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Comparative effects of the ACTH 4–9 analogue (ORG 2766), ACTH 4–10 and [d -Phe7] ACTH 4ndash;10 on medial septal self-stimulation behaviour in rats
Neurophurmucology Vol. 21. pp. 909 to 916. 1982 Printed in Great Britain. All rights reserved
0028-3908/82/090909-08$03.00/O Copyright 0 1982 Pergamon Press Ltd
COMPARATIVE EFFECTS OF THE ACTH 4-9 ANALOGUE (ORG 2766), ACTH 4-10 AND CD-PHE7] ACTH 4-10 ON MEDIAL SEPTAL SELF-STIMULATION BEHA?‘IOUR IN RATS M.
FEKETE*.
B. BOHUS,
Rudolf Magnus
L. VAN WOLFSWINKEL.J. M. VAN REE and D. DE WI&
Institute
for Pharmacology, Medical Faculty, Utrecht. The Netherlands (Arcrptrd
University
of Utrecht.
3 March 1982)
Summary-Experiments were performed to examine the effects of various analogues of ACTH on electrical self-stimulation behaviour elicited from the medial septal area using an ascending or descending sequence of stimulus intensities within a session. When an ascending sequence of threshold multiples was used ACTH 4-10 and the ACTH 4-9 analoeue (ORG 2766) enhanced lever oressine for low intensity stimulation but attenuated self-stimulation at greater current intensities. The analogue ORG 2766 appeared to be a thousand times more potent than ACTH 4-10; [D-Phe7] ACTH 4-10 inhibited the response rate at threshold level but was inactive at greater current intensities. The same effect was found following administration of ORG 2766 in a dose which was 20 times greater (1 pg/rat) than that used in the first experiments. Lever pressing was not affected by treatment with ACTH 4410 or ORG 2766 when a descending sequence of stimulus intensities was used within a session. Thus. ACTH-related peptides may affect motivational processes involved in self-stimulation rather than the reward of the stimulation per se. It is suggested that although ORG 2766 mimicked the action of ACTH 4-10 this synthetic peptide may have additional behavioural properties.
Fragments
of
analogues
of
corticotropin
(ACTH)
have lost most if not all of their steroidogenic, melanocyte-stimulating, lipolytic and opiate-like effects. may retain or have even increased behavioural potency (Greven and De Wied, 1973, 1977; De Wied, Witter and Greven, 1975; De Wied, Bohus, Van Ree and Urban, 1978). An example of such a peptide is the ACTH 4-9 analogue (H-Met/O,/-Glu-His-Phe-D-LysPhe-OH; ORG 2766), which has retained less than l/lOOth of the in vitro corticotropic activity, less than l/lOOOth of the melanocyte-stimulating activity (Witter, Greven and De Wied, 1975), about 1/25th of the in vitro lipolytic activity (Opmeer, Van Ree and De Wied, 1978) as compared to ACTH 4-10 and no opiate-like activity (Terenius, Gispen and De Wied, 1975). This analogue is however a thousand times more potent than ACTH 4-10 on extinction of pole-jumping active avoidance behaviour (Greven and De Wied, 1973, 1977; De Wied et al., 1975, 1978; Witter et al., 1975; De Wied, 1977). which
Address all correspondence: Prof. Dr D. De Wied. Rudolf Magnus Institute for Pharmacology. Vondellaan 6, Utrecht. The Netherlands. Key words: Self-stimulation behaviour. medial septum, ACTH 4410. ACTH 4-9 analogue, [D-Phe’] ACTH 4-10. * On leave of absence from the Department of Pathophysiology, University Medical School, Szeged, Hungary. t Reprint requests should be addressed to Prof. Dr D. De Wied. 909
Jouhaneau-Bowers and Le Magnen (1979) have shown that ACTH 1-24 and ACTH 4-10 are positively reinforcing in a self-administration task. Treatment with ACTH 4-10 also resulted in an increase in the response rate of self-stimulation behaviour elicited from the medial septum at small current intensity and in a decrease in response rate at large current intensities, when an ascending sequence of threshold multiples was used within a session (Nyakas, Bohus and De Wied, 1980). Katz (1980) reported that ORG 2766 in large amounts (20pg/kg) increased the response rate of self-stimulation behaviour elicited from the medial forebrain bundle as measured in overnight response records. To investigate whether the markedly increased potency of ORG 2766 as found on extinction of polejumping active avoidance behaviour can be generalized to rewarded behaviour, the present experiments were designed to compare the effects of different doses of ACTH 4-10 and ORG 2766 on self-stimulation behaviour elicited from the medial septum using an ascending or descending sequence of threshold multiples. The effect of CD-Phe’] ACTH 4-10 was also studied in the same paradigm. This peptide contains a D-enantiomer amino acid like ORG 2766 and was found in previous studies to exert an effect on extinction of active avoidance behaviour opposite to that of ACTH l-10 or ACTH 4-10 (Bohus and De Wied, 1966; Van Wimersma Greidanus and De Wied, 1971;
910
Wiegant, Colbern, Gispen, 1978).
M. Van Wimersma
Greidanus
FEKETEpr al.
and
METHODS
Ten male Wistar rats of an inbred strain (CPB-TNO. Zeist. The Netherlands) were used which weighed 200.-220g at the time of operation. They were housed in single cages and kept at room temperature (20-21’C). All animals had access to commercial food and tap water tm ~i~~~~~~,and were kept on an controlled illumination schedule (light between 6 a.m. and 8 p.m.). Sl4rgury Twisted bipolar stimulation electrodes, made of stainless steel wire of 200 p diameter, were implanted stereotaxically under anesthesia (0.1 ml Hypnorm” s.c.). The unisolated tips of the electrodes were aimed at the rostra1 part of the medial septum according to the atlas of De Groot (1967). The coordinates were as follows: AP = 7.4; L = 0.2; D = 0.7.
