated by a 25 W red light bulb (0.4-1 Ix) mounted 60 cm above the test cage. The behaviors of the animals were recorded on video tape (Sony U-matic). During a test session, only animals tested were present in the inner chamber. Video equipment except for cameras was in the outer room. After weaning on day 19 of life, litters were distributed over 2 cages of 4 rats per cage. To habituate the animals to transportation and injections, all animals were brought to the outer room on the two days preceding the experiment where they received a subcutaneous injection of 0.1 ml saline.

149

On the day of the experiment, the animals were socially isolated in macrolon cages measuring 22 × 13 × 20 cm (1 × w × h) for 0, 3.5 or 24 h prior to the experiment. All testing was done under dim light-unfamiliar test conditions [23,50,79], which means testing under red light and in a novel test cage. The test consisted of either placing a single animal (no play) or a pair of similarly treated animals (play) into the test cage for 15 min. The animals of a pair were not littermates and did not differ by more than 10 g in body weight. Analysis from the video tape recordings was performed afterwards without knowledge of the treatment of the rats. For the pairs of animals tested in dyadic encounters, the frequencies of the following behaviors were scored per pair per 15 min: pinning, which is defined as one of the animals lying with its dorsal surface on the floor of the test cage with the other animal standing over it; boxing-wrestling, a group of behaviors including boxing, wrestling and pouncing; following-chasing, moving in the direction of or pursuing the test partner, who moves away; social exploration, sniffing any part of the body of the test partner, including the anogenital area; contact behavior, which includes crawling over and under the test partner and social grooming. For the singly tested animals, the following behaviors were scored per 15 min: frequencies of rearing (standing up on hindlegs), comer visits (no. of times a corner of the test cage is investigated), and durations of digging, walking, self-grooming and

sniffing walls or floor. 2.3. Autoradiographic procedure Since it is difficult to implant intravenous catheters in 21-day-old rats (body weight was approximately 40 g), and subcutaneous and intravenous injection of tracer have been shown to yield similar autoradiographic pictures of rat brain opioid binding [54], tracer was administered subcutaneously. Thus, immediately after the behavioral test, the animals were subcutaneously injected with 150 /zCi/kg (37 Ci/mmol; 86 m C i / m g ) [3H]diprenorphine (Amersham), an opioid receptor antagonist without marked selectivity for tz-, 6-, or Kopioid receptors. The animals were killed by decapitation 30 rain after tracer injection. Brains were rapidly removed, frozen in isopentane (2-methyl-butane) cooled with dry ice and stored at - 8 0 ° C. Sectioning was performed at - 2 0 ° C on a Reichert cryostat after brains were allowed to equilibrate to the temperature of the cryostat. Sections were thaw-mounted onto chrom-alum-gelatin coated slides and dried on air at room temperature. Slides and radioactivity standards (Amersham [3H]microscales) were then apposed to tritium sensitive film ([3H] Hyperfilm, Amersham) in light tight cassettes for 16 weeks. Films were developed

150

L.J.M.J. Vanderschuren et al. / Brain Research 680 (1995) 148-156

in Kodak D-19 developer at 20°C for 5 min. Developing was terminated using 5% acetic acid, and films were fixed using Kodak Rapid Fixer (15 min). Films were then washed in water and hung to dry. Slides were thionin stained for use for structure identification during image analysis.

not normally distributed, pinning data were analysed using a Kruskal-Wallis non-parametric test. The other variables were analysed using analysis of variance (ANOVA). Regional R O D measurements were subjected to an arcsin transformation to ensure linearity, followed by two-way analyses of variance (ANOVA) for social isolation and social play.

2.4. Image analysis 44 regions of interest were selected according to the atlas of Paxinos and Watson [58]. Autoradiograms were analyzed on an image analyzer-microdensitometer (MCID, Imaging Research, St. Catherines, Ontario, Canada). Optical density (OD) measurements from regions of interest were converted to a relative optical density (ROD) scale [25]. This method normalizes the mean OD value of the region to a cumulative frequency histogram of OD pixels derived from the entire section from which the region was measured. The R O D scale ranges from 0.0, representing the lightest, to 1.0, representing the darkest pixel of the section. This procedure has been shown to provide a more sensitive and reliable measure of changes in OD than standardizing to regions of very high or low (e.g. white matter) binding [25].

