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A method for the study of swimming stress and stress-induced antinociception in preweanling rats
JPM Vol. 29, No. 3 June 1993:139-141
ORIGINAL
ARTICLES
A Method for the Study of Swimming Stress and Stress-Induced Antinociception in Preweanling Rats B a l a Y. M u h a m m a d a n d I a n K i t c h e n
Receptors and Cellular Regulation Research Group, School of Biological Sciences, University of Surrey, Guildford, Surrey, England, U.K.
Stress-induced antinociception (SIA) which is well characterized in the adult rat can also be observed in young rats, and, by varying swimming times, it can be dissociated into opioid and nonopioid forms. However, swimming ability in the rat does not fully develop until the third postnatal week and this has precluded the study of swim SIA in neonates. We report here the development of a harness device to aid swimming in young rats which we have successfully employed down to the age of 2 days without distress to the animals. Further we have also shown the development of the opioid form of swim SIA in the rat using this device. Swim SIA is absent at days 2 and 5, but at postnatal day 10 a small level of SIA is evident which is reversed by naloxone (10 mg/kg). Swim SIA develops rapidly thereafter, and the adult profile is observed by day 25.
Keywords: Stress-induced antinociception; Swimming stress; Opioid; Naloxone; Development; Ontogeny
Introduction Stress-induced antinociception (SIA) is an adaptive physiological response which can be induced by a number of different stressors including cold, immobilization, electroshock, and swimming (Bodnar, 1984). The latter stressor has been particularly useful as by varying the duration or temperature of swimming it is possible to dissociate opioid and nonopioid forms of the behavior, on the basis of naloxone reversal or naloxone insensitivity, respectively. We recently reported that swim SIA can be observed in young (20 days old) rats and that the neurochemistry and r e c e p t o r control of this response differs at this preweanling age in comparison with the adult (Jackson and Kitchen, 1989a; Kitchen and Pinker, 1990). Because swimming behavior in the rat does not properly develop until days 12-15
Address reprint requests to Dr. I. Kitchen, Receptors and Cellular Regulation Research Group, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, England, U.K. Received October 1992; revised and accepted December 1992. Journal of Pharmacological and Toxicological Methods 29, 139-141 (1993) © 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010
(Schapiro et al., 1990; Salas, 1972), it has proved difficult to study swim SIA in preweanl~ng animals. Further, in contrast to swim stress, other stressors, such as electroshock and cold exposure, are more severe and for ethical reasons are thus less desirable. We have, therefore, attempted to develop a methodology for using warm-water swimming stress in rat pups and report here the development of a swimming harness that allows the study of swim SIA down to the age of 2 days without distress to the animals and describe how the opioid form of swim SIA develops.
Methods Animals and Nociceptive Testing Wistar albino rats (University of Surrey strain) of mixed sexes were used in all experiments and maintained in litters of eight pups. All rats were maintained at 21 ° _+ I°C in a constant 12 hr light/dark cycle (lights on at 0:7 hr 00 min), and experimental procedures were carried out in a quiet, windowless, air-controlled laboratory. Pups up to the age of 20 days remained with the
1056-8719/93/$6.00
140
mother except during naloxone (10 mg/kg) administration, swimming, and nociceptive testing to minimize stress due to maternal deprivation. All procedures were carried out between 11 hr 00 min and 16 hr 00 min. Nociceptive responses were measured by the warmwater tail-immersion test adapted for use in neonates to give equivalent basal nociceptive measures (Kitchen et al., 1984; De Cabo et al., 1992) using the following temperatures (43.5°C, days 2 and 5; 47.5°C, day 10; 50°C, days 15, 20, and 25). The choice of temperatures were based on previous studies (De Cabo et al., 1992) which have shown that at ages below 10 days, deviation of _+0.5°C from 43.5°C produces either instant removal of the tail or absence of response. Use of 47.5°C at day 10 and 50°C at later ages was aimed at producing an equivalent basal response time (De Cabo et al., 1992), since in animals > 10 days of age consistent nociceptive responses are not achievable at the lower temperature (43.5°C). Nociceptive responses were recorded immediately before i.p. administration of naloxone or saline, 10 min before a 3-min period of swimming stress and at 1, 5, 10, 15, and 30 min following swim stress.
