Life Sciences, Vol . 24, pp , 439-448 Printed in the U .S .A .
Pergamon Press
THS PITQITARY GLAND MEDL11TE5 ACQTE AND CHRONIC PAIN RESPONSIVENESS IN STRESSSD AND NON-STRESSED RATS Shimon Amir and Zalman Amit Center for Research on Drug Dependence Department of Psychology Concordia IIniversity 1455 de Malsonneuve Blvd . Went Montreal, Quebec H3G 1MB (Received in final form December 19, 1978) Su~ The effect of hypophysectomy on the responeivenesa of rate to acute (hot plate teat) and chronic (formalin test) pain was studied. Hypophysectomy did not alter hot plate behavior but increased the duration of the paw withdrawal in the formalin teat . Prreaposure of rats to immobilisation stress caused a transient yet significant increase in escape latencies from a hot plate se well ss s significant decrease in the duration of paw withdrawal in the formalin test . Hypophysectomy blocked the effect of stress on these behavioral manifestations of acute and chronic pain . The effect of hypophysectomy was not reversed by adrenocorticotropin pretreatment . These results suggest that centrally acting pituitary hormones may have a requisite function in normal and adaptive pain control in mammals. There ie accumulating evidence that pituitary hormones modulate the function of the central nervous system and directly influence animal behavior in addition to producing their better known endocrine effect (1-3) . Recently, there has been observations that fragments of the pituitary polypeptide hormone beta-lipotropin (i .e . endorphins) interact with opiate receptors in the brain and have potent analgesic profile similar to that of morphine (4-7) . Furthermore, fragments of the anterior pituitary polypnptide hormone adrenocorticotropin (ACTS), which share a common precursor with beta-lipotropia (g, 9), were found to have affinity for opiate receptor systems and to alter morphine analgesia in e systematic manner (10-13) . The findings that betaendorphin (beta-lipotropin 61-91), the most potent analgesic polypeptide hormone (14), and ACTH are released from the pituitary concomitantly during stress (15) and have affinity for central opiate receptors suggest the possibility that functional interrelationships may exist among these pituitary peptides, opiate receptor systems in the brain, and the meaifeatation of pain related behaviors in mammals . If the release of pharmacologically active polypeptides from the pituitary gland is indicative of requisite function in normal or adaptive pain control, hypophysectomy may deprive opiate receptors in pain controlling 0300-9653/79/0129-0439$02 .00/0 Copyright (c) 1979 Pergamon Press
44 0
Pituitary Gland Mediation of Pain REsponse
Vol . 24, No . 5,
1979
system of their endogenous ligands and possibly result in modification of physiological or behavioral response to pain . Previous findings indicate that acupuncture analgesia depend on the functional integrity of the pituitary gland (16) . Furthermore, exposure of animals to stress, which normally activates the pituitary gland (15, 17,18), alters nociception threshold in various pain producing situations (19-24) . On the other hand, attempts to implicate the pituitary gland in the behavioral response to noxious stimuli in normally behaving animals has yielded ambiguous results (25-27) . In the present series of experiments we ezamined the role of the pituitary gland in mediating the behavioral response of rata to acute (hot plate teat) and chronic (formalin test) pain under normal, or following a stress producing situation . Method Animals : Male Wiatar rate approximately 75 days old were used . Hypophysectomias were performed by the breeder (Canadian Breeding Farm and Laboratories, St . Constant, Quebec), four weeks before testing, using the transauricul ar method of Falconi and Rosei (28) . The animals that underwent hypophyaectomy were selected by the breeder from larger groups of animals in a random manger . All operated and son-operated animals were assigned to the different ezperimeatal and control groups in a random manner upon arrival from the breeder . The completeness of hypophysectomy was evidenced by failure of the operated