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An ACTH4–9 analog (ORG 2766) speeds recovery from septal hyperemotionality in the rat
BEHAVIORAL AND NEURAL BIOLOGY
39, 52-59 (1983)
An ACTH4_9Analog (ORG 2766) Speeds Recoveryfrom Septal Hyperemotionalityin the Rat ROBERT L . ISAACSON
Department of Psychology and Center for Neurobehavioral Sciences, State University of New York at Binghamton, Binghamton, New York 13901 AND ALEX POPLAWSKY
Department of Psychology, Bloomsburg State College, BIoomsburg, Pennsylvania 17815 The amount of hyperemotionality initially demonstrated after septal area lesions was reduced, and the rate at which the hyperemotionality attenuated over repeated testing, was enhanced by the administration of an ACTH4_9 analog, ORG 2766. This ACTH fragment was given for 4 consecutive days after surgery but terminated before testing began. Two weeks after the daily tests of emotionality, the animals were trained in a two-way active avoidance task. The typical increase in avoidance behavior seen in animals with septal lesions was observed in the lesioned animals tested with ORG 2766, but the usual high number of intertrial responses was greatly reduced in these animals. The results indicate that even after a brief series of ORG 2766 administration, there are changes in emotionality that may last for an extended period of time after the cessation of treatment.
The possibility that ACTH or certain of its fragments could produce beneficial effects after damage to the nervous system has been investigated intermittantly over the last 30 years. However, only modest beneficial results have been found. For example, some limited restoration of function has been reported in a minority of animals after spinal cord damage (Windle, Clemente, & Chambers, 1952; Clemente & Windle, 1954; McMasters, 1962). Histological evaluations of the changes at the site of damage have indicated reduced collagen deposits, fibroblast proliferation, and connective tissue invasion following ACTH treatment (Fertig, Kiernan, & Seyan, 1971; Berry, Knowles, Willis, Riches, Morgans, & Steers, 1979). The effects of the neuropeptide treatment, in experiments in which they have been observed, seem to occur in the first few days after the damage. 52
0163-1047/83 $3.00 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
ACTH AND RECOVERY FROM SEPTAL LESIONS
53
The systemic administration of ACTHj_39 enhances functional and structural changes after the crushing of peripheral nerve in the rat. In ACTHLtreated animals and in animals with elevated ACTH levels subsequent to adrenalectomy, normal and heat-evoked foot movements return more quickly after nerve crush than in control animals (Strand & Kung, 1980; Saint-C6me, Acker, & Strand, 1982). After the crush, sprouting axons grow more rapidly than in saline-treated control animals, develop larger motor endplates, and have more preterminal branching. No neuropeptide effects are found on types of muscle fibers or protein synthesis in the muscles. The enhanced regeneration following increased ACTH availability is hypothesized to result from enhanced synthesis or preferential delivery of neurotransmitter or neurotrophic substances to the sprouting axons (Strand & Smith, 1980). Recent investigations confirmed the fact that ACTH administration facilitates functional recovery after peripheral nerve crush and that the administration of certain N-terminal ACTH fragments can produce similar effects (Bijlsma, Jennekens, Schotman, & Gispen, 1981). One effect of ACTH and its effective fragments seems to be an enhancement of the number of regenerating fibers (Bijlsma, Jennekens, Schotman, & Gispen, 1983). Fragments containing the 4-9 amino acid sequence have the ability to hasten recovery after peripheral nerve damage. The ACTHI~_24 fragment did not. The effective fragments included ones with behavioral but not corticosteroid potency, including the modified ACTH4_9 fragment ORG 2766. The neuropeptide, ORG 2766 (H-Met/0J-Glu-His-Phe-D-Lys-Phe-OH), is a structural variant of ACTH4_ 9 and has been reported to be one thousand times more potent than other N-terminal fragments as measured by its effects on the extinction of an active avoidance response (de Wied, Witter, & Greven, 1975; Greven & de Wied, 1977). Overall, ORG 2766 shares many behavioral effects with ACTH4_J0 and ACTH4_7 fragments, although there is evidence that it may have some unique properties in some testing situations (Greven & de Wied, 1973; Opmeer, Van Ree, & de Wied, 1978; Terenius, Gispen, & de Wied, 1975; Fekete & de Wied, 1982). Many behavioral changes found after damage to the central nervous system, both for which some "recovery" may occur and those that have progressive debilitation effects, are due to secondary reactions occurring in remaining neural systems (Isaacson, 1975; Schoenfeld & Hamilton, 1977). Given that N-terminal ACTH fragments can facilitate the regeneration of peripheral nerve fibers and hasten functional recovery, an issue of considerable theoretical and practical importance is whether or not the administration of ACTH fragments could influence the course of central nervous system damage. In an attempt to do this, we elected to study the behavioral effects of ORG 2766 administered for several days after experimentally induced destruction of the septal area in rats. In particular
54
ISAACSON AND POPLAWSKY
we studied its effects on the enhanced reactivity initially found after the lesion and the facilitated performance in an active avoidance task which is found at all times after the lesion. The hyperreactivity, often called "septal rage," is largely an emotional change, but the change in the avoidance task is best understood as a change in the performance of a learned behavior. The hyperreactivity induced by the lesion declines progressively over time and even more rapidly when the animals are repeatedly tested (Brady & Nauta, 1953; Reynolds, 1965). The facilitation of performance of an active avoidance response, on the other hand, appears to be a permanent consequence of the septal lesion (Poplawsky, 1978). This suggests that after the damage the progressive changes in emotionality could represent functional or structural alterations that might be susceptible to alteration by ACTH or its effective fragments. The purpose of this study was to investigate the effects of ORG 2766 on the time course of the emotionality changes following septal lesions from postoperative Day 3 to Day 12, and later on performance in a twoway avoidance task.