Experiments were carried out in a Skinner box (30 x 24 x 38 cm) which had a metal 5 x 2 x 1 cm lever protruding from the wall positioned 6cm above the grid floor and a white light. Each lever press delivered a 0.5 set train of biphasic square wave of pulses through the electrode on a continuous reinforcement schedule. Each train consisted of pulses of a frequency of IOOHz, with a pulse width of 0.5 msec and a delay of 0.5 msec between positive and negative pulses. The intensity of the stimulation was varied according to the experimental procedure and was repeatedly checked using an oscilloscope to measure the voltage drop across a 10 kOhm resistor. During the period of stimulation the Light was turned off and keeping the lever pressed did not produce an additional train of pulses. The rats were allowed to self-stimulate for 6-7 days for 20 min daily between 4 and 8 p.m. at a current level above the expected threshold. The threshold current which evoked selfstimulation was measured on the next three days. The current intensity was decreased by steps of IO-20 to ObtA. The current was then increased stepwise and decreased again the same way and there were at least six repeats. The threshold intensity was the smallest current which evoked a significant difference (P < 0.05; Student’s r-test) in the rate of lever pressing between current on and off periods. In the first and second experiments self-stimulation behaviour was studied by using an ascending sequence of threshold multiples within a session (0.0. 0.5, 0.8, 1.0, 1.2, 1.5, 2.0. 3.0, 0.0 times threshold. respectively). The current was increased every 8 min. Three trains of impulses were given externally before the start of each threshold multiples. The rate of lever pressing during the last 6min of each period was
recorded. In order to reach a stabilized self-stimulation pattern the rats were trained for 4 days for 72min daily with the ascending multiples of the threshold current before the peptides were applied. In the third experiment self-stimulation behaviour of the same animals was studied using a descending sequence of threshold multiples within a session (0.0. 3.0, 2.0, 1.5. 1.2, 1.0, 0.8, 0.5, 0.0 times threshold. respectively). In order to reach a stabilized self-stimulation pattern the rats were trained 7 days for 72 min daily with the descending multiples of the threshold current before peptides were applied.
The peptides ACTH 4-10 (H-Met-Glu-His-PheArg-Trp-Gly-OH), [D-Phe’] ACTH 4- IO (H-MetGlu-His-I)-Phe-Arg-Trp-Gly-OH) and ACTH 4--9 analogue (H-Met/O,/-Glu-His-Phe-D-Lys-Phe-OH: ORG 2766) were dissolved in one drop of IO _ ’ N HCI then diluted with 0.9% saline (pH: 6.5.--6.7). All injections were given subcutaneously I hr prior to the behavioural test in a volume of 0.5 ml. Control rats received the same volume of the vehicle. Vehicle injection was administered before the control sessions. The treatments were given in blocks of 4 days during which peptide and vehicle administration were alternated. Each rat received ail three peptides and the different doses in a random order and there were at least two repeats for each treatment.
Placement of the electrodes was examined histologically. The brains were removed and placed in IOY~:;, formalin solution. The localization of the electrodes was determined on 100 p thick frozen sections.
If an animal received the same treatment more than once the mean of the values obtained was calculated. Statistical comparisons of the number of lever presses at each current intensity following vehicle or peptide treatment were performed by a two-way analysis of variance for paired samples. A level of P i 0.05 was accepted as indicating a statistical significant effect. This analysis was followed by paired comparison (Student’s t-test). RESULTS
Locations of the tips of the etectrodes in the medial septum are shown in Figure 1. Septal electrodes stimulated the main body of the medial septal nuclei but extended anteriorly in the diagonal band of Brocca. Current intensity (threshold) inducing self-stimulation behaviour was in the range of 70-370 PA. Following the stabilization of self-stimulation performance no differetlce was found in response rate between saline treatment days, but significant differences (P < 0.005) were observed between animals. This lat-
ACTH 449 analog and self-stimulation
911
8.2
-41 0
I
2
Fig. 1. Localization of the tip of the electrodes in the medial septum. Numbers on the right of the drawings represent the coronal sections according to the atlas of De Groot (1967). ter difference was not related to the localization of the electrodes. In the first experiment. the effects of 15 and 50 lg of ACTH 4-10 and 15 and 50 ng of ORG 2766 were studied on the response rate of self-stimulation using the ascending sequence of threshold multipIes (Fig. 2). The self-stimulation rate of rats depended upon the intensity of stimulating current: at the higher level of current intensity the larger response rate was observed. Both peptides increased the rate of self-stimulation at 0.5 times the threshold current. while the larger doses also increased that at 0.8 and 1.0 times the threshold current. The analogue ORG 2766 (50ng) attenuated the rate of self-stimulation at 2.0 times the threshold current. while the larger dose of both peptides decreased self-stimulation rate at 3.0 times the threshold current. Peptide treatment did not affect response rates at 1.2 and 1.5 times the threshold current or at 0.0 current. On a weight basis ORC 2766 appeared to be thousand times more potent than ACTH 4-10. In the second experiment the effect of 50 pg ACTH 4-10, 50 ng and 1 pg of ORG 2766, IS and 50 pg of (n-Phe’] ACTH 4-10 was compared with saline
given one day before and one day after treatment with peptide. As can be seen in Table 1, ACTH 4-10 increased the response rate of self-stimulation around threshold (at 0.8, 1.0. 1.2 times the threshold current), while this peptide decreased that at 2.0 and 3.0 times the threshold current, but there were no signi~cant differences between the effects of saline given 1 day before or 1 day after treatment with ACTH 410. Fifty ng of ORG 2766 showed the same effects as ACTH 4-10 (Table 1). It increased the response rate at 0.5.0.8, I.0 and 1.2 times the threshold current and decreased the rate at higher multiples (2.0 and 3.0 times threshold). SeIf-stimulation performance of peptide-treated rats returned to the control ievef on the day after treatment. Accordingly, a carry over effect of the peptide could be excluded. A 20 times larger dose of ORG 2766 (1 pg/rat) had an effect opposite to that found after 15 or 50ng and inhibited self-stimulation around threshold currents (0.8, 1.0 and 1.2 times threshoid current). In this dose ORG 2766 had no effect at greater current intensities. Self-stimulation performance of peptide-treated rats returned to the control level on the day after the treatment (Table 1).