rats to gain weight during the four weeks between surgery and testing and by the absence of pituitary gland under the diaphragms sense observed at sacrifice, at the termination of the experiment . The hypophysectomized rata were housed singly is a temperature regulated room (25° C) with free access to Purina lab chow and 5Z sucrose solution . Since no difference in hot-plate and formalin teat behaviors were noted in preliminary experiments between nonoperated and shamroperated rate, only non-operated control rats were used in the present experiments. These animals were housed under similar conditions with food and tap water ad-lib . All animals were used only once in the present experiments . Thn hot plate test . A modified hot plate apparatus was used to assess the behavioral response of animals to acute, escapable pain . Animals were individually placed on an aluminum plate constantly heated to 51° C and their paw lick and escape latencies were determined . Animals were confined to the hot plate by means of a pleaiglass cylinder (20 cm in diem., 25 cm tall) and were required to jump and hang over the edge of the cylinder for 5 sec . in order to meet escape criteria . Animals were removed from the hot plate if they failed to escape within 120 sec . Hypophysectomized rats were treated with saline (1 ml/kg, i .p ., n = 12) or a rapid-acting form of ACTH (ACTHAH, lyophilized porcine ACTH from AR1~IIß; 12 .5 mg/kg, i .p . ; n ~ 6) 10 min. before the first testing trial . A similar dose of ACTH has been found to reverse the ; nh~ bitory effect of hypophysectomy on morphine drinking in rats (29) . Control animals were treated with saline (n = 10) or ACTH (n ~ 6) in a similar manner . All animals ware tested five times, with 15 min . intervals between testing trials . The effect of immobilization stress on the hot plate induced paw lick and escape responses of hypophysectomised and non-operated, control animals was studied in a separate ezperiment . Immobilization stress was produced by placing each animal in a well ventilated, sung fit ple:iglass restraining apparatus 30 min . prior to the first testing trial . This procedure was found previously to cause a significant increase in escape latencies from a hot plate (24) . Groups consisted of hypophysectomized : saline-treated (n = 12), hypophysectomized : ACTH-treated (n ~ 6), control : saline-treated (a = 12), and control: ACTH-treated rate (n = 6) . All animals were injected 10 min. before the termination of the restraining period . Testing protocol was
Vol,
24, No . 5,
1979
Pituitary Gland Mediation of Pain Response
44 1
similar to that described before . The formalin teat . A modified version of the formalin test (30) was used to Each assess the behavioral respoaae of animals to chronic, inescapable pain . animal received a subdersial injection of 0.05 ml of sterile SZ formalin into the dorsal surface of the left forepaw and immediately placed on a suspended plexiglass floor. A large mirror was mounted at 45o angle beneath the floor Each animal was to allow unhindered observation of the pave of the animals. observed for 15 min. and the following behavioral categories was assessed : A.
B. C.
D.
This response occurs immediately following Latency to the first paw lick . the formalin injection and is often accompanied by shaking or biting of The response does not occur when isotonic saline rather the injected paw. than formalin is injected . Number of paw licks. This response category does not include grooming, during which both forepaws are elevated and 'dashed" . Duration of paw withdrawal . The formalin injected paw is elevated and not in contact with the floor. The uainjected paw is placed firmly on the floor . This response category does not include rearing during which both forepaws are elevated and not in contact with the floor. Rotor activity . Motor activity (including locomotion, grooming, rearing, etc .) was recorded by the use of three sensitive accelerometers that were mounted on the bottom aide of the suspended plexiglass floor . The acceler ometers were connected to a digital integration unit that transformed activity signals into numbers .