METHODS Thirty-one adult female albino rats of the Sprague-Dawley strain (Taconic Farms, N.Y.) with an average weight of 250 g were used in this experiment. Each rat was housed individually with free access to food and water. The colony room was on a 12/12-hr light-dark cycle with lights on at 7:00 AM. The test apparatus used in the avoidance task was an automated dualcompartment shuttlebox contained within a sound-attenuated cubicle. The shuttlebox was a Lehigh Valley Model 146-04 enclosed in that company's sound attenuating chamber, Model 132-06. A 28 V dc light bulb was mounted 14 cm above the shock grids in the center of the far wall of each compartment. Shock was delivered to the floor grid by a Coulbourn shocker-distributor, Model E13-16. One rat from the group with septal lesions given ORG 2766 was eliminated from the avoidance task analysis because of an equipment failure during its testing. A barrier separated the two compartments. The "toggle floor" of the chamber reported the animals' location both during and between trials. Sessions were controlled automatically by solid state circuitry. Prior to surgery each rat was rated for emotionality and given a score of 0-5 on five rating scales: reaction to object presentation, response to tap on back, resistance to capture, resistance to handling, and vocalization to capture and handling (King, 1958). Rats were matched on preoperative emotionality scores and assigned to the following groups: rats with septal lesions and vehicle injections (Groups SEP; n = 10), rats with septal lesions and ORG 2766 injections (Group SEP 2766; n = l 1), and rats with control operations and vehicle injections (Group CON; n = 10).
ACTH AND RECOVERY FROM SEPTAL LESIONS
55
The rats were anesthetized with ethyl ether and placed in a Kopf stereotaxic instrument. Anodal electrolytic lesions were accomplished stereotaxically using a stainless-steel electrode insulated by epoxy enamel except for 0.5 mm closest to the tip and a current of 1.5 mA was passed for 20 sec. The coordinates for the lesion were 9.5 mm anterior to frontal plane zero, _+0.75 mm lateral to midline, and 5.25 mm below brain surface. The incisor bar was adjusted until lambda and bregma were on the same horizontal plane. The control operation consisted of the same procedures without current being passed through the electrode. Rats in Group SEP 2766 were injected subcutaneously with 0.5 ml of 1 ~g ACTH4_9 analog (H-MET/02/Glu-His-Phe-D-Lys-Phe-OH; ORG 2766) dissolved in a 0.9% saline solution on the day of surgery and on postsurgical Days 1, 2, and 3. Groups SEP and CON received subcutaneous injections of the saline solution on these 4 days. All rats were lightly anesthetized with ether before injections were administered. The rats were rated for emotionality on postsurgical Day 3 (before the last injection) until postsurgery Day 12 (10 days). The experimenter was unaware of the experimental groups during emotionality scoring. On postsurgery Day 35, the rats were tested in the two-way shuttle avoidance chamber for 120 trials. A trial was started by presenting the conditioned stimulus (cue lights on) approximately 30 sec after each rat was placed in the dark shuttlebox. If the rat did not cross into the other half of the chamber within 10 sec of the light onset, a continuous shock of 1 mA was applied to the grid floor of the side of the apparatus. Avoidance responses, escape responses, and intertrial crossing were recorded. Responses made during the intertrial interval were not punished. Avoidance responses were crossings into the other compartment of the shuttlebox during the 10 sec following light onset. Escape responses were crossing made between 10 and 13 sec after light onset. Both types of responses terminated the light and shock (if applied). If the rat did not make a response after 3 sec of shock, the 13th sec after light onset, the trial was terminated. The intertrial interval was determined by a random time schedule of 30 sec with a minimum interval of 7.5 sec. Approximately 10 weeks after surgery, all rats were placed under deep chloral hydrate anesthesia (400 mg/kg; ip) and perfused intracardially with a physiological saline solution followed by a 10% Formalin-saline solution. Brains were extracted, and frozen brain sections were made every 80 b~, saving every second section through the extent of the lesion. The sections were stained with cresyl violet and lesion damage was evaluated.