r-:
1 -
d 6
ACTH 4-9 analog and
sejr-stipulation
9t3
Fifty /cg of [I,-Phe’] ACTH 4-10 decreased the rate of self-stimulation around threshold (Table I). There was a significant difference at the threshofd current, and no ditferences at greafer current intensities. When the I5 krg dose of [rz-Phe’] ACTH 4-10 was given the response rate tended to be decreased around threshold current, but this difference was not statistically significant. In addition there were no differences between the effects of saline given one day before or one day after treatment with [r>-Phe?] ACTH 4-10. In the third experiment the effect of 50 irg of ACTH 4-10 and 50ng of ORG 2766 was studied on the response rate of self-stimulation using a descending sequence of stimulus intensities within a session. Response rate at the given threshold multiples was not significantly different from that found at the ascending sequence in saline sessions. The various peptides induced no significant differences at either of the current intensities used (Table 2). After completion of this experiment. the rats were using the ascending sequence of tested again threshold multiple within a session. After a stabilized self-stimu~atjon pattern was obtained, the effect of 50 gig ACTH 4-10 and 50 ng URG 2766 was assessed (repeat of experiment I). The data obtained were quite similar to those found previously, in that the peptides increased responding at low intensity stimulation and attenuated self-stimulation of greater current intensities. DlSCUSSlON
The present experiments show that both ACTH 4-10 and ORG 2766 dose-dependently enhanced selfstimulation brhaviour e&cited from the medial septum at tow response rates, while attenuating performance at greater current intensities, when ascending series of stimulus intensities were used within a session. The analogue ORG 2766 was a thousand times more effective than ACTH 4-10. This greater potency of ORG 2766 which has been found earlier in pace-jumping active avoidance behaviour (Greven and De Wied, 1973, 1977; De Wied et &., 1975, 1978; Witter rt nl., 1975) and in passive avoidance behaviour (Greven and De Wied, 1973, 1977; Rigter, Janssens-Elbertse and Van Riezen, 1976; Fekete and De Wied, 1982). may thus be generalized to rewarded beh&our. However, the long-lasting effect at 24 hr as found in active and passive avoidance b&&our (Fekete and De Wied. unpublished). was not observed in the self-stimulation paradigm since the performance of peptide-treated rats returned to the control level on the day after treatment. These results are in agreement with those of Nyakas of a!.. fl98O) who found tbe same effects of ACTH 4-10 with a similar self-stimulation paradigm as used in the present study. Katz. (1980) demonstrated that in,jection of ORG 2766 was followed by facilitation of intracranial self-stimulation in the medial forebrain bundle. This author used much
914 Self-
stimulat8on
Fig. 2. Effects of two different doses of ORG 2766 and ACTH 4-10 on the response rate of selfstimulation behaviour elicited from the medial septum using an ascending sequence of stimulus intensities (0.0, 0.5, 0.8, 1.0. 1.2, 1.5, 2.0, 3.0, 0.0). The threshold multiples are given in the upper part of
eachblock.~P~0.05;~0P~0.02;~~@P~0.01;~0~P~O.l;n=6. larger doses of ORG 2766 (5. 10 and ZO.~rg/kg. i.p.) the electrode was placed in a neuroanatomic~~ily different structure. Nyakas (31~1.. (1980) failed to find an effect of ACTH 4- IO on self-stimulation behaviour in the medial forebrain bundle when an ascending sequence of threshold multiples was used within a session. However, the rate of self-stimulation was increased by ACTH 4-10 when only one current was within each session and the medial forebrain bundle was stimulated at the threshold level but not at 1.2 times the threshold. The present experiments confirm earlier findings that ACTH 4-10 facilitates self-stimulation behaviour when the intensity of stimulation was low and decreased the behaviour when the intensity was high (Nyakas rt ul., 1980). Although this effect could be explained by assuming a rate-dependent effect of peptide treatment. this suggestion is not supported by the other findings. In fact, there was no effect of ACTH 4-10 or ORG 2766 on self-stimulatjon behaviour in the medial septum when a descending sequence of threshold multiples was used within a session. This lack of effect is not likely to be the result of repeated testing and treatment of the animals, since the peptide effect using the ascending sequence was repeated after and