The groups tested in this ezperimeat consisted of hypophysectomized: saline-treated (n ~ 8), hypophysectomized : ACTH-treated (n = 6), control: saline-treated (n = 8), and control : ACTH-treated (n -- 6) animals. The effect of immobilization stress on the responeiveaese to chronic, inescapable pain of hypophysectomized and non-operated control animals was studied in a separate ezperimeat . The groups studied consisted of hypophysect saline-treated (n = 6) . Hypophysectomized : ACTH-treated (n = 6), omized : ACTH-treated (a = 6) animals. controls : saline-treated (n ~ 6), and controls : The restraining and injection procedures were similar to those described before . All animals were injected with formalin immediately following the termination of the restraining period and were observed for 15 min . se described . In all eaperimente, testing always took place between 11 a.m . and 3 p .m . All the data derived from the hot plate ezperimeate were aaslyzed using a three way analysis of variance with two between-subjects factors (Group : hypophyaectomy or control ; Drug : Saline or ACTH) and one between subject variable (trials) . The data from the formalin test ezperiments were analysed using a two way analysis of variance with two between subjects factors (Group and Drug) . Results The hot plate test. Fig . 1 shove the affect of hypophysectomy on paw lick and escape latencies from a hot plate of saline treated and ACTH (12 .5 s8/kg) Analysis of variance computed on the paw lick data (Fig . 1-A) treated rata . oa each of the five testing trials revealed no significant differences in paw Furthermore, lick latencies between hypophysectomized and control eaimals. ACTH pretreatment did not affect the initial (zero time) paw lick response of hypophysectomized or control animals . However in both groups paw lick latencies of the ACTH-treated animals were sigaificantlq lover than those of the saline-treated groups in the second (F (1,21) = 7 .15, pt0.02) sad the third (F (1,12) = 7 .35, pc0.02)te~ting trials .
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Vol . 24, No . 5, 1979
Pituitary Gland Mediation of Pain Response
No significant differences in eacape latencies were noted between the hypophysectomized and the control animals or between ACTH-treated and salinetreated rata (Fig . 1-B) . However, a significant time a ACTH interaction was observed (F (4,120) ~ 2 .52, p0 .1) . Pre-exposure of animals to immobilization stress caused a transient increase in the eacape latencies of the non-operated control animals but had no effect in the hypophysectomized rats (Fig . 2-B) . Analysis of the escape data of animals pre-exposed to mobilisation stress revealed a significant effect of hypophysectomy (F (1,32) = 35 .46, p
Pergamon Press
THS PITQITARY GLAND MEDL11TE5 ACQTE AND CHRONIC PAIN RESPONSIVENESS IN STRESSSD AND NON-STRESSED RATS Shimon Amir and Zalman Amit Center for Research on Drug Dependence Department of Psychology Concordia IIniversity 1455 de Malsonneuve Blvd . Went Montreal, Quebec H3G 1MB (Received in final form December 19, 1978) Su~ The effect of hypophysectomy on the responeivenesa of rate to acute (hot plate teat) and chronic (formalin test) pain was studied. Hypophysectomy did not alter hot plate behavior but increased the duration of the paw withdrawal in the formalin teat . Prreaposure of rats to immobilisation stress caused a transient yet significant increase in escape latencies from a hot plate se well ss s significant decrease in the duration of paw withdrawal in the formalin test . Hypophysectomy blocked the effect of stress on these behavioral manifestations of acute and chronic pain . The effect of hypophysectomy was not reversed by adrenocorticotropin pretreatment . These results suggest that centrally acting pituitary hormones may have a requisite function in normal and adaptive pain control in mammals. There ie accumulating evidence that pituitary hormones modulate the function of the central nervous system and directly influence animal behavior in addition to producing their better known endocrine effect (1-3) . Recently, there has been observations that fragments of the pituitary polypeptide hormone beta-lipotropin (i .e . endorphins) interact with opiate receptors in the brain and have potent analgesic profile similar to that of morphine (4-7) . Furthermore, fragments of the anterior pituitary polypnptide hormone adrenocorticotropin (ACTS), which share a common precursor with beta-lipotropia (g, 9), were found to have affinity for opiate receptor systems and to alter morphine analgesia in e systematic manner (10-13) . The findings that betaendorphin (beta-lipotropin 61-91), the most potent analgesic polypeptide hormone (14), and ACTH are released from the pituitary concomitantly during stress (15) and have affinity for central opiate receptors suggest the possibility that functional interrelationships may exist among these pituitary peptides, opiate receptor systems in the brain, and the meaifeatation of pain related behaviors in mammals . If the release of pharmacologically active polypeptides from the pituitary gland is indicative of requisite function in normal or adaptive pain control, hypophysectomy may deprive opiate receptors in pain controlling 0300-9653/79/0129-0439$02 .00/0 Copyright (c) 1979 Pergamon Press