2.5. Statistical analyses Behavioral data were analysed as follows: group medians (for pinning) or group means + S.E.M. (other variables) were calculated. Since pinning levels were

Pinning 40

3. Results

Effects of social isolation on social (animals tested in dyadic encounters) and non-social (singly tested animals) behaviors are presented in Figs. 1 and 2, respectively. Frequencies of all social behaviors measured were markedly increased after prolonged social isolation [pinning: X 2 = 6.71, P < 0.05; boxing-wrestling: Fe.8 = 45.28, P < 0.001; following-chasing: F2,s = 19.94, P < 0.01; social exploration: Fe, 8 = 10.91, P < 0.01; contact behavior: F:. 8 = 113.46, P < 0.001] (Fig. 1). In contrast, social isolation did not affect non-social, exploratory behaviors in singly tested animals. Neither the frequencies of rearing [F2.17 = 0.04, n.s.] or corner visits [F2,17 = 0.32, n.s.] nor the durations of digging [F2,|7 = 0.09, n.s.], walking [F2,17 = 0.66, n.s.], self-grooming [F2,17 = 0.28, n.s.] or sniffing walls-floor [F2.17 = 0.21, n.s.] were altered by increased periods of social isolation before testing (Fig. 2). The arcsin corrected R O D values for the brain areas investigated are presented in Table 1. In general, changes in brain opioid receptor binding as a result of

Following-chasing

Boxing-wresOing 100

60 !

30

75

40

20

50 20

10

25 0

0

0

Contact behavior

Social exploration

•

0 hours isolation

150

[]

3.5 hours isolation

60

100

[]

30

50

24 hours isolation

0

0

120

200

90

Fig. 1. Effects of social isolation on the frequencies of different variables of social behavior in animals tested in dyadic encounters. 21-day-old rats were socially isolated for 0, 3.5 or 24 h, and subsequently placed in the test cage for 15 min with a similarly treated test partner. Behavior was recorded on videotape and scored afterwards. Medians (pinning) and m e a n s + S.E.M. (other behaviors) are presented.

L.J.M.J. Vanderschuren et al. / Brain Research 680 (1995) 148-156

testing under the different conditions were rather small. In the rostral claustrum, social isolation (especially 3.5 h) decreased binding [F(social isolation)2,28 = 6.75, P < 0.01]. Upon social play, binding was increased in the paraventricular hypothalamic nucleus, irrespective of duration of social isolation preceding the test [F(social play)1,34 = 9.66, P < 0.01]. In the paratenial thalamic nucleus social play decreased opioid receptor binding, especially after 0 and 24 h of social isolation [F(social isolation x social plaY)2,34= 5.32, P < 0.01; F(social play)l,34 = 5.48, P < 0.05]. In the rostral nucleus accumbens, social play decreased binding in animals isolated for 24 h [F(social isolation × social play)2.31 = 3.40, P < 0.05]. In the dorsolateral thalamus effects similar to the rostral accumbens were seen [F(social isolation × social play) 2,35 = 3.85, P < 0.05]. In the caudal claustrum [F(social isolation × social play) 2,35 3.37, P < 0.05], globus pallidus [F(social isolation x social play)2,33= 4.64, P < 0.01] and arcuate nucleus [F(social isolation x social play)2.32= 6.15, P < 0.01] interactions between social isolation and social play were found. In globus pallidus and arcuate nucleus, social play caused a decrease in binding in non-isolated animals, an increase in 3.5 h isolated animals, while in animals isolated for 24 h social play had no effect on binding. In caudal claustrum a similar effect in the opposite direction was found: social play caused an increase in binding in non-isolated animals, a decrease in 3.5 h isolated animals, and no difference in animals isolated for 24 h. =