completion of the descending sequence experiments in the same rats. It seems that an initial low response rate is an essential prerequisite for the modulation of behavioural performance by ACTH-related peptides (Nyakas CTal., 1980). This may explain why the effect of ACTH-like peptides is absent when a descending sequence of stimulus intensities is used within a session, when the initial response rate is high. According to Nyakas et ul.. (1980) ACTH-related peptides tend to stabilize behavioural performance under conditions of modification of reward. The present observations with a descending sequence of stimulus intensities indicate that the suggestion of Nyakas et trl. (1980) is valid only when the initial response level is low, due to a low level of incentive motivation. This agrees rather well with the suggestion that ACTHrelated peptides are involved in motivational processes (De Wied, 1977). Interestingly, administration of [D-Phe’j ACTH &IO which has an opposite action to ACTH 4-10 on extinction of active avoidance behaviour (Bohus and De Wied, 1966; Greven and De Wied, 1973; Van Wimersma Greidanus and De Wied, 1971; Wiegant et ul., 1978; Fekete and De Wied, unpublished) and on hippocampal theta activity (Urban, Lopes de Silva,
ACTH 4--9 analog and selk.timulation
915
M. FI-KI:TF er trl
916
Storm van Leeuwen and De Wied, 1974) inhibited self-stimulation at threshold current, but had no effect at greater current intensities. The opposite effect of [I,-Phe’] ACTH 4-10 fragments may be due to an antagonistic effect of this peptide towards ACTH and related peptides of brain origin. This suggestion stems from observations by Gent and Normanton (1980) who showed that a continuous microiontophoretic application of [o-Phe’] ACTH 4-10 reduced the increased firing rate elicited by ACTH I-24 recorded extracellularly in the medullary reticular formation. The large dose of ORG 2766 (1 Lcgirat) had a similar effect on self-stimulation behaviour as did [r>-Phe’] ACTH 4410. This effect cannot be explained by the presence of the I>-enantiomer Lysine since only substitution of I>-Phenylalanine in ACTH-like peptides in position 7 reversed the effect on pole-jumping avoidance behaviour (De Wied rr (I/.. 1975). A similar dual effect of ORG 2766 has been found in passive avoidance behaviour since ng amounts facilitated while pg amounts attenuated passive avoidance responsing (Fekete and De Wied, unpublished). Studies are in progress to investigate the behavioural effects of relatively large doses of ORG 2766 in more detail. AcknoM~lrdyernents--the peptides were kindly supplied by Organon International BV. Oss, The Netherlands. This research was supported by a grant of the Stichting Pharmacologisch Studiefonds. Utrecht. The Netherlands. REFERENCES
Bohus, B. and De Wied, D. (1966). Inhibitory and facilitatory effect of two related peptides on extinction of avoidance behavior. Science 153: 318-320. De Groot. J. (1967). The Rat Forehrui,i in Stereotu.xic Coordinates. North Holland. Amsterdam. De Wied. D. (1967). Peptides and behavior. Life Sc,i. 20: 195 204. De Wied, D.. Witter. A. and Greven. H. M. (1975). Behaviourally active ACTH analogues. Biochrm. Phorntuc. 24: 1463m 1468. De Wied, D., Bohus, B.. Van Ree, J. M. and Urban. I. (1978). Behavioral and electrophysiological effects of peptides related to lipotropin (/I-LPH). J. Pharmuc. r\-p. Thrr. 204: 57@580. Gent. J. P. and Normanton, J. R. (1980). Antagonism of the excitatory action of ACTH (l-24) by t>-Phe’ ACTH
(4 IO) on single neurones in the rat medulla. Br. J. Phtrrmuc. 70: 8OP-8 I P. Greven, H. M. and De Wied, D. (1973). The influence of peptides derived from corticotrophin (ACTH) on performance. Structure activity studies. In: Drug/ Eficrs o)i Nruroendocrine Rrqulation (Zimmermann, E.. Gispen. W. H., Marks. B. H. and De Wied, D.. Eds), Progress in Brain Research. Vol. 39, pp. 429-442. Amsterdam. Elsevier. Greven. H. M. and De Wied. D. (1977). Influence of peptides structurally related to ACTH and MSH on active avoidance behavior in rats. In: f~ronticr.s of Normow Rcwrrrch (Van Wimersma Greidanus. T. B.. Ed.), Vol. 4. pp. 140- 152. Karger. Basel. Jouhaneau-Bowers. M. and Le Magnen. J. 11979). ACTH self-administration in rats. Phclrrnccc. Bir)c~/icrii. Bchtrr. IO: 325m 32X. Katz. R. J. (1980). Effects of an ACTH 449 r&ted peptide upon intracranial self-stimulation and general activity in the rat. P.s~c~hoph~~r~~~u~~~~(~~~!~71 : 67 70. Nyakas, C.. Bohus. B. and De Wied, D. (1980). Etrects of ACTH 4410 on self-stimulation behavior in the rat. P/I!,sib/. Brhun. 24: 759-764. Opmeer. F. A.. Van Ree. J. M. and De Wied, D. (1978). ACTH-induced lipolysis in rat adipocytes: structureactivity relationships. Nuutlyn-Schmiedrhrr!ls Arch. Phtrrmuc. 302:
31 -36.
Rigter. H., Janssens-Elbertse, R. and Van Riezen. H. (1976). Reversal of amnesia by an orally active ACTH 4 9 analogue (ORG 2766). Phurmtrc. Bioc,hem. Behar. 5: Suppl. 1. 53358. Terenius, L.. Gispen, W. H. and De Wied, D. (1975). ACTH-like peptides and opiate receptors in the rat brain: structure- activity studies. Elrr. J. Phurmuc. 33: 3955399. Urban, 1.. Lopes de Silva. F. H., Storm van Leeuwen. W. and De Wied. D. (1974). A frequency shift in the hippocampa theta activity: an electrical correlate of central action of ACTH analogues in the dog? Bruin Rrs. 69: 361-365.
Van Wimersma Greidanus, T. B. and De Wied. D. (1971). Effects of systemic and intracerebral administration of two opposite acting ACTH-related peptides on extinction of conditioned avoidance behavior. Ne~~rorrldoc,rirroloqy
7: 291L301.
Wiegant, V. M.. Colbern, D.. Van Wimersma
Greidanus. T. B. and Gispen, W. H. (1978). Differential behavioral effects of ACTH 4- 10 and (D-Phe’) ACTH 4 IO. Bruin Res. Bull.
3: 167T170.