44 0
Pituitary Gland Mediation of Pain REsponse
Vol . 24, No . 5,
1979
system of their endogenous ligands and possibly result in modification of physiological or behavioral response to pain . Previous findings indicate that acupuncture analgesia depend on the functional integrity of the pituitary gland (16) . Furthermore, exposure of animals to stress, which normally activates the pituitary gland (15, 17,18), alters nociception threshold in various pain producing situations (19-24) . On the other hand, attempts to implicate the pituitary gland in the behavioral response to noxious stimuli in normally behaving animals has yielded ambiguous results (25-27) . In the present series of experiments we ezamined the role of the pituitary gland in mediating the behavioral response of rata to acute (hot plate teat) and chronic (formalin test) pain under normal, or following a stress producing situation . Method Animals : Male Wiatar rate approximately 75 days old were used . Hypophysectomias were performed by the breeder (Canadian Breeding Farm and Laboratories, St . Constant, Quebec), four weeks before testing, using the transauricul ar method of Falconi and Rosei (28) . The animals that underwent hypophyaectomy were selected by the breeder from larger groups of animals in a random manger . All operated and son-operated animals were assigned to the different ezperimeatal and control groups in a random manner upon arrival from the breeder . The completeness of hypophysectomy was evidenced by failure of the operated rats to gain weight during the four weeks between surgery and testing and by the absence of pituitary gland under the diaphragms sense observed at sacrifice, at the termination of the experiment . The hypophysectomized rata were housed singly is a temperature regulated room (25° C) with free access to Purina lab chow and 5Z sucrose solution . Since no difference in hot-plate and formalin teat behaviors were noted in preliminary experiments between nonoperated and shamroperated rate, only non-operated control rats were used in the present experiments. These animals were housed under similar conditions with food and tap water ad-lib . All animals were used only once in the present experiments . Thn hot plate test . A modified hot plate apparatus was used to assess the behavioral response of animals to acute, escapable pain . Animals were individually placed on an aluminum plate constantly heated to 51° C and their paw lick and escape latencies were determined . Animals were confined to the hot plate by means of a pleaiglass cylinder (20 cm in diem., 25 cm tall) and were required to jump and hang over the edge of the cylinder for 5 sec . in order to meet escape criteria . Animals were removed from the hot plate if they failed to escape within 120 sec . Hypophysectomized rats were treated with saline (1 ml/kg, i .p ., n = 12) or a rapid-acting form of ACTH (ACTHAH, lyophilized porcine ACTH from AR1~IIß; 12 .5 mg/kg, i .p . ; n ~ 6) 10 min. before the first testing trial . A similar dose of ACTH has been found to reverse the ; nh~ bitory effect of hypophysectomy on morphine drinking in rats (29) . Control animals were treated with saline (n = 10) or ACTH (n ~ 6) in a similar manner . All animals ware tested five times, with 15 min . intervals between testing trials . The effect of immobilization stress on the hot plate induced paw lick and escape responses of hypophysectomised and non-operated, control animals was studied in a separate ezperiment . Immobilization stress was produced by placing each animal in a well ventilated, sung fit ple:iglass restraining apparatus 30 min . prior to the first testing trial . This procedure was found previously to cause a significant increase in escape latencies from a hot plate (24) . Groups consisted of hypophysectomized : saline-treated (n = 12), hypophysectomized : ACTH-treated (n ~ 6), control : saline-treated (a = 12), and control: ACTH-treated rate (n = 6) . All animals were injected 10 min. before the termination of the restraining period . Testing protocol was
Vol,
24, No . 5,
1979
Pituitary Gland Mediation of Pain Response
44 1
similar to that described before . The formalin teat . A modified version of the formalin test (30) was used to Each assess the behavioral respoaae of animals to chronic, inescapable pain . animal received a subdersial injection of 0.05 ml of sterile SZ formalin into the dorsal surface of the left forepaw and immediately placed on a suspended plexiglass floor. A large mirror was mounted at 45o angle beneath the floor Each animal was to allow unhindered observation of the pave of the animals. observed for 15 min. and the following behavioral categories was assessed : A.