RESULTS Histological evaluations revealed that for most rats the septal lesions extended from the genu of the corpus callosum to the columns of the
56
ISAACSON AND POPLAWSKY
fornix (anterior-posterior) and from the corpus callosum to the anterior commissure (dorsal-ventral). However, six of these rats also had damage to the anterior commissure, and a few had only small damage to the posterior third of the septum. Similar septal lesions were found in groups SEP and SEP 2766. A 3 x 10 mixed-design analysis of variance was used to analyze the emotionality ratings. The specific factors were groups (SEP, SEP 2766, and CON) and the l0 scoring days. A 3 x 2 mixed-design was used to analyze avoidances and intertrial crossings, where the between-factor was the three groups and the within-factor was two blocks of 60 trials. Figure 1 presents the mean emotionality scores for the three groups of rats over 10 postoperative observation days. There was a significant interaction between groups and test days (F(18,252) -- 7.62, p < .01). The group of rats with septal damage and the ACTH fragment had lower emotionality scores (p < .05) than the group with septal lesions and saline injections on Days 3, 4, 5, 6, and 7 after surgery. The ACTHtreated lesion group also reached control levels of emotionality by postoperation Day 12. The analyses of the data from the avoidance task showed that rats in both groups with septal damage had high levels of avoidances (82 and 83%, respectively) compared to the control group ( 5 2 % ) , (F(2,27) • 8.97, p < ,01). However, the untreated septal lesion animals made significantly more intertrial crossings than the nonlesioned control group (a mean of 100 as compared to 38). The group of rats with 16
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13
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12
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II
I,o :S hi Z
8
I0 9
7 6 5 4 3 2
I ;5
4
5 6 7 8 9 I0 II DAYS A F T E R SURGERY
12
FIG. 1. Mean emotionality scores of'animals with septal lesions treated with saline (triangles), septal lesions treated with ORG 2766 (open circles), and control animals (closed circles) over repeated tests beginning the third day after surgery.
ACTH AND RECOVERY FROM SEPTAL LESIONS
57
septal damage treated with ORG 2766 had approximately the same number of intertrial crossings as the nonlesioned group. DISCUSSION The repeated administration of the ACTH4_9 fragment, ORG 2766, ameliorates some of the usual behavioral consequences of septal area damage and hastens the return to near normal emotional responsiveness, since the septal lesion group receiving ORG 2766 reached control levels of emotionality by postoperative Day 6, while the lesioned animals without the neuropeptide fragment reached control levels on postoperative Day 12. Other studies have also reported that the hyperemotionality following septal lesions dissipated by the 12th postoperative day (e.g., Brady & Nauta, 1953). These results indicate that rats with septal lesions treated with ORG 2766 both had lower emotionality scores early in testing and reached normal levels of emotionality sooner than untreated rats with septal lesions. This could be interpreted as an acceleration of normal readjustive activities occurring in remaining brain systems. However, not all lesion-induced changes are affected by the administration of ORG 2766. Only those behaviors which are of an emotional nature or ones likely related to emotional responsiveness (intertrial crossings) were affected. The commonly observed facilitation of the acquisition of a two-way active avoidance problem was not changed. This lesioninduced behavior, in contrast to septal hyperemotionality, does not seem to change in a progressive fashion after the lesion. On the other .hand, intertrial crossings do decrease over testing sessions in the avoidance problem (Poplawsky, 1978). In the present study the intertrial crossings for the lesioned group receiving ORG 2766 was almost half that of nontreated group on the first avoidance test day. This suggests that prolonged alterations in emotionality or hyperreactivity are affected by treatment with ORG 2766 but the associational processes underlying avoidance conditioning were not. The mechanisms whereby ORG 2766 influences the lesion-induced behaviors are uncertain. It is unlikely that it does so by affecting endocrine systems since septal hyperreactivity is not itself the consequence of endocrine changes. Hyperemotionality is frequently observed as soon as the animal comes out of the anesthesia. Furthermore, castration, adrenalectomy, or hypophysectomy fail to attenuate the septal syndrome (Montgomery, Berkut, Grubb, & Westbrook, 1971; Seggie, 1971; Phillips & Lieblich, 1972). It is possible that the peptide fragment produces its effects by alteration of glial or connective tissue responses to damage, although if this is the case it would have to be assumed that such responses account for the reduction of septal emotionality rather than its production. Assuming a similarity in effect between ORG 2766 and the ACTH4_I0 analog, it is POssible that the beneficial effect could be due to an enhanced
58
ISAACSON AND POPLAWSKY