4. Discussion

Binding patterns obtained from in vivo opioid binding studies using [3H]diprenorphine closely resemble those from [3H]naloxone in vitro [53]. In addition, [3H]diprenorphine in vivo binding levels can be displaced by co-administration of excess cold naloxone [10,54,63,80] suggesting that [3H]diprenorphine is retained at opioid receptors in vivo and thus that the signal obtained is specific for opioid receptors. The in vivo autoradiographic technique allows for a detailed analysis of changes in brain opioid activity with high anatomical resolution [54]. Depolarization of a hippocampal slice in vitro has been shown to reduce [3H]diprenorphine binding in a transient, calcium- and peptidase inbititor-dependent way, suggesting that release of endogenous opioid peptides accounted for the differences in binding [49]. The presence of a microcompartment around the opioid synapse in which continuous binding and rebinding of ligand takes place has been proposed to explain exogenous ligand exclusion from receptors previously occupied with endogenous ligand [24,60,62]. Although it can not be ruled out that observed differences in binding are caused by factors other than increases in opioid peptide release (e.g. temporary changes in receptor sensitivity), changes in in vivo opioid receptor binding elicited by short-term manipulations (for example, the social interaction test in the present study) are likely to be due to the release of endogenous opioid peptides.

Digging

Corner visits 30

50

40

40

30

20

151

30 20 20

10

10

10

0

0

0

Sniffingfloor-walls

Self-grooming

Wslking 100

200

200

75

150

150

50

100

100

25

50

50

0

0

0

Fig. 2. Effects of social isolation on the frequencies of corner visits and rearing and the durations (in seconds) of digging, walking, self-grooming and sniffing walls or floor in singly tested rats. 21-day-old rats were socially isolated for 0, 3.5 or 24 h, and subsequently placed singly in the test cage for 15 min. Behavior was recorded on videotape and scored afterwards. Means + S.E.M. are presented. For legends see Fig. 1

L.J.M.J. Vanderschuren et al. / Brain Research 680 (1995) 148-156

152 The periods

effects

of

social

play

behavior

after

o f s o c i a l i s o l a t i o n o n in v i v o o p i o i d

binding were subtle. In the paraventricular

various receptor

nucleus of

the

hypothalamus,

creased

opioid

receptor

binding

was

in-

in a n i m a l s t e s t e d i n a s o c i a l s i t u a t i o n , r e g a r d -

less of social isolation. This phenomenon

might reflect

Table 1 Duration of social isolation

3.5 h

0h Play

No play

Play

24 h No play

Play

No play

Cortex Medial prefrontal cortex Frontal cortex Cingulate cortex Parietal cortex Piriform cortex

50.1 53.3 46.9 37.8 29.6

_+ 1.1 ± 0.9 ± 0.5 ± 0.8 ± 1.8

47.9 52.7 46.3 38.5 28.0

± ± ± ± ±

1.0 1.1 1.9 1.5 1.8

50.8 53.6 48.4 37.1 28.9

+ ± ± ± ±

1.3 0.5 0.8 1.3 1.5

49.6 53.6 48.4 38.9 29.7

± ± ± ± ±

0.9 0.8 1.1 1.6 1.6

50.5 54.0 48.9 38.1 26.9

± + ± ± ±

1.7 0.9 1.9 1.1 2.0

49.4 53.4 48.4 37.4 28.1

± ± ± ± ±

1.3 0.6 2.3 1.2 0.7

55.0 47.5 61.8 52.1 55.6 50.2 47.1 50.8 61.3 29.7 60.5 40.1 46.1 42.3

± + ± + ± ± ± ± ± ± ± ± ± ±

1.8 2.1 1.3 0.6 1.3 0.6 1.2 1.7 1.2 0.9 1.1 1.3 2.7 1.0

57.1 43.4 62.7 52.1 53.9 50.2 47.8 53.2 63.9 28.4 60.3 44.0 47.9 44.3

± 0.8 ± 1.4 ± 0.7 ± 1.0 ± 0.4 ± 0.9 ± 0.9 _+ 0.5 ± 1.0 ± 1.1 ± 0.8 ± 0.7 ± 1.0 ± 1.3

53.4 44.4 62.0 52.0 54.5 50.3 47.4 51.7 64.9 27.9 60.2 43.1 51.0 45.2

± ± ± ± ± ± ± + ± ± ± ± ± ±

0.9 1.5 0.9 0.6 0.8 0.5 0.8 1.6 0.8 1.8 0.6 1.7 1.8 2.2

50.8 47.4 59.8 51.0 53.2 49.5 46.5 51.4 63.6 30.8 61.1 37.7 45.5 43.0

± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.3 1.0 1.1 0.4 1.2 1.1 1.0 1.8 1.8 1.1 1.0 1.6 1.0 1.0

