Social and exploratory behaviour in the rat after septal administration of ORG 2766 and ACTH4–10

Psychoneuroendocrinology,Vol. 8, No. 3, pp. 343- 350, 1983.

0306- 4530/83$3.00 + 0.00 © 1983PergamonPressLtd.

Printed in Great Britain.

SOCIAL AND EXPLORATORY

BEHAVIOUR

SEPTAL ADMINISTRATION

IN THE RAT AFTER

O F O R G 2766 A N D A C T H 4 ,~

ANTHONY CLARKE and SANDRA E. FILE Department of Pharmacology, The School of Pharmacy, University of London, 29/39 Brunswick Square, London W C I N lAX, U.K.

(Received 10 March 1982; in final form 18 January 1983) SUMMARY The time spent in active social interaction by pairs of male rats in a dimly-lit, familiar test arena, was decreased significantly after intraseptal injection of A C T H 4_ to ( 2 5 0 - 500 ng). In contrast, the time spent in active social interaction was increased significantly after intraseptal injection of the tri-substituted ACTH4_ 9 analogue O R G 2766 ( 2 5 0 - 500 pg). Neither A C T H 4_ J0 (250 ng) nor O R G 2766 (250 pg) affected exploration or locomotor activity measured in a holeboard, after intraseptal injection. Intraseptally administered ACTH4_I0 (250 ng) increased aggression measured in a colony intruder model, but O R G 2766 (250 pg) was without effect.

INTRODUCTION ADRENOCORTICOTROPHIC hormone (ACTH), as well as acting to stimulate the synthesis and release of corticosteroids from the adrenal cortex, has been reported to have behavioural actions, e.g. to prolong extinction in several paradigms (de Wied, 1974: Levine & Jones, 1965; Guth et al., 1971a,b). These responses are thought to be mediated through direct effects of the peptide itself, since corticosteroids do not elicit the same effects, and fragments of ACTH, such as ACTH 4_ ~0, which are not steroidogenic, elicit identical responses to those of the full molecule (de Wied et al., 1975; de Wied, 1977; Colbern et al., 1977). The importance of centrally-mediated effects of ACTH has been heightened in recent years by reports that ACTH and related peptides are present within neurone terminals in discrete extra-pituitary loci, including the septum (Watson et al., 1978), amygdala (Pacold et al., 1978), hippocampus, cortex (Krieger et al., 1977a,b), and periaqueductal grey and reticular formation (Krieger & Liotta, 1979). Furthermore, lesion and deafferentation studies suggest strongly that most ACTH found in neurone terminals is derived from cell bodies located in the arcuate nuclei of the hypothalamus (Watson et al., 1978; Krieger et al., 1979; Pelletier et al., 1980). In an attempt to elucidate the segment of ACTH responsible for its behavioural effects, several fragments have been synthesised and tested (e.g. de Wied, 1974; de Wied et al., 1975). ORG 2766 is an a n a l o g u e of ACTH4_ 9 with three s u b s t i t u t i o n s [4-Met(0)-8-D : Lys-9-Phe] that is particularly long-lasting in its behavioural effects (Fekete & de Wied, 1982). In many behavioural situations, both ACTH 4_ to and ORG 2766 have the same action, e.g. to prolong extinction (Greven & de Wied, 1977), to increase the rate of intracranial self-stimulation (Nyakas et al., 1980; Katz, 1980), to induce analgesia 343