catecholamine turnover (Versteeg & Wurtman, 1975), producing an effect similar to the beneficial effect produced by the administration of L-dopa (Gage & Olton, 1975; Marotta, Logan, potegal, Glusman, & Gardner, 1977). However, if this is the case, the effect far outlasts the time period at which the neuropeptide had been acting and the catecholamine turnover enhanced, since the reduction of intertrial crossings was found at more than 20 days after the end of the neuropeptide treatment. It is also possible that the ACTH4_9 analog produced its effects by action on the generation of proliferation of axonal sprouts in remaining brain tissue, as has been demonstrated in peripheral nerve. The fact that a relatively brief period of administration of ORG 2766 has both immediate and prolonged effects on emotionality and reactivity has practical and theoretical importance. Some of the effects reported following ORG 2766 in people, such as increased sociability, gregariousness, and reduced anxiety (Gaillard & Varey, 1979; Sandman, Walker, & Lawton, 1980), may be related to the emotional changes found in the present experiments. REFERENCES Berry, M., Knowles, J., Willis, P., Riches, A. C., Morgans, G. P., & Steers, D. (1979). A reappraisal of the effects of ACTH on the response of the central nervous system to injury. Journal of Anatomy, 128, 859-871. Bijlsma, W. A., Jennekens, F. G. I., Schotman, P., & Gispen, W. H. (1981). Effects of corticotrophin (ACTH) on recovery of sensorimotor function in the rat: Structureactivity study. European Journal of Pharmacology, 76, 73-79. Bijlsma, W. A., Jennekens, F. G. 1., Schotman, P., & Gispen, W. H. (1983). Stimulation by ACTH4_~0 of nerve fiber regeneration following sciatic nerve crush. Muscle and Nerve, 6, 102-110. Brady, J. V., & Nauta, W. H. J. (1953). Subcortical mechanisms in emotional behaviour: Affective changes following septal forebrain lesions in the albino rat. Journal of Comparative and Physiological Psychology, 46, 339-346. Clemente, C. D., & Windle, W. F. (1954). Regeneration of severed nerve fibers in the spinal cord of the adult cat. Journal of Comparative Neurology, 101, 691-731. de Wied, D., Witter, A., & Greven, H. M. (1975). Behaviorally active ACTH analogues. Biochemical Pharmacology, 24, 1463-1468. Fekete, M., & de Wied, D. (1982). Potency and duration of action of the ACTH4_9 analog (ORG 2766) as compared to ACTH,_lo and D-Phe 7 ACTH4 10 on active and passive avoidance behavior of rats. Pharmacology Biochemistry and Behavior, 16, 387-392. Fertig, A., Kiernan, J. A., & Seyan, S. S. A. S. (1971). Enhancement ofaxonal regeneration in the brain of the rat by corticotrophin and triiodorthyronine. Experimental Neurology, 33, 372-385. Gage, F. H., I I I , & Olton, D. S. (1975). Hippocampal influence on septal hyperreactivity. Brain Research, 98, 311-325. Gaillard, A. W. K., & Varey, C. A. (1979). Some effects of an ACTH4_9 analog (ORG 2766) on human performance. Physiology and Behavior, 23, 79-84. Greven, H. M., & de Wied, D. (1973). The influence of peptides derived from corticotrophin (ACTH) on performance. Structure activity studies. In E. Zimmerman, W. H. Gispen,
ACTH AND RECOVERY FROM SEPTAL LESIONS
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B. H. Marks, & D. de Wied (Eds.), Progress in Brain Research, Vol. 39, pp. 429442. Amsterdam: Elsevier. Greven, H. M., & de Wied, D. (1977). Influence of peptides structurally related to ACTH and MSH on active avoidance behaviour in rats. In T. V. van Wimersma Greidanus (Ed.), Frontiers of Hormone Research, Vol. 4, pp. 140-152. Basel: Karger. Isaacson, R. L. (1975). The myth of recovery from early brain damage. In N. R. Ellis (Ed.), Aberrant Development in Infancy: Human and Animal Studies, pp. 1-26. Potomac, Md. King, F. A. (1958). Effects of septal and amygdala lesions on emotional behavior and conditioned avoidance responses in the rat. Journal of Nervous and Mental Diseases, 126, 57-63. McMasters, R. E. (1962). Regeneration of the spinal cord in the rat. Effects of Piromen and ACTH upon the regenerative capacity. Journal of Comparative Neurology, 119, 113-125. Marotta, R. F., Logan, N., Potegal, M., Glusman, M., & Gardner, E. L. (1977). Dopamine agonists induce recovery from surgically-induced septal rage. Nature, (London), 269, 513-515. Montgomery, R. L., Berkut, M. K., Grubb, E. F., & Westbrook, D. L. (1971). Hormonal influence on behavior in brain lesioned male rats. Physiology and Behavior, 7, 107111. Opmeer, F. A., van Ree, J. M., & de Wied, D. (1978). ACTH-induced lipolysis in rat adipocytes: Structure-activity relationship. Naunyn-Schmiedeberg's Archives of Pharmacology, (1972). 302, 31-36. Phillips, A. G., & Lieblich, 1. (1972). Developmental and hormonal aspects of hyperemotionality produced by septal lesions in male rats. Physiology and Behavior, 9, 237-242. Poplawsky, A. (1978). Long-term maintenance of shuttlebo× avoidance behavior before and after septal lesions. Physiological Psychology, 6, 294-299. Reynolds, R. (1965). Equivalence of radio frequency and electrolytic lesions in producing septal rage. Psychonomic Science, 2, 35-36. Saint-C6me, C., Acker, G. R., & Strand, F. L. (1982). Peptide influences on the development and regeneration of motor performance. Peptides, 3, 439-449. Sandman, C. A., Walker, B. B., & Lawton, C. A. (1980). An analog of MSH/ACTH 4-9 enhances interpersonal and environmental awareness in mentally retarded adults. Peptides, 1, 109-114. Schoenfeld, T. A., & Hamilton, L. W. (1977). Secondary brain changes following lesions: A new paradigm for lesion experimentation. Physiology and Behavior, 18, 951-967. Seggie, J. (1971). Effect of adrenalectomy or gonadectomy on affective behavior changes following septal lesions in the rat. Journal of comparative and physiological Psychology, 74, 11-19. Strand, F. L., & Kung, T. T. (1980). ACTH accelerates recovery of neuromuscular function following crushing of peripheral nerve. Peptides, 1, 135-138. Strand, F. L., & Smith, C. M. (1980). LPH, ACTH, MSH, and motor systems. Pharmacology and Therapeutics, 11, 509-533. Terenius, L., Gispen, W. H., & de Wied, D. (1975). ACTH-like peptides and opiate receptors in the rat brain: Structure-activity studies. European Journal of Pharrnacology, 33, 395-399. Versteeg, D. H. G., & Wurtman, R. J. (1975). Effect of ACTH4_m on the rate of synthesis of [3H]catecholamines in the brains of intact, hypophysectomized, and adrenalectomized rats. Brain Research, 93, 552-557. Windle, W. F., Clemente, C. D., & Chambers, W. W. (1952). Inhibition of formation of a glial barrier as a means of permitting a peripheral nerve to grow into the brain. Journal of comparative Neurology, 96, 359-370.