Witter. A., Greven. H. M. and De Wied, D. (1975). Corrclation between structure. behavioral activity and rate of biotransformation of some ACTH 4- 9 analogs. .I. Phtrrmw r.~p. Ther. 193: X53-860.
0028-3908/82/090909-08$03.00/O Copyright 0 1982 Pergamon Press Ltd
COMPARATIVE EFFECTS OF THE ACTH 4-9 ANALOGUE (ORG 2766), ACTH 4-10 AND CD-PHE7] ACTH 4-10 ON MEDIAL SEPTAL SELF-STIMULATION BEHA?‘IOUR IN RATS M.
FEKETE*.
B. BOHUS,
Rudolf Magnus
L. VAN WOLFSWINKEL.J. M. VAN REE and D. DE WI&
Institute
for Pharmacology, Medical Faculty, Utrecht. The Netherlands (Arcrptrd
University
of Utrecht.
3 March 1982)
Summary-Experiments were performed to examine the effects of various analogues of ACTH on electrical self-stimulation behaviour elicited from the medial septal area using an ascending or descending sequence of stimulus intensities within a session. When an ascending sequence of threshold multiples was used ACTH 4-10 and the ACTH 4-9 analoeue (ORG 2766) enhanced lever oressine for low intensity stimulation but attenuated self-stimulation at greater current intensities. The analogue ORG 2766 appeared to be a thousand times more potent than ACTH 4-10; [D-Phe7] ACTH 4-10 inhibited the response rate at threshold level but was inactive at greater current intensities. The same effect was found following administration of ORG 2766 in a dose which was 20 times greater (1 pg/rat) than that used in the first experiments. Lever pressing was not affected by treatment with ACTH 4410 or ORG 2766 when a descending sequence of stimulus intensities was used within a session. Thus. ACTH-related peptides may affect motivational processes involved in self-stimulation rather than the reward of the stimulation per se. It is suggested that although ORG 2766 mimicked the action of ACTH 4-10 this synthetic peptide may have additional behavioural properties.
Fragments
of
analogues
of
corticotropin
(ACTH)
have lost most if not all of their steroidogenic, melanocyte-stimulating, lipolytic and opiate-like effects. may retain or have even increased behavioural potency (Greven and De Wied, 1973, 1977; De Wied, Witter and Greven, 1975; De Wied, Bohus, Van Ree and Urban, 1978). An example of such a peptide is the ACTH 4-9 analogue (H-Met/O,/-Glu-His-Phe-D-LysPhe-OH; ORG 2766), which has retained less than l/lOOth of the in vitro corticotropic activity, less than l/lOOOth of the melanocyte-stimulating activity (Witter, Greven and De Wied, 1975), about 1/25th of the in vitro lipolytic activity (Opmeer, Van Ree and De Wied, 1978) as compared to ACTH 4-10 and no opiate-like activity (Terenius, Gispen and De Wied, 1975). This analogue is however a thousand times more potent than ACTH 4-10 on extinction of pole-jumping active avoidance behaviour (Greven and De Wied, 1973, 1977; De Wied et al., 1975, 1978; Witter et al., 1975; De Wied, 1977). which
Address all correspondence: Prof. Dr D. De Wied. Rudolf Magnus Institute for Pharmacology. Vondellaan 6, Utrecht. The Netherlands. Key words: Self-stimulation behaviour. medial septum, ACTH 4410. ACTH 4-9 analogue, [D-Phe’] ACTH 4-10. * On leave of absence from the Department of Pathophysiology, University Medical School, Szeged, Hungary. t Reprint requests should be addressed to Prof. Dr D. De Wied. 909
Jouhaneau-Bowers and Le Magnen (1979) have shown that ACTH 1-24 and ACTH 4-10 are positively reinforcing in a self-administration task. Treatment with ACTH 4-10 also resulted in an increase in the response rate of self-stimulation behaviour elicited from the medial septum at small current intensity and in a decrease in response rate at large current intensities, when an ascending sequence of threshold multiples was used within a session (Nyakas, Bohus and De Wied, 1980). Katz (1980) reported that ORG 2766 in large amounts (20pg/kg) increased the response rate of self-stimulation behaviour elicited from the medial forebrain bundle as measured in overnight response records. To investigate whether the markedly increased potency of ORG 2766 as found on extinction of polejumping active avoidance behaviour can be generalized to rewarded behaviour, the present experiments were designed to compare the effects of different doses of ACTH 4-10 and ORG 2766 on self-stimulation behaviour elicited from the medial septum using an ascending or descending sequence of threshold multiples. The effect of CD-Phe’] ACTH 4-10 was also studied in the same paradigm. This peptide contains a D-enantiomer amino acid like ORG 2766 and was found in previous studies to exert an effect on extinction of active avoidance behaviour opposite to that of ACTH l-10 or ACTH 4-10 (Bohus and De Wied, 1966; Van Wimersma Greidanus and De Wied, 1971;
910
Wiegant, Colbern, Gispen, 1978).
M. Van Wimersma
Greidanus
FEKETEpr al.
and
METHODS
Ten male Wistar rats of an inbred strain (CPB-TNO. Zeist. The Netherlands) were used which weighed 200.-220g at the time of operation. They were housed in single cages and kept at room temperature (20-21’C). All animals had access to commercial food and tap water tm ~i~~~~~~,and were kept on an controlled illumination schedule (light between 6 a.m. and 8 p.m.). Sl4rgury Twisted bipolar stimulation electrodes, made of stainless steel wire of 200 p diameter, were implanted stereotaxically under anesthesia (0.1 ml Hypnorm” s.c.). The unisolated tips of the electrodes were aimed at the rostra1 part of the medial septum according to the atlas of De Groot (1967). The coordinates were as follows: AP = 7.4; L = 0.2; D = 0.7.