344

ANTHONY C[.ARKE and SANDRA E. FILE

after injection into sites in the periaqueductal grey (Jacquet, 1978, 1979; Walker et al., 1980, 1981), and to prevent experimentally-induced amnesia (Rigter et al., 1974, 1976; Rigter & van Riezen, 1975). In humans, too, there is evidence that both A C T H 4 ~o and ORG 2766 can have similar actions, since both can increase selective attention (Miller et al., 1974; Sandman & Kastin, 1977a,b; Sandman et al., 1977; Gaillard & Varey, 1979; Walker & Sandman, 1979). However, these peptides do not always have the same effect, and, whereas intraventricularly-administered A C T H results in the development of a characteristic vigorous grooming response (Ferrari et al., 1963; Izumi et al., 1973), ORG 2766 does not. These A C T H fragments also differ in their effects on social behaviour in rats. The time spent in active social interaction by two male rats in an arena in which neither has established territory is decreased by peripherally or centrally administered ACTH~ 24 and ACTH~ ~0 (File & Vellucci, 1978; File, 1979; File & Clarke, 1980) and by peripherally a d m i n i s t e r e d c~-MSH (File, 1981), whereas it is increased after peripheral or intraventricular administration of ORG 2766 (File, 1981; File & Clarke, 1981a). In this test, as well as having an effect in the opposite direction to A C T H 4 ~0, ORG 2766 was also about 4000 times more potent. This order of magnitude has been found in other behaviours, for example extinction of a conditioned avoidance response (Greven & de Wied, 1977; Pigache & Rigter, 1981), but it is not always found: nearer equal potency between A C T H 4 ~ and ORG 2766 can be seen in amnesia paradigms (Rigter et al., 1974, 1976), in tests of analgesia (Jacquet, 1978; Walker et al., 1981), in measures of intracranial self-stimulation (Nyakas et al., 1980; Katz, 1980) and in the reduction of exploratory head-dipping after either peripheral or intraventricular administration (File, 1978; File & Clarke, 1981b; lsaacson & Green, 1978). The purpose of the present experiments was to explore further the similarities and differences between ACTH4_~0 and ORG 2766, following injection directly into the septum. This area was chosen as a possible site of action of the peptides because previous reports have shown that the septum shows a marked accummulation of radiolabelled ORG 2766 after intraventricular injection (Verhoeff et al., 1977a,b; Rees et al., 1980). The doses of peptides were chosen on the basis of their effects following intraventricular injection. Social interaction between pairs of male rats was studied in a dimly-lit test arena which was familiar to the rats, but in which neither had established territory (File, 1980). Under such conditions most of the social behaviour seen is investigatory in nature. In order to study the possible peptide effects on aggressive behaviours, rats also were placed singly as intruders into a long-established colony of ten resident male rats (File, 1982). Finally, the effects of intraseptally administered peptides were studied on exploratory behaviour. This was tested in a holeboard, since this provides a reliable measure of directed exploration (head-dipping), as well as an independent measure of locomotor activity (File & Wardili, 1975a,b). METHODS Animals and surgery Male hooded rats (Rattus norvegicus) from Olac Ltd. Bicester were used throughout. At the time of surgery, the rats weighed 120- 180 g. Bilateral septal cannulae were aimed at the border between the lateral and fimbrial nuclei of the septum, and were implanted under halothane-induced anaesthesia [1% (v/v) in oxygenr; (co-

SEPTAL ORG 2766 AND ACTH 4_ 10 AND BEHAVIOUR

345

ordinates from ear bar: A / P 6.8, L _ I. V 4.2). The lateral/fimbrial area was chosen since these two nuclei show the highest accumulation of 'H-ORG 2766 (Verhoeff et al., 1977b). All cannulae (outside diameter 0.82 mm) were made of surgical steel and were held in place with acrylic dental cement set onto four screws placed into the skull. All animals were allowed two weeks post-operative recovery time and were housed in groups of six in a room maintained at 25°C in a I 1 hr light : 13 hr dark cycle (lights on at 0700 hr). Food and water were available ad libitum. At the time of testing, the animals weighed 2 2 0 - 300 g.

Drugs All drugs were dissolved in an artificial cerebrospinal fluid (CSF; Bradbury & Davson, 1964); the intraseptal injection volume was 0.5 ~l. Injections were given immediately before testing and were delivered over 30 sec.