39, 52-59 (1983)
An ACTH4_9Analog (ORG 2766) Speeds Recoveryfrom Septal Hyperemotionalityin the Rat ROBERT L . ISAACSON
Department of Psychology and Center for Neurobehavioral Sciences, State University of New York at Binghamton, Binghamton, New York 13901 AND ALEX POPLAWSKY
Department of Psychology, Bloomsburg State College, BIoomsburg, Pennsylvania 17815 The amount of hyperemotionality initially demonstrated after septal area lesions was reduced, and the rate at which the hyperemotionality attenuated over repeated testing, was enhanced by the administration of an ACTH4_9 analog, ORG 2766. This ACTH fragment was given for 4 consecutive days after surgery but terminated before testing began. Two weeks after the daily tests of emotionality, the animals were trained in a two-way active avoidance task. The typical increase in avoidance behavior seen in animals with septal lesions was observed in the lesioned animals tested with ORG 2766, but the usual high number of intertrial responses was greatly reduced in these animals. The results indicate that even after a brief series of ORG 2766 administration, there are changes in emotionality that may last for an extended period of time after the cessation of treatment.
The possibility that ACTH or certain of its fragments could produce beneficial effects after damage to the nervous system has been investigated intermittantly over the last 30 years. However, only modest beneficial results have been found. For example, some limited restoration of function has been reported in a minority of animals after spinal cord damage (Windle, Clemente, & Chambers, 1952; Clemente & Windle, 1954; McMasters, 1962). Histological evaluations of the changes at the site of damage have indicated reduced collagen deposits, fibroblast proliferation, and connective tissue invasion following ACTH treatment (Fertig, Kiernan, & Seyan, 1971; Berry, Knowles, Willis, Riches, Morgans, & Steers, 1979). The effects of the neuropeptide treatment, in experiments in which they have been observed, seem to occur in the first few days after the damage. 52
0163-1047/83 $3.00 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
ACTH AND RECOVERY FROM SEPTAL LESIONS
53
The systemic administration of ACTHj_39 enhances functional and structural changes after the crushing of peripheral nerve in the rat. In ACTHLtreated animals and in animals with elevated ACTH levels subsequent to adrenalectomy, normal and heat-evoked foot movements return more quickly after nerve crush than in control animals (Strand & Kung, 1980; Saint-C6me, Acker, & Strand, 1982). After the crush, sprouting axons grow more rapidly than in saline-treated control animals, develop larger motor endplates, and have more preterminal branching. No neuropeptide effects are found on types of muscle fibers or protein synthesis in the muscles. The enhanced regeneration following increased ACTH availability is hypothesized to result from enhanced synthesis or preferential delivery of neurotransmitter or neurotrophic substances to the sprouting axons (Strand & Smith, 1980). Recent investigations confirmed the fact that ACTH administration facilitates functional recovery after peripheral nerve crush and that the administration of certain N-terminal ACTH fragments can produce similar effects (Bijlsma, Jennekens, Schotman, & Gispen, 1981). One effect of ACTH and its effective fragments seems to be an enhancement of the number of regenerating fibers (Bijlsma, Jennekens, Schotman, & Gispen, 1983). Fragments containing the 4-9 amino acid sequence have the ability to hasten recovery after peripheral nerve damage. The ACTHI~_24 fragment did not. The effective fragments included ones with behavioral but not corticosteroid potency, including the modified ACTH4_9 fragment ORG 2766. The neuropeptide, ORG 2766 (H-Met/0J-Glu-His-Phe-D-Lys-Phe-OH), is a structural variant of ACTH4_ 9 and has been reported to be one thousand times more potent than other N-terminal fragments as measured by its effects on the extinction of an active avoidance response (de Wied, Witter, & Greven, 1975; Greven & de Wied, 1977). Overall, ORG 2766 shares many behavioral effects with ACTH4_J0 and ACTH4_7 fragments, although there is evidence that it may have some unique properties in some testing situations (Greven & de Wied, 1973; Opmeer, Van Ree, & de Wied, 1978; Terenius, Gispen, & de Wied, 1975; Fekete & de Wied, 1982). Many behavioral changes found after damage to the central nervous system, both for which some "recovery" may occur and those that have progressive debilitation effects, are due to secondary reactions occurring in remaining neural systems (Isaacson, 1975; Schoenfeld & Hamilton, 1977). Given that N-terminal ACTH fragments can facilitate the regeneration of peripheral nerve fibers and hasten functional recovery, an issue of considerable theoretical and practical importance is whether or not the administration of ACTH fragments could influence the course of central nervous system damage. In an attempt to do this, we elected to study the behavioral effects of ORG 2766 administered for several days after experimentally induced destruction of the septal area in rats. In particular
54
ISAACSON AND POPLAWSKY
we studied its effects on the enhanced reactivity initially found after the lesion and the facilitated performance in an active avoidance task which is found at all times after the lesion. The hyperreactivity, often called "septal rage," is largely an emotional change, but the change in the avoidance task is best understood as a change in the performance of a learned behavior. The hyperreactivity induced by the lesion declines progressively over time and even more rapidly when the animals are repeatedly tested (Brady & Nauta, 1953; Reynolds, 1965). The facilitation of performance of an active avoidance response, on the other hand, appears to be a permanent consequence of the septal lesion (Poplawsky, 1978). This suggests that after the damage the progressive changes in emotionality could represent functional or structural alterations that might be susceptible to alteration by ACTH or its effective fragments. The purpose of this study was to investigate the effects of ORG 2766 on the time course of the emotionality changes following septal lesions from postoperative Day 3 to Day 12, and later on performance in a twoway avoidance task.