53.3 45.5 58.7 52.3 54.0 49.8 47.1 52.6 64.0 30.4 59.8 41.8 51.5 45.8

± ± + + ± ± ± + ± ± ± + + ±

1.2 1.0 0.7 2.0 2.0 0.6 0.6 1.2 1.0 1.9 1.0 1.4 2.2 1.1

53.5 45.4 61.3 51.6 54.8 49.5 45.8 51.3 60.9 30.6 58.7 42.3 46.4 44.0

± ± ± ± ± ± + + ± ± ± ± ± ±

(I.9 0.7 0.9 0.8 1.6 0.6 1.9 1.0 1.9 1.4 2.2 2.0 1.2 1.6

47.8 47.2 47.3 57.5 47.0 50.8

± + ± + ± ±

0.7 1.1 1.6 2.4 1.4 1.3

46.1 47.7 46.3 60.8 47.9 48.3

± ± ± ± ± ±

1.2 2.1 2.0 2.0 1.6 0.9

46.9 46.2 44.9 60.3 46.6 47.9

± ± ± ± ± +

1.0 0.3 0.6 1.8 1.8 0.7

46.3 45.3 44.1 60.5 46.4 49.6

± + ± ± ± ±

1.3 2.1 2.0 1.7 1.2 2.0

45.0 46.2 46.7 61.5 46.0 48.5

± ± ± ± ± ±

0.6 1.0 2.0 1.4 1.4 1.5

45.6 45.0 46.1 62.0 48.2 50.5

± ± ± ± ± ±

2.1 1.0 1.7 1.8 0.9 2.1

47.0 61.8 74.5 52.7 51.3 35.1

+ ± ± ± ± +

1.6 1.4 1.6 1.2 1.7 1.7

47.5 59.9 64.2 54.0 52.6 40.6

± ± ± ± ± ±

2.3 1.7 3.0 1.4 1.1 0.8

47.4 62.0 73.6 54.5 54.3 39.2

_+ 3.2 ± 1.9 ± 2.0 ± 0.9 ± 1.0 ± 0.8

49.3 60.6 68.2 51.4 52.0 36.6

± ± ± ± ± ±

1.8 2.8 3.0 0.9 2.0 1.6

48.6 58.9 68.8 54.1 50.5 37.6

± ± ± ± ± ±

2.9 1.6 1.6 1.0 0.9 1.1

45.5 59.8 64.4 52.4 50.5 37.0

± ± ± ± ± ±

1.7 3.9 3.4 1.2 0.7 1.0

46.9 74.4 69.0 69.7 56.2

± ± ± ± ±

1.0 0.9 1.3 0.9 2.3

45.7 73.6 69.4 72.3 52.7

± ± ± ± ±

0.4 0.5 1.0 1.0 2.4

46.9 75.7 70.9 71.8 54.2

± ± ± ± ±

0.9 1.3 0.6 1.4 0.8

46.9 75.8 68.6 69.8 52.6

± ± ± ± ±

1.1 0.4 1.1 0.5 1.6

44.0 74.6 71.4 68.5 53.0

± ± ± ± ±

0.7 0.4 0.9 1.3 0.9

47.4 74.4 70.8 73.8 53.7

± ± ± ± ±

0.8 1.6 1.1 1.2 1.7

35.3 43.7 41.7 23.5

+_ 1.5 ± 1.3 ± 1.0 ± 1.8

34.0 43.4 42.4 22.0

± + ± +

0.5 0.5 0.5 0.7

34.5 43.5 40.6 23.6

_+ 0.7 ± 0.8 ± 0.6 ± 1.3

36.0 44.0 41.5 24.6

± ± ± ±

0.5 0.9 0.5 0.7

34.4 43.6 42.7 22.2

± ± ± ±

1.0 1.0 1.3 0.7

36.0 43.1 43.3 26.2

± ± ± ±

1.3 0.7 0.6 1.7

61.1 58.2 47.9 58.7

± 1.0 ± 0.7 ± 0.6 _+ 0.7

63.3 58.5 48.3 56.5

+_ 0.8 ± 1.3 ± 1.1 _+ 1.1

63.6 60.9 47.2 57.0

± ± ± ±

62.2 60.4 47.3 58.7

± ± ± ±

1.7 1.2 1.8 0.8

60.4 61.1 46.6 56.7

± ± ± ±

1.2 0.8 1.5 1.3

62.1 57.0 48.6 56.8

± ± ± ±

0.6 1.8 2.0 1.3

Forebrain Claustrum (rostral) ++ Claustrum (caudal) # Nucleus accumbens (rostral) ~ Nucleus accumbens (core) Nucleus accumbens (shell) Caudate putamen Olfactory tubercle V. pallidum-Islands of Calteja Medial septum Lateral septum Diagonal band of Broca Globus pallidus ## Ventral pallidum Bed nucleus stria terminalis