B. C.
D.
This response occurs immediately following Latency to the first paw lick . the formalin injection and is often accompanied by shaking or biting of The response does not occur when isotonic saline rather the injected paw. than formalin is injected . Number of paw licks. This response category does not include grooming, during which both forepaws are elevated and 'dashed" . Duration of paw withdrawal . The formalin injected paw is elevated and not in contact with the floor. The uainjected paw is placed firmly on the floor . This response category does not include rearing during which both forepaws are elevated and not in contact with the floor. Rotor activity . Motor activity (including locomotion, grooming, rearing, etc .) was recorded by the use of three sensitive accelerometers that were mounted on the bottom aide of the suspended plexiglass floor . The acceler ometers were connected to a digital integration unit that transformed activity signals into numbers .
The groups tested in this ezperimeat consisted of hypophysectomized: saline-treated (n ~ 8), hypophysectomized : ACTH-treated (n = 6), control: saline-treated (n = 8), and control : ACTH-treated (n -- 6) animals. The effect of immobilization stress on the responeiveaese to chronic, inescapable pain of hypophysectomized and non-operated control animals was studied in a separate ezperimeat . The groups studied consisted of hypophysect saline-treated (n = 6) . Hypophysectomized : ACTH-treated (n = 6), omized : ACTH-treated (a = 6) animals. controls : saline-treated (n ~ 6), and controls : The restraining and injection procedures were similar to those described before . All animals were injected with formalin immediately following the termination of the restraining period and were observed for 15 min . se described . In all eaperimente, testing always took place between 11 a.m . and 3 p .m . All the data derived from the hot plate ezperimeate were aaslyzed using a three way analysis of variance with two between-subjects factors (Group : hypophyaectomy or control ; Drug : Saline or ACTH) and one between subject variable (trials) . The data from the formalin test ezperiments were analysed using a two way analysis of variance with two between subjects factors (Group and Drug) . Results The hot plate test. Fig . 1 shove the affect of hypophysectomy on paw lick and escape latencies from a hot plate of saline treated and ACTH (12 .5 s8/kg) Analysis of variance computed on the paw lick data (Fig . 1-A) treated rata . oa each of the five testing trials revealed no significant differences in paw Furthermore, lick latencies between hypophysectomized and control eaimals. ACTH pretreatment did not affect the initial (zero time) paw lick response of hypophysectomized or control animals . However in both groups paw lick latencies of the ACTH-treated animals were sigaificantlq lover than those of the saline-treated groups in the second (F (1,21) = 7 .15, pt0.02) sad the third (F (1,12) = 7 .35, pc0.02)te~ting trials .
44 2
Vol . 24, No . 5, 1979
Pituitary Gland Mediation of Pain Response
No significant differences in eacape latencies were noted between the hypophysectomized and the control animals or between ACTH-treated and salinetreated rata (Fig . 1-B) . However, a significant time a ACTH interaction was observed (F (4,120) ~ 2 .52, p0 .1) . Pre-exposure of animals to immobilization stress caused a transient increase in the eacape latencies of the non-operated control animals but had no effect in the hypophysectomized rats (Fig . 2-B) . Analysis of the escape data of animals pre-exposed to mobilisation stress revealed a significant effect of hypophysectomy (F (1,32) = 35 .46, p