METHODS Thirty-one adult female albino rats of the Sprague-Dawley strain (Taconic Farms, N.Y.) with an average weight of 250 g were used in this experiment. Each rat was housed individually with free access to food and water. The colony room was on a 12/12-hr light-dark cycle with lights on at 7:00 AM. The test apparatus used in the avoidance task was an automated dualcompartment shuttlebox contained within a sound-attenuated cubicle. The shuttlebox was a Lehigh Valley Model 146-04 enclosed in that company's sound attenuating chamber, Model 132-06. A 28 V dc light bulb was mounted 14 cm above the shock grids in the center of the far wall of each compartment. Shock was delivered to the floor grid by a Coulbourn shocker-distributor, Model E13-16. One rat from the group with septal lesions given ORG 2766 was eliminated from the avoidance task analysis because of an equipment failure during its testing. A barrier separated the two compartments. The "toggle floor" of the chamber reported the animals' location both during and between trials. Sessions were controlled automatically by solid state circuitry. Prior to surgery each rat was rated for emotionality and given a score of 0-5 on five rating scales: reaction to object presentation, response to tap on back, resistance to capture, resistance to handling, and vocalization to capture and handling (King, 1958). Rats were matched on preoperative emotionality scores and assigned to the following groups: rats with septal lesions and vehicle injections (Groups SEP; n = 10), rats with septal lesions and ORG 2766 injections (Group SEP 2766; n = l 1), and rats with control operations and vehicle injections (Group CON; n = 10).
ACTH AND RECOVERY FROM SEPTAL LESIONS
55
The rats were anesthetized with ethyl ether and placed in a Kopf stereotaxic instrument. Anodal electrolytic lesions were accomplished stereotaxically using a stainless-steel electrode insulated by epoxy enamel except for 0.5 mm closest to the tip and a current of 1.5 mA was passed for 20 sec. The coordinates for the lesion were 9.5 mm anterior to frontal plane zero, _+0.75 mm lateral to midline, and 5.25 mm below brain surface. The incisor bar was adjusted until lambda and bregma were on the same horizontal plane. The control operation consisted of the same procedures without current being passed through the electrode. Rats in Group SEP 2766 were injected subcutaneously with 0.5 ml of 1 ~g ACTH4_9 analog (H-MET/02/Glu-His-Phe-D-Lys-Phe-OH; ORG 2766) dissolved in a 0.9% saline solution on the day of surgery and on postsurgical Days 1, 2, and 3. Groups SEP and CON received subcutaneous injections of the saline solution on these 4 days. All rats were lightly anesthetized with ether before injections were administered. The rats were rated for emotionality on postsurgical Day 3 (before the last injection) until postsurgery Day 12 (10 days). The experimenter was unaware of the experimental groups during emotionality scoring. On postsurgery Day 35, the rats were tested in the two-way shuttle avoidance chamber for 120 trials. A trial was started by presenting the conditioned stimulus (cue lights on) approximately 30 sec after each rat was placed in the dark shuttlebox. If the rat did not cross into the other half of the chamber within 10 sec of the light onset, a continuous shock of 1 mA was applied to the grid floor of the side of the apparatus. Avoidance responses, escape responses, and intertrial crossing were recorded. Responses made during the intertrial interval were not punished. Avoidance responses were crossings into the other compartment of the shuttlebox during the 10 sec following light onset. Escape responses were crossing made between 10 and 13 sec after light onset. Both types of responses terminated the light and shock (if applied). If the rat did not make a response after 3 sec of shock, the 13th sec after light onset, the trial was terminated. The intertrial interval was determined by a random time schedule of 30 sec with a minimum interval of 7.5 sec. Approximately 10 weeks after surgery, all rats were placed under deep chloral hydrate anesthesia (400 mg/kg; ip) and perfused intracardially with a physiological saline solution followed by a 10% Formalin-saline solution. Brains were extracted, and frozen brain sections were made every 80 b~, saving every second section through the extent of the lesion. The sections were stained with cresyl violet and lesion damage was evaluated.
RESULTS Histological evaluations revealed that for most rats the septal lesions extended from the genu of the corpus callosum to the columns of the
56
ISAACSON AND POPLAWSKY
fornix (anterior-posterior) and from the corpus callosum to the anterior commissure (dorsal-ventral). However, six of these rats also had damage to the anterior commissure, and a few had only small damage to the posterior third of the septum. Similar septal lesions were found in groups SEP and SEP 2766. A 3 x 10 mixed-design analysis of variance was used to analyze the emotionality ratings. The specific factors were groups (SEP, SEP 2766, and CON) and the l0 scoring days. A 3 x 2 mixed-design was used to analyze avoidances and intertrial crossings, where the between-factor was the three groups and the within-factor was two blocks of 60 trials. Figure 1 presents the mean emotionality scores for the three groups of rats over 10 postoperative observation days. There was a significant interaction between groups and test days (F(18,252) -- 7.62, p < .01). The group of rats with septal damage and the ACTH fragment had lower emotionality scores (p < .05) than the group with septal lesions and saline injections on Days 3, 4, 5, 6, and 7 after surgery. The ACTHtreated lesion group also reached control levels of emotionality by postoperation Day 12. The analyses of the data from the avoidance task showed that rats in both groups with septal damage had high levels of avoidances (82 and 83%, respectively) compared to the control group ( 5 2 % ) , (F(2,27) • 8.97, p < ,01). However, the untreated septal lesion animals made significantly more intertrial crossings than the nonlesioned control group (a mean of 100 as compared to 38). The group of rats with 16
A
15 14 LIJ O::
13
8 u)
12
>I--
II
I,o :S hi Z
8
I0 9
7 6 5 4 3 2
I ;5
4
5 6 7 8 9 I0 II DAYS A F T E R SURGERY
12
FIG. 1. Mean emotionality scores of'animals with septal lesions treated with saline (triangles), septal lesions treated with ORG 2766 (open circles), and control animals (closed circles) over repeated tests beginning the third day after surgery.