Apparatus Social interaction. The test box was 60 × 60 × 36 cm with wooden walls and floor. Rats in the box could be observed via a closed-circuit television system. An automated measure of locomotor activity derived from infrared beams that crossed the box; a count was scored each time a beam was interrupted. The light level used was 23 scotopic lux. Holeboard. The holeboard was 60 × 60 × 36 cm with wooden walls and floor, with four equally-spaced holes 3.8 cm in diameter placed around the centre of the floor. An automated measure of locomotor activity derived from infra-red beams that crossed the box; a count was scored each time a beam was broken. Exploration was measured automatically, as the number and duration of head-dips made, via infra-red beams placed below each hole. The light level in the test box was 23 scotopic lux. Rat colony The colony box was made of clear Perspex and had three stories; each measured 90 × 60 cm. The middle and lower stories were 20 cm high, while the upper story was 15 cm high. Water was available on the upper and lower floors; food was available oa the upper floor only. Rats in the box had free access to all floors, During testing, the light levels (in scotopic lux) were upper 115, middle 41.5 and lower 3.32. The colony consisted of 10 animals and was established for six months before the beginning of these experiments. The colony had experienced intruders before the commencement of this study (File, 1982). Procedure Social interaction. Five days prior to testing, all animals were housed singly. Rats were randomly assigned to a partner with whom they were to be tested (pairs of rats did not differ in weight by more than l0 g), and the pairs of rats were randomly assigned to the following drug groups: ACTH 4_ J0 ( 100, 250 or 500 ng) or ORG 2766 ( 100, 250 or 500 pg). There were seven pairs of rats in each drug group, except the ACTH 4_ 10 250 ng group in which there were eight. The pairs were tested on each of two consecutive days; once after administration of vehicle (CSF), and once after injection of peptide. The rats were tested in a counterbalanced design, with half of the pairs receiving peptide first. Both rats in a pair always received the same treatment. On the day immediately before the first day of testing, all of the rats were placed singly into the test box for I 0 rain in order to,familiarise them with the experimental conditions. On the two test days, the rats were given their peptide or vehicle injection, then placed immediately into the test box. The time spent in social interaction was then scored via a closed-circuit television system by an observer who had no knowledge of the drug state of the animals. The b~haviours scored as active social interaction were sniffing, following, grooming, kicking, mounting, jumping on, wrestling and boxing with, and crawling under or over the partner. Simple proximity was not scored, and passive contact (time spent lying with body in contact with the partner) was scored separately. Testing took place between 0700 and 1100 hr, in an order randomised between the various drug/CSF groups, and each test lasted 7.5 min. For full details of the validation and methodology of the Social Interaction test, see File (1980). At the end of each test, the test box was wiped with detergent and dried. Holeboard. When all social interaction testing was complete, the rats were group housed again, and then randomly assigned to be tested immediately after injection of vehicle (CSF) or peptide (ACTH.~_ ~0 250 ng or ORG 2766 250 pg). Rats were tested between 0700 and 1100 hr on one day only, in an order randomised between the various peptide/CSF groups. Rats were placed singly into the centre of the test box, and behaviours were monitored automatically for I0 rain. Each test was divided into four 2.5 min trials so that a measure of withinsession habituation could be taken. At the end of each test, the test box was wiped with detergent and dried. Rat colony intruder test. Rats were randomly assigned to be tested immediately after injection of vehicle (CSF) or peptide (ACTH4_ l0 250 ng or ORG 2766 250 pg). Rats were placed singly onto the middle floor of the colony, in an order randomised between the various peptide/CSF groups, and behaviour was monitored for 10 rain. The behaviour of the intruder was scored separately from that of residents. The frequency of the following behaviours was scored: sniff, anogenital sniff, attack, groom, wrestle, approach, being sniffed, being

346

A N r ~ O N Y CLARKE a n d SANDRA E . FI[.E

anogenitally sniffed, being groomed, boxing, submitting and squeaking. Testing took place between 1330 and 1430 hr. Statistics The social interaction and locomotor activity scores were analysed with paired t-tests. The holeboard data were analysed with two-way split-plot analyses of variance, with drug groups as independent variables and the four within-session trials as repeated measures. The rat colony intruder scores were analysed x~ith Mann - Whitney U-tests. All statistical analyses were performed on ra~ scores. For the purposes of clarity, the data in Fig. 1 have been expressed as a percentage of control scores. This was derived by calculating the peptide score for each pair of rats as a percentage of its paired control score. Plotted data are the means of the peptide percentage scores. RESULTS

Social interaction These results are shown in Fig. 1. The m e a n time spent in active social interaction was decreased significantly by A C T H 4 ~{} at doses o f 250 ng and 500 ng It(7) = 4.25, p < 0.005, and t(6) = 4.45, p < 0.005, respectively]. These reductions in social interaction were not secondary to sedation, since none o f the l o c o m o t o r activity scores was significantly different from control levels.

In contrast, O R G 2766 increased the mean time spent in social interaction at doses o f 250 and 500 pg It(6) = 2.38, p < 0.05, and t(6) = 4.23, p < 0.005, respectively]. Again, none o f the l o c o m o t o r activity scores differed significantly from control levels. ZOO" O r,Z

.J

"G

ne W t.Z

~ 500

°mwII l im

10O25O500 10O25050O

ACTH4_I0(ncj) ORG2766(log) FIG. 1. Mean time spent in active social interaction (expressed as a percentage of paired control) by pairs of male rats after intraseptal administration of ACTH4 z0 ( 1 0 0 - 5 0 0 ng) and ORG 2766 ( 1 0 0 - 5 0 0 pg). (*p < 0.05; **p < 0.005).