Amygdala Cortical nucleus Medial nucleus Central nucleus Basolateral nucleus Lateral nucleus Basomedial nucleus

Hypothalarnus Medial preoptic area Lateral preoptic area Paraventricular nucleus * * Lateral nucleus Ventromedial nucleus Arcuate nucleus '~#

Thalamus Dorsolateral nucleus ~ Dorsomedial nucleus Reuniens nucleus Paratenial nucleus * #'~ Parafascicular area

Hippocampus CA1 area CA3 area Dentate gyrus Subiculum

Midbrain Periaqueductal gray Ventral tegmental area Substantia nigra pars reticulata Substantia nigra pars compacta

0.7 1.1 0.9 1.1

Effects of social play behavior and social isolation on opioid receptor binding in various brain areas. Means + S.E.M. of arcsin transformed R O D values are presented. Play = tested in dyadic encounter, No play = singly tested. No. of animals was 6 for each group. Effect of social play: * P < 0.05, * * P _< 0.01; Effect of social isolation: ++ P _< 0.01; Interaction between social isolation and social play: ~ P _< 0.05, #~' P _< 0.01

L.J.M.J. Vanderschuren et al./ Brain Research 680 (1995) 148-156

an effect of novelty stress on opioid binding. In reaction to stress, alterations in opioidergic tone in the paraventricular nucleus have been described [44,63] and injection of /z-opioid receptor agonists into the paraventricular nucleus of the hypothalamus seems to counteract some of the effects of stress [38]. Electrical stimulation of the paraventricular nucleus induces self-grooming in rats [42], and opioids have been implicated in several aspects of self-grooming, a form of behavior that has been associated with periods of dearousal after stress [67]. Indeed, the animals tested in the non-social situation spent a significant part of the test period (approximately 15%) grooming themselves. This suggests that placement in a novel environment induces opioid peptide release, which is attenuated in a social condition. Presence of a conspecific (social buffering) has been shown to alleviate the consequences of stress [72]. Decreases in binding due to social play, especially after 24 h of social isolation, when levels of social play were high, were found in the rostral nucleus accumbens, and the paratenial and dorsolateral thalamic nuclei. The paratenial nucleus has been shown to support opioid-dependent self-stimulation [70,75], while the nucleus accumbens has also been shown to be important for opioid-dependent reward processes [40,61,64,82]. The nucleus paratenialis sends projections to the accumbens [7,15,36], and receives projections from the ventral pallidum [17], which has reciprocal connections with the accumbens [29,30,65,83]. The existence of cortico-striato-pallido-thalamic circuits, integrating limbic and motor functions has been suggested [18,30]. The role of the dorsolateral thalamus in this respect is not clear, albeit that the neighbouring dorsomedial thalamus receives projections from the ventral pallidum and indirectly innervates the accumbens [28]. The claustrum might also be linked to the circuit mentioned above, since it projects to mediodorsal thalamus [27], medial prefrontal cortex [39], paratenial nucleus [17], and in turn receives projections from the paratenial nucleus [8]. The claustrum, supposedly a link between cortical and limbic sites [81], has been suggested to play a role in movement organization, and injection of dopaminergic drugs into claustrum influences locomotion [13]. Opioid systems in the globus pallidus, which receives major GABAergic-enkephalinergic projections from the caudate-putamen [22,26,65], have been suggested to be involved in the regulation of motor activity [21]. The differences in binding that were found (in non-isolated animals social play decreased and in 3.5 h-isolated animals social play increased binding) can, however, not easily be linked to changes in motor activity, suggesting that perhaps other phenomena might cause the differences in binding in globus pallidus. In the arcuate nucleus of the hypothalamus, opioid binding decreased as a result of social