ACTH AND RECOVERY FROM SEPTAL LESIONS
57
septal damage treated with ORG 2766 had approximately the same number of intertrial crossings as the nonlesioned group. DISCUSSION The repeated administration of the ACTH4_9 fragment, ORG 2766, ameliorates some of the usual behavioral consequences of septal area damage and hastens the return to near normal emotional responsiveness, since the septal lesion group receiving ORG 2766 reached control levels of emotionality by postoperative Day 6, while the lesioned animals without the neuropeptide fragment reached control levels on postoperative Day 12. Other studies have also reported that the hyperemotionality following septal lesions dissipated by the 12th postoperative day (e.g., Brady & Nauta, 1953). These results indicate that rats with septal lesions treated with ORG 2766 both had lower emotionality scores early in testing and reached normal levels of emotionality sooner than untreated rats with septal lesions. This could be interpreted as an acceleration of normal readjustive activities occurring in remaining brain systems. However, not all lesion-induced changes are affected by the administration of ORG 2766. Only those behaviors which are of an emotional nature or ones likely related to emotional responsiveness (intertrial crossings) were affected. The commonly observed facilitation of the acquisition of a two-way active avoidance problem was not changed. This lesioninduced behavior, in contrast to septal hyperemotionality, does not seem to change in a progressive fashion after the lesion. On the other .hand, intertrial crossings do decrease over testing sessions in the avoidance problem (Poplawsky, 1978). In the present study the intertrial crossings for the lesioned group receiving ORG 2766 was almost half that of nontreated group on the first avoidance test day. This suggests that prolonged alterations in emotionality or hyperreactivity are affected by treatment with ORG 2766 but the associational processes underlying avoidance conditioning were not. The mechanisms whereby ORG 2766 influences the lesion-induced behaviors are uncertain. It is unlikely that it does so by affecting endocrine systems since septal hyperreactivity is not itself the consequence of endocrine changes. Hyperemotionality is frequently observed as soon as the animal comes out of the anesthesia. Furthermore, castration, adrenalectomy, or hypophysectomy fail to attenuate the septal syndrome (Montgomery, Berkut, Grubb, & Westbrook, 1971; Seggie, 1971; Phillips & Lieblich, 1972). It is possible that the peptide fragment produces its effects by alteration of glial or connective tissue responses to damage, although if this is the case it would have to be assumed that such responses account for the reduction of septal emotionality rather than its production. Assuming a similarity in effect between ORG 2766 and the ACTH4_I0 analog, it is POssible that the beneficial effect could be due to an enhanced
58
ISAACSON AND POPLAWSKY
catecholamine turnover (Versteeg & Wurtman, 1975), producing an effect similar to the beneficial effect produced by the administration of L-dopa (Gage & Olton, 1975; Marotta, Logan, potegal, Glusman, & Gardner, 1977). However, if this is the case, the effect far outlasts the time period at which the neuropeptide had been acting and the catecholamine turnover enhanced, since the reduction of intertrial crossings was found at more than 20 days after the end of the neuropeptide treatment. It is also possible that the ACTH4_9 analog produced its effects by action on the generation of proliferation of axonal sprouts in remaining brain tissue, as has been demonstrated in peripheral nerve. The fact that a relatively brief period of administration of ORG 2766 has both immediate and prolonged effects on emotionality and reactivity has practical and theoretical importance. Some of the effects reported following ORG 2766 in people, such as increased sociability, gregariousness, and reduced anxiety (Gaillard & Varey, 1979; Sandman, Walker, & Lawton, 1980), may be related to the emotional changes found in the present experiments. REFERENCES Berry, M., Knowles, J., Willis, P., Riches, A. C., Morgans, G. P., & Steers, D. (1979). A reappraisal of the effects of ACTH on the response of the central nervous system to injury. Journal of Anatomy, 128, 859-871. Bijlsma, W. A., Jennekens, F. G. I., Schotman, P., & Gispen, W. H. (1981). Effects of corticotrophin (ACTH) on recovery of sensorimotor function in the rat: Structureactivity study. European Journal of Pharmacology, 76, 73-79. Bijlsma, W. A., Jennekens, F. G. 1., Schotman, P., & Gispen, W. H. (1983). Stimulation by ACTH4_~0 of nerve fiber regeneration following sciatic nerve crush. Muscle and Nerve, 6, 102-110. Brady, J. V., & Nauta, W. H. J. (1953). Subcortical mechanisms in