Experiments were carried out in a Skinner box (30 x 24 x 38 cm) which had a metal 5 x 2 x 1 cm lever protruding from the wall positioned 6cm above the grid floor and a white light. Each lever press delivered a 0.5 set train of biphasic square wave of pulses through the electrode on a continuous reinforcement schedule. Each train consisted of pulses of a frequency of IOOHz, with a pulse width of 0.5 msec and a delay of 0.5 msec between positive and negative pulses. The intensity of the stimulation was varied according to the experimental procedure and was repeatedly checked using an oscilloscope to measure the voltage drop across a 10 kOhm resistor. During the period of stimulation the Light was turned off and keeping the lever pressed did not produce an additional train of pulses. The rats were allowed to self-stimulate for 6-7 days for 20 min daily between 4 and 8 p.m. at a current level above the expected threshold. The threshold current which evoked selfstimulation was measured on the next three days. The current intensity was decreased by steps of IO-20 to ObtA. The current was then increased stepwise and decreased again the same way and there were at least six repeats. The threshold intensity was the smallest current which evoked a significant difference (P < 0.05; Student’s r-test) in the rate of lever pressing between current on and off periods. In the first and second experiments self-stimulation behaviour was studied by using an ascending sequence of threshold multiples within a session (0.0. 0.5, 0.8, 1.0, 1.2, 1.5, 2.0. 3.0, 0.0 times threshold. respectively). The current was increased every 8 min. Three trains of impulses were given externally before the start of each threshold multiples. The rate of lever pressing during the last 6min of each period was
recorded. In order to reach a stabilized self-stimulation pattern the rats were trained for 4 days for 72min daily with the ascending multiples of the threshold current before the peptides were applied. In the third experiment self-stimulation behaviour of the same animals was studied using a descending sequence of threshold multiples within a session (0.0. 3.0, 2.0, 1.5. 1.2, 1.0, 0.8, 0.5, 0.0 times threshold. respectively). In order to reach a stabilized self-stimulation pattern the rats were trained 7 days for 72 min daily with the descending multiples of the threshold current before peptides were applied.
The peptides ACTH 4-10 (H-Met-Glu-His-PheArg-Trp-Gly-OH), [D-Phe’] ACTH 4- IO (H-MetGlu-His-I)-Phe-Arg-Trp-Gly-OH) and ACTH 4--9 analogue (H-Met/O,/-Glu-His-Phe-D-Lys-Phe-OH: ORG 2766) were dissolved in one drop of IO _ ’ N HCI then diluted with 0.9% saline (pH: 6.5.--6.7). All injections were given subcutaneously I hr prior to the behavioural test in a volume of 0.5 ml. Control rats received the same volume of the vehicle. Vehicle injection was administered before the control sessions. The treatments were given in blocks of 4 days during which peptide and vehicle administration were alternated. Each rat received ail three peptides and the different doses in a random order and there were at least two repeats for each treatment.
Placement of the electrodes was examined histologically. The brains were removed and placed in IOY~:;, formalin solution. The localization of the electrodes was determined on 100 p thick frozen sections.
If an animal received the same treatment more than once the mean of the values obtained was calculated. Statistical comparisons of the number of lever presses at each current intensity following vehicle or peptide treatment were performed by a two-way analysis of variance for paired samples. A level of P i 0.05 was accepted as indicating a statistical significant effect. This analysis was followed by paired comparison (Student’s t-test). RESULTS
Locations of the tips of the etectrodes in the medial septum are shown in Figure 1. Septal electrodes stimulated the main body of the medial septal nuclei but extended anteriorly in the diagonal band of Brocca. Current intensity (threshold) inducing self-stimulation behaviour was in the range of 70-370 PA. Following the stabilization of self-stimulation performance no differetlce was found in response rate between saline treatment days, but significant differences (P < 0.005) were observed between animals. This lat-
ACTH 449 analog and self-stimulation
911
8.2
-41 0
I
2
Fig. 1. Localization of the tip of the electrodes in the medial septum. Numbers on the right of the drawings represent the coronal sections according to the atlas of De Groot (1967). ter difference was not related to the localization of the electrodes. In the first experiment. the effects of 15 and 50 lg of ACTH 4-10 and 15 and 50 ng of ORG 2766 were studied on the response rate of self-stimulation using the ascending sequence of threshold multipIes (Fig. 2). The self-stimulation rate of rats depended upon the intensity of stimulating current: at the higher level of current intensity the larger response rate was observed. Both peptides increased the rate of self-stimulation at 0.5 times the threshold current. while the larger doses also increased that at 0.8 and 1.0 times the threshold current. The analogue ORG 2766 (50ng) attenuated the rate of self-stimulation at 2.0 times the threshold current. while the larger dose of both peptides decreased self-stimulation rate at 3.0 times the threshold current. Peptide treatment did not affect response rates at 1.2 and 1.5 times the threshold current or at 0.0 current. On a weight basis ORC 2766 appeared to be thousand times more potent than ACTH 4-10. In the second experiment the effect of 50 pg ACTH 4-10, 50 ng and 1 pg of ORG 2766, IS and 50 pg of (n-Phe’] ACTH 4-10 was compared with saline
given one day before and one day after treatment with peptide. As can be seen in Table 1, ACTH 4-10 increased the response rate of self-stimulation around threshold (at 0.8, 1.0. 1.2 times the threshold current), while this peptide decreased that at 2.0 and 3.0 times the threshold current, but there were no signi~cant differences between the effects of saline given 1 day before or 1 day after treatment with ACTH 410. Fifty ng of ORG 2766 showed the same effects as ACTH 4-10 (Table 1). It increased the response rate at 0.5.0.8, I.0 and 1.2 times the threshold current and decreased the rate at higher multiples (2.0 and 3.0 times threshold). SeIf-stimulation performance of peptide-treated rats returned to the control ievef on the day after treatment. Accordingly, a carry over effect of the peptide could be excluded. A 20 times larger dose of ORG 2766 (1 pg/rat) had an effect opposite to that found after 15 or 50ng and inhibited self-stimulation around threshold currents (0.8, 1.0 and 1.2 times threshoid current). In this dose ORG 2766 had no effect at greater current intensities. Self-stimulation performance of peptide-treated rats returned to the control level on the day after the treatment (Table 1).