Holeboard The results are shown in Table 1. Directed exploration was not affected by treatment with either peptide, since neither the number o f head-dips nor the time spent head-dipping was altered significantly by ACTH4_,0 ( F 1,14 = 0.36 and F 1,14 = 1.10) or O R G 2766 ( F 1,12 = 1.50 and F 1,12 = 2.37). The peptides also failed to alter l o c o m o t o r activity significantly ( A C T H 4 ~0: F 1,14 = 0.10 and O R G 2766: F 1,12 = 0.31).

SEPTAL ORG 2766 AND ACTH 4_ Io AND BEHAVIOUR

347

T h e r e w a s a s i g n i f i c a n t r e s p o n s e - d e c r e m e n t a c r o s s t h e f o u r t r i a l s in t h e n u m b e r o f h e a d d i p s m a d e ( A C T H 4_ 10: F 3,42 = 2 . 8 0 , p < 0 . 0 5 , a n d O R G 2766: F 3,36 = 3.67, p < 0.02), t h e t i m e s p e n t h e a d - d i p p i n g ( A C T H 4_ ~o: F 3 , 4 2

= 7.09, p < 0.001, a n d O R G 2 7 6 6 : F 3 , 3 6

= 5 . 9 5 , p < 0 . 0 0 2 ) , a n d l o c o m o t o r a c t i v i t y ( A C T H 4_ ~0: F 3,42 = 4 , 0 2 , p < 0 . 0 2 , a n d ORG

2766: F 3,36

=

5.65, p <

0.003).

However,

as n o s i g n i f i c a n t d r u g

x

trial

i n t e r a c t i o n s o c c u r r e d , it c a n b e c o n c l u d e d t h a t n e i t h e r A C T H 4_ lO n o r O R G 2766 a f f e c t e d t h i s s h o r t - t e r m h a b i t u a t i o n at t h e d o s e s t e s t e d . TABLE I. THE EFFECTSOF INTRASEPTALLYADMINISTEREDACTH 4_ Io (250 ng) AND ORG 2766 (250 pg) ON THE FREQUENCY AND DURATION OF HEAD-DIPS~ AND ON LOCOMOTOR ACTIVITY MEASURED IN A HOLEBOARD

Consecutive 2.5 min trials 1

Number of Head-dips Duration of Head-dipping (sec) Locomotor Activity

Control ACTH4- l0 ORG 2766 Control ACTH4_I0 ORG2766 Control ACTH4_IO ORG2766

7.75 5.88 10.7 43.6 26.5 48.1 94.8 86.8 115.9

+_ 2.20 _+ 1.85 ,+ 2.80 ,+ 12.2 .+ 6.30 .+ 7.60 .+ 9.70 .+ 11.4 .+ I3.7

2

6.38 4.88 7.56 24.9 16.3 25.4 82.9 77.0 102.1

3

_+ 1.86 _+ 1.33 .+ 1,30 _+ 7.40 _+ 3.60 _+ 3.74 .+ 14.3 _+ 12.9 _+ 4.10

5.25 _+ 1.08 4.00 -+ 1.29 6.86_+ 2.60 20.5 .+ 4.90 14.5 _+ 2.50 20.6 _+ 5.60 70.8 _+ 17.6 69.6 .+ 9.60 96.4 _+ 10.7

4

3.88 4.25 5.57 15.9 16.4 10.6 64.0 69.3 92.6

_+ 1.24 _+ 1.25 .+ 1.70 _+ 6.40 ,+ 4.30 _+ 3.20 -+ 12.9 _+ 12.6 _+20.0

The data are expressed as the mean _ S.E.M.

Rat colony intruder test T h e o v e r a l l f r e q u e n c y o f b e h a v i o u r s s c o r e d w a s i n c r e a s e d s i g n i f i c a n t l y b y A C T H 4_ ~0 ( U -- 24, p < 0.05 o n e - t a i l e d ) , a l t h o u g h n o n e o f t h e i n d i v i d u a l p a r a m e t e r s w a s a l t e r e d . O R G 2766 d i d n o t a l t e r a n y o f t h e p a r a m e t e r s ( s e e T a b l e II). TABLE II. THE EFFECTSOF INTRASEPTALLYADMINISTEREDACTH 4_ 1o (250 ng) AND ORG 2766 (250 pg) ON THE MEAN FREQUENCY OF BEHAVIOURS IN THE RAT COLONY INTRUDER TEST

Behaviour Sniff Anogenital sniff Attack Groom Wrestle Approach Being sniffed Being anogenitally sniffed Being groomed Boxing Submitting Squeaking Total behaviours

Control

ACTH4- l0

ORG 2766

0.6 0.0 1.0 0.1 0.9 0.7 1.4

0.6 0.6 1.1 0.1 1.2 0.3 2.3

0.9 0.0 1.2 0.3 0.6 0.4 2.0

0.2 0.6 0.0 0.4 1.8 8.8

0.3 1.7 0.3 0.9 2.8 13.2"

0.3 1.8 0. ! 0.9 1.3 9.9

*p = 0.05 ( M a n n - Whitney U test, one-tailed).