153

play in non-isolated animals, while in animals isolated for 3.5 h a reverse effect was seen. This finding can not easily be explained. The arcuate nucleus contains the majority of POMC perikarya in the brain [1,19,37], and alterations in opioid binding in the arcuate nucleus might reflect an opioid feedback mechanism. The finding that social play changes opioid receptor binding in the paratenial nucleus is in accordance with an earlier study on social interaction and in vivo opioid binding [56], supporting an important role for this area in opioid-regulated social play behavior. However, the paratenial nucleus is the only area that showed decreases in binding due to social play in both studies. These discrepancies can be explained by methodological issues. In the present study, tracer was administered after the social interaction, while in the earlier study tracer was administered before social interaction. Timing of tracer administration has been shown to cause different results [63], with post-behavior labelling being a more sensitive method [10,63,68]. Another difference is the way in which binding was analyzed: in the present study ROD values were analyzed [25], whereas in the earlier study [56] rough OD values were used. There are also some differences in the behavioral procedure: animals of different strains and ages were used, and, more important, in the present study very subtle manipulations, in terms of social isolation (0-24 h, as compared to 10 days of social isolation prior to testing) were employed. In contrast to the effects of opioid drugs on social play, effects of social play on opioid receptor binding were quite small. Treatment with morphine, fentanyl or /3-endorphin markedly increases, while treatment with naloxone, naltrexone, /3-funaltrexamine or U50,488H d e c r e a s e s social play b e h a v i o r [4,35,51,57,77,78]. Several explanations can be found for this apparent discrepancy. Small changes in opioidergic tone might result in relatively large alterations in behavior. It could, for instance, be that large differences in binding in subsets of neurons within an analyzed area occur. These differences will then be confounded by the fact that the area analyzed is larger than the area in which the changes take place. Note that in comparison with other behavioral paradigms in which changes in in vivo opioid binding have been analyzed (electrical brain stimulation [9,68,69], water deprivation-induced drinking [10], cold-water immersion or footshock stress [63]), the manipulations employed in the present study are quite subtle. It has also been suggested that sensitivities of opioid receptors in immature animals are different from adult animals [16,34,66]. In 21-day-old animals /~-, ~5-, and K-opioid receptors are already functional with respect to opioid effects on neurotransmitter release and adenylate cyclase activity [20], and although 21-day-old animals show strong resemblances with adult animals, the pat-

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terns of opioid p e p t i d e a n d opioid r e c e p t o r p r e s e n c e in the rat b r a i n have not yet r e a c h e d their adult p a t t e r n [41,43,46]. I n conclusion, the p r e s e n t results suggest that during social play in juvenile rats, changes in b r a i n opioid activity take place, probably in the form of release of e n d o g e n o u s opioid peptides. A l t h o u g h the differences d e t e c t e d in the p r e s e n t study were modest, they can be fitted into a theoretical framework; social play behavior in juvenile rats can be s t i m u l a t e d by t r e a t m e n t with opioid r e c e p t o r agonists a n d suppressed using opioid receptor antagonists [4,35,51,57,77,78]. Previously, it has b e e n shown that of the social behaviors m e a s u r e d in the p r e s e n t study, mainly social behaviors related to play are subject to opioidergic r e g u l a t i o n a n d that this r e g u l a t i o n is exerted t h r o u g h /z- a n d K-opioid receptors [77,78]. Thus, it is suggested that the differences in b i n d i n g observed in the p r e s e n t study are due to p,a n d K-opioid r e c e p t o r s t i m u l a t i o n caused by social behaviors related to play. A t least some of the b r a i n areas in which changes in opioidergic activity were f o u n d (e.g. n u c l e u s a c c u m b e n s , n u c l e u s paratenialis) are involved in o p i o i d - d e p e n d e n t reward processes. Thus, together with previous behavioral results [52,77], it is suggested that opioid systems are involved in the r e g u l a t i o n of the rewarding aspects of play. To investigate w h e t h e r areas in which differences in b i n d i n g were f o u n d in the p r e s e n t study are i n d e e d involved in the r e g u l a t i o n of social play behavior, behavioral studies in which opioid drugs are a d m i n i s t e r e d into these areas could be employed.

Acknowledgements This study was s u p p o r t e d by a g r a n t from the Korczak F o u n d a t i o n for autism a n d related disorders. T h e skilful assistence of Scott A. F u l l e r a n d H e n k A. S p i e r e n b u r g is gratefully acknowledged.

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