r-:
1 -
d 6
ACTH 4-9 analog and
sejr-stipulation
9t3
Fifty /cg of [I,-Phe’] ACTH 4-10 decreased the rate of self-stimulation around threshold (Table I). There was a significant difference at the threshofd current, and no ditferences at greafer current intensities. When the I5 krg dose of [rz-Phe’] ACTH 4-10 was given the response rate tended to be decreased around threshold current, but this difference was not statistically significant. In addition there were no differences between the effects of saline given one day before or one day after treatment with [r>-Phe?] ACTH 4-10. In the third experiment the effect of 50 irg of ACTH 4-10 and 50ng of ORG 2766 was studied on the response rate of self-stimulation using a descending sequence of stimulus intensities within a session. Response rate at the given threshold multiples was not significantly different from that found at the ascending sequence in saline sessions. The various peptides induced no significant differences at either of the current intensities used (Table 2). After completion of this experiment. the rats were using the ascending sequence of tested again threshold multiple within a session. After a stabilized self-stimu~atjon pattern was obtained, the effect of 50 gig ACTH 4-10 and 50 ng URG 2766 was assessed (repeat of experiment I). The data obtained were quite similar to those found previously, in that the peptides increased responding at low intensity stimulation and attenuated self-stimulation of greater current intensities. DlSCUSSlON
The present experiments show that both ACTH 4-10 and ORG 2766 dose-dependently enhanced selfstimulation brhaviour e&cited from the medial septum at tow response rates, while attenuating performance at greater current intensities, when ascending series of stimulus intensities were used within a session. The analogue ORG 2766 was a thousand times more effective than ACTH 4-10. This greater potency of ORG 2766 which has been found earlier in pace-jumping active avoidance behaviour (Greven and De Wied, 1973, 1977; De Wied et &., 1975, 1978; Witter rt nl., 1975) and in passive avoidance behaviour (Greven and De Wied, 1973, 1977; Rigter, Janssens-Elbertse and Van Riezen, 1976; Fekete and De Wied, 1982). may thus be generalized to rewarded beh&our. However, the long-lasting effect at 24 hr as found in active and passive avoidance b&&our (Fekete and De Wied. unpublished). was not observed in the self-stimulation paradigm since the performance of peptide-treated rats returned to the control level on the day after treatment. These results are in agreement with those of Nyakas of a!.. fl98O) who found tbe same effects of ACTH 4-10 with a similar self-stimulation paradigm as used in the present study. Katz. (1980) demonstrated that in,jection of ORG 2766 was followed by facilitation of intracranial self-stimulation in the medial forebrain bundle. This author used much
914 Self-
stimulat8on
Fig. 2. Effects of two different doses of ORG 2766 and ACTH 4-10 on the response rate of selfstimulation behaviour elicited from the medial septum using an ascending sequence of stimulus intensities (0.0, 0.5, 0.8, 1.0. 1.2, 1.5, 2.0, 3.0, 0.0). The threshold multiples are given in the upper part of
eachblock.~P~0.05;~0P~0.02;~~@P~0.01;~0~P~O.l;n=6. larger doses of ORG 2766 (5. 10 and ZO.~rg/kg. i.p.) the electrode was placed in a neuroanatomic~~ily different structure. Nyakas (31~1.. (1980) failed to find an effect of ACTH 4- IO on self-stimulation behaviour in the medial forebrain bundle when an ascending sequence of threshold multiples was used within a session. However, the rate of self-stimulation was increased by ACTH 4-10 when only one current was within each session and the medial forebrain bundle was stimulated at the threshold level but not at 1.2 times the threshold. The present experiments confirm earlier findings that ACTH 4-10 facilitates self-stimulation behaviour when the intensity of stimulation was low and decreased the behaviour when the intensity was high (Nyakas rt ul., 1980). Although this effect could be explained by assuming a rate-dependent effect of peptide treatment. this suggestion is not supported by the other findings. In fact, there was no effect of ACTH 4-10 or ORG 2766 on self-stimulatjon behaviour in the medial septum when a descending sequence of threshold multiples was used within a session. This lack of effect is not likely to be the result of repeated testing and treatment of the animals, since the peptide effect using the ascending sequence was repeated after and
completion of the descending sequence experiments in the same rats. It seems that an initial low response rate is an essential prerequisite for the modulation of behavioural performance by ACTH-related peptides (Nyakas CTal., 1980). This may explain why the effect of ACTH-like peptides is absent when a descending sequence of stimulus intensities is used within a session, when the initial response rate is high. According to Nyakas et ul.. (1980) ACTH-related peptides tend to stabilize behavioural performance under conditions of modification of reward. The present observations with a descending sequence of stimulus intensities indicate that the suggestion of Nyakas et trl. (1980) is valid only when the initial response level is low, due to a low level of incentive motivation. This agrees rather well with the suggestion that ACTHrelated peptides are involved in motivational processes (De Wied, 1977). Interestingly, administration of [D-Phe’j ACTH &IO which has an opposite action to ACTH 4-10 on extinction of active avoidance behaviour (Bohus and De Wied, 1966; Greven and De Wied, 1973; Van Wimersma Greidanus and De Wied, 1971; Wiegant et ul., 1978; Fekete and De Wied, unpublished) and on hippocampal theta activity (Urban, Lopes de Silva,