348

ANTHONY CLARKE and SANDRA E. FJtE DISCUSSION

The effects on social interaction following septal administration of ACTH~ ~0 and ORG 2766 parallel those seen after peripheral or intraventricular administration: a decrease after ACTH4_,~ and an increase after ORG 2766. The increase in social interaction after ORG 2766 also parallels the changes found after administration of this peptide in man (Pigache & Rigter, 1981; Sandman et al., 1980). The results from the colony intruder test suggest that the changes in social interaction might be specific to investigatory behaviours, and indeed changes in aggressiveness in man have not been reported following administration of these peptides. The contrast in the effects of the two peptides raises the intriguing possibility that ORG 2766 is acting as an antagonist at sites where ACTH (endogenous and exogenous) is an agonist. In this experiment we did not extend our investigations with ORG 2766 into the ng dose range. It is possible that at such doses different behavioural effects might be found and that this synthetic analogue might act as a partial agonist. The results from other behavioural situations where both peptides have the same effect makes this more likely. Because we have found opposite behavioural effects with A C T H ~ ,~ and ORG 2766, our results cannot be explained by changes in selective attention or in learning and memory, since in these behavioural tests both peptides have effects in the same direction (Miller et al., 1974; Gaillard & Varey, 1979; Sandman & Kastin, 1977a,b; Beckwith & Sandman, 1978; Walker & Sandman, 1979; Gaillard & Varey, 1979; Gold & McGaugh, 1977; Gold e t a l . , 1977; Klein, 1972; Rigter et al., 1974; Rigter & van Riezen, 1975; Kastin et al., 1971; Miller et al., 1974). However, it is unnecessary to demand a single unifying hypothesis for all the actions of a drug or peptide; quite different neural pathways are likely to subserve different behaviours. If the synthetic analogue is an antagonist at least at some of the receptors for A C T H , then opposite effects would be expected; for example, ACTH increasing fear or anxiety (Weiss et al., 1969, 1970; File & Vellucci, 1978), i.e. producing a dysphoric state, and ORG 2766 producing a euphoric state, as suggested from work in humans (Pigache & Rigter, 1981). An increased state of arousal could accompany mood changes in either direction; thus, our results are not incompatible with suggestions that ACTH produces a generalised change in arousal (Bohus, 1975). A.C. was in receipt of a Medical Research Council research studentship. S.E.F. is a Wellcome Trust Senior Lecturer. This work was supported by a Medical Research Council grant held by S.E.F. The video apparatus was purchased with a grant from the Central Research Fund, University of London. We are grateful to Organon for the gift of these peptides. REFERENCES BEC~WITH, B. E. & SANDMAN, C. A. (1978) Behavioral influences of the neuropeptides ACTH and MSH: a methodological review. Neurosci. Biobehav. Rev. 2, 3 1 1 - 338. Bonus, B. (1975). Pituitary peptides and autonomic responses. In Hormones Homeostasis and the Brain. Gispen, W. H., van Wimersma Greidanus, T. J. F., Bohus, B. & de Wied, D. (Eds.). Prog. Brain Res. 42, 275 - 283. BRADBURY, M. W. B. & DAVSON, R. J. (1964) The transport of urea, creatinine and certain monosaccharides between blood and fluid perfusing the cerebral ventricutar system of rabbits. J. Physiol. 170, 195- 211. COLBERN, D., ISAACSON,R. k., BOHUS, B. & GISPEN, W. H. (1977) Limbic-midbrain lesions and ACTH-induced excessive grooming. Life Sci. 2 1 , 3 9 3 - 4 0 2 . DF WlED, D. (1974) Pituitary-adrenal system hormones and behaviour. In The Neurosciences: Third Stttdy I~rogram, Schmitt, F. O. & Worden, F. G. (Eds.), pp. 653-666. MIT Press, Boston. DE WlED, D. (1977) Minireview: peptides and behavior. Life Sci. 20, 195- 204.

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