emotional behaviour: Affective changes following septal forebrain lesions in the albino rat. Journal of Comparative and Physiological Psychology, 46, 339-346. Clemente, C. D., & Windle, W. F. (1954). Regeneration of severed nerve fibers in the spinal cord of the adult cat. Journal of Comparative Neurology, 101, 691-731. de Wied, D., Witter, A., & Greven, H. M. (1975). Behaviorally active ACTH analogues. Biochemical Pharmacology, 24, 1463-1468. Fekete, M., & de Wied, D. (1982). Potency and duration of action of the ACTH4_9 analog (ORG 2766) as compared to ACTH,_lo and D-Phe 7 ACTH4 10 on active and passive avoidance behavior of rats. Pharmacology Biochemistry and Behavior, 16, 387-392. Fertig, A., Kiernan, J. A., & Seyan, S. S. A. S. (1971). Enhancement ofaxonal regeneration in the brain of the rat by corticotrophin and triiodorthyronine. Experimental Neurology, 33, 372-385. Gage, F. H., I I I , & Olton, D. S. (1975). Hippocampal influence on septal hyperreactivity. Brain Research, 98, 311-325. Gaillard, A. W. K., & Varey, C. A. (1979). Some effects of an ACTH4_9 analog (ORG 2766) on human performance. Physiology and Behavior, 23, 79-84. Greven, H. M., & de Wied, D. (1973). The influence of peptides derived from corticotrophin (ACTH) on performance. Structure activity studies. In E. Zimmerman, W. H. Gispen,
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B. H. Marks, & D. de Wied (Eds.), Progress in Brain Research, Vol. 39, pp. 429442. Amsterdam: Elsevier. Greven, H. M., & de Wied, D. (1977). Influence of peptides structurally related to ACTH and MSH on active avoidance behaviour in rats. In T. V. van Wimersma Greidanus (Ed.), Frontiers of Hormone Research, Vol. 4, pp. 140-152. Basel: Karger. Isaacson, R. L. (1975). The myth of recovery from early brain damage. In N. R. Ellis (Ed.), Aberrant Development in Infancy: Human and Animal Studies, pp. 1-26. Potomac, Md. King, F. A. (1958). Effects of septal and amygdala lesions on emotional behavior and conditioned avoidance responses in the rat. Journal of Nervous and Mental Diseases, 126, 57-63. McMasters, R. E. (1962). Regeneration of the spinal cord in the rat. Effects of Piromen and ACTH upon the regenerative capacity. Journal of Comparative Neurology, 119, 113-125. Marotta, R. F., Logan, N., Potegal, M., Glusman, M., & Gardner, E. L. (1977). Dopamine agonists induce recovery from surgically-induced septal rage. Nature, (London), 269, 513-515. Montgomery, R. L., Berkut, M. K., Grubb, E. F., & Westbrook, D. L. (1971). Hormonal influence on behavior in brain lesioned male rats. Physiology and Behavior, 7, 107111. Opmeer, F. A., van Ree, J. M., & de Wied, D. (1978). ACTH-induced lipolysis in rat adipocytes: Structure-activity relationship. Naunyn-Schmiedeberg's Archives of Pharmacology, (1972). 302, 31-36. Phillips, A. G., & Lieblich, 1. (1972). Developmental and hormonal aspects of hyperemotionality produced by septal lesions in male rats. Physiology and Behavior, 9, 237-242. Poplawsky, A. (1978). Long-term maintenance of shuttlebo× avoidance behavior before and after septal lesions. Physiological Psychology, 6, 294-299. Reynolds, R. (1965). Equivalence of radio frequency and electrolytic lesions in producing septal rage. Psychonomic Science, 2, 35-36. Saint-C6me, C., Acker, G. R., & Strand, F. L. (1982). Peptide influences on the development and regeneration of motor performance. Peptides, 3, 439-449. Sandman, C. A., Walker, B. B., & Lawton, C. A. (1980). An analog of MSH/ACTH 4-9 enhances interpersonal and environmental awareness in mentally retarded adults. Peptides, 1, 109-114. Schoenfeld, T. A., & Hamilton, L. W. (1977). Secondary brain changes following lesions: A new paradigm for lesion experimentation. Physiology and Behavior, 18, 951-967. Seggie, J. (1971). Effect of adrenalectomy or gonadectomy on affective behavior changes following septal lesions in the rat. Journal of comparative and physiological Psychology, 74, 11-19. Strand, F. L., & Kung, T. T. (1980). ACTH accelerates recovery of neuromuscular function following crushing of peripheral nerve. Peptides, 1, 135-138. Strand, F. L., & Smith, C. M. (1980). LPH, ACTH, MSH, and motor systems. Pharmacology and Therapeutics, 11, 509-533. Terenius, L., Gispen, W. H., & de Wied, D. (1975). ACTH-like peptides and opiate receptors in the rat brain: Structure-activity studies. European Journal of Pharrnacology, 33, 395-399. Versteeg, D. H. G., & Wurtman, R. J. (1975). Effect of ACTH4_m on the rate of synthesis of [3H]catecholamines in the brains of intact, hypophysectomized, and adrenalectomized rats. Brain Research, 93, 552-557. Windle, W. F., Clemente, C. D., & Chambers, W. W. (1952). Inhibition of formation of a glial barrier as a means of permitting a peripheral nerve to grow into the brain. Journal of comparative Neurology, 96, 359-370.