ACTH 4--9 analog and selk.timulation
915
M. FI-KI:TF er trl
916
Storm van Leeuwen and De Wied, 1974) inhibited self-stimulation at threshold current, but had no effect at greater current intensities. The opposite effect of [I,-Phe’] ACTH 4-10 fragments may be due to an antagonistic effect of this peptide towards ACTH and related peptides of brain origin. This suggestion stems from observations by Gent and Normanton (1980) who showed that a continuous microiontophoretic application of [o-Phe’] ACTH 4-10 reduced the increased firing rate elicited by ACTH I-24 recorded extracellularly in the medullary reticular formation. The large dose of ORG 2766 (1 Lcgirat) had a similar effect on self-stimulation behaviour as did [r>-Phe’] ACTH 4410. This effect cannot be explained by the presence of the I>-enantiomer Lysine since only substitution of I>-Phenylalanine in ACTH-like peptides in position 7 reversed the effect on pole-jumping avoidance behaviour (De Wied rr (I/.. 1975). A similar dual effect of ORG 2766 has been found in passive avoidance behaviour since ng amounts facilitated while pg amounts attenuated passive avoidance responsing (Fekete and De Wied, unpublished). Studies are in progress to investigate the behavioural effects of relatively large doses of ORG 2766 in more detail. AcknoM~lrdyernents--the peptides were kindly supplied by Organon International BV. Oss, The Netherlands. This research was supported by a grant of the Stichting Pharmacologisch Studiefonds. Utrecht. The Netherlands. REFERENCES
Bohus, B. and De Wied, D. (1966). Inhibitory and facilitatory effect of two related peptides on extinction of avoidance behavior. Science 153: 318-320. De Groot. J. (1967). The Rat Forehrui,i in Stereotu.xic Coordinates. North Holland. Amsterdam. De Wied. D. (1967). Peptides and behavior. Life Sc,i. 20: 195 204. De Wied, D.. Witter. A. and Greven. H. M. (1975). Behaviourally active ACTH analogues. Biochrm. Phorntuc. 24: 1463m 1468. De Wied, D., Bohus, B.. Van Ree, J. M. and Urban. I. (1978). Behavioral and electrophysiological effects of peptides related to lipotropin (/I-LPH). J. Pharmuc. r\-p. Thrr. 204: 57@580. Gent. J. P. and Normanton, J. R. (1980). Antagonism of the excitatory action of ACTH (l-24) by t>-Phe’ ACTH
(4 IO) on single neurones in the rat medulla. Br. J. Phtrrmuc. 70: 8OP-8 I P. Greven, H. M. and De Wied, D. (1973). The influence of peptides derived from corticotrophin (ACTH) on performance. Structure activity studies. In: Drug/ Eficrs o)i Nruroendocrine Rrqulation (Zimmermann, E.. Gispen. W. H., Marks. B. H. and De Wied, D.. Eds), Progress in Brain Research. Vol. 39, pp. 429-442. Amsterdam. Elsevier. Greven. H. M. and De Wied. D. (1977). Influence of peptides structurally related to ACTH and MSH on active avoidance behavior in rats. In: f~ronticr.s of Normow Rcwrrrch (Van Wimersma Greidanus. T. B.. Ed.), Vol. 4. pp. 140- 152. Karger. Basel. Jouhaneau-Bowers. M. and Le Magnen. J. 11979). ACTH self-administration in rats. Phclrrnccc. Bir)c~/icrii. Bchtrr. IO: 325m 32X. Katz. R. J. (1980). Effects of an ACTH 449 r&ted peptide upon intracranial self-stimulation and general activity in the rat. P.s~c~hoph~~r~~~u~~~~(~~~!~71 : 67 70. Nyakas, C.. Bohus. B. and De Wied, D. (1980). Etrects of ACTH 4410 on self-stimulation behavior in the rat. P/I!,sib/. Brhun. 24: 759-764. Opmeer. F. A.. Van Ree. J. M. and De Wied, D. (1978). ACTH-induced lipolysis in rat adipocytes: structureactivity relationships. Nuutlyn-Schmiedrhrr!ls Arch. Phtrrmuc. 302:
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Rigter. H., Janssens-Elbertse, R. and Van Riezen. H. (1976). Reversal of amnesia by an orally active ACTH 4 9 analogue (ORG 2766). Phurmtrc. Bioc,hem. Behar. 5: Suppl. 1. 53358. Terenius, L.. Gispen, W. H. and De Wied, D. (1975). ACTH-like peptides and opiate receptors in the rat brain: structure- activity studies. Elrr. J. Phurmuc. 33: 3955399. Urban, 1.. Lopes de Silva. F. H., Storm van Leeuwen. W. and De Wied. D. (1974). A frequency shift in the hippocampa theta activity: an electrical correlate of central action of ACTH analogues in the dog? Bruin Rrs. 69: 361-365.
Van Wimersma Greidanus, T. B. and De Wied. D. (1971). Effects of systemic and intracerebral administration of two opposite acting ACTH-related peptides on extinction of conditioned avoidance behavior. Ne~~rorrldoc,rirroloqy
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Wiegant, V. M.. Colbern, D.. Van Wimersma
Greidanus. T. B. and Gispen, W. H. (1978). Differential behavioral effects of ACTH 4- 10 and (D-Phe’) ACTH 4 IO. Bruin Res. Bull.
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Witter. A., Greven. H. M. and De Wied, D. (1975). Corrclation between structure. behavioral activity and rate of biotransformation of some ACTH 4- 9 analogs. .I. Phtrrmw r.~p. Ther. 193: X53-860.