Swimming Aid Devices and Swim-Stress Procedures In confirmation of the work of Schapiro et al. (1970), 15-day-old animals were able to swim properly, unaided. However, 10-day-old rats exhibited swimming behavior for < 1 min and were unable to continue to float. Flotation devices consisting of rings of polystyrene (akin to the rubber ring used for children) were initially assessed in 10-day-old rats. These proved unsatisfactory as they prevented freedom of movement causing the pups to struggle and within 1 min the rats became immobile. The alternative approach which proved successful was to attach a harness to the trunk of the pups attached by a hook and a single string to a horizontal bar connected to a retort stand placed centrally over the swimming tank. 5-mm plastic cable ties with self-locking fasteners (R. S. Components, London, England, U.K.) were used for the harness device which allowed infinite adjustment from 1 to 3.5 cm diameter. The harness was adjusted so that both front and hind paws were free and no pressure was exerted on the thorax or abdomen. The degree of immersion of the animals could be finely adjusted using a rack and pinion adjuster on the stand. Neonatal rats were immersed sufficiently to induce a paddling type of response, and the harness allowed appreciable movement around the swimming tank while maintaining a fixed ventral posture. Neonate rats (2-20 days) were made to swim individually in a plastic tank (29 x 22 × 28 cm deep) containing water at 20° _+ 10°C for a period
JPM Vol. 29, No. 3 June 1993:139-141
of 3 min as previously described for 20-day-old animals (Jackson and Kitchen, 1989a). At the end of the swimming period, rats were removed from the harness by cutting the cable ties with a pair of scissors and returned to the home cage before subsequent nociceptive testing.
Results The swimming harness enabled 3-min periods of swimming to be observed in 2-, 5-, and 10-day-old rats without distress. Figure I shows the ontogenetic profile of swim-stress-induced antinociception from days
Figure 1. Effect of 3 min of swimming stress on nociceptive response in (a) 2-, (b) 5-, (c) 10-, (d) 15-, (e) 20-, and (f) 25day-old rats. Values represent means, and vertical bars, SE mean for groups of six animals. (O) Saline-injected, unstressed. (0) Saline-injected, swim-stressed. (D) Naloxone l0 mg/kg, unstressed. (I) Naloxone l0 mg/kg, swimstressed. Pre- Pretest responses 10 min before swim stress. Measures at 30 min were equivalent to those at 15 min at all ages and are omitted for clarity of presentation. Treatment groups were compared using ANOVA and posthoc comparisons made by Duncans New Multiple Range Test. *p < 0.05 swim-stressed versus appropriate unstressed control. +p < 0.05 naloxone-treated, swim-stressed versus saline-treated swim-stressed. a
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B. Y. MUHAMMADAND I. KITCHEN STRESS-INDUCEDANTINOCICEPTIONIN NEONATES
2 to 25. T h e r e was no indication of antinociception in 2- or 5-day-old rats after swimming stress. At day 10, h o w e v e r , swimming p r o d u c e d a small but significant increase in tail-immersion latency 5 min after swim stress; an effect which was c o m p l e t e l y r e v e r s e d by naloxone (10 mg/kg). At day 15, the level of S I A was greater and also significantly attenuated by the opioid antagonist. At this age, a greater level o f S I A could be achieved (5-min r e s p o n s e latency; 6.59 _+ 0.5 sec.) if the immersion t e m p e r a t u r e was reduced to 47.5°C. N a l o x o n e (10 mg/kg) c o m p l e t e l y antagonized SIA using these p a r a m e t e r s (5-min r e s p o n s e latency; 3.05 _+ 0.55 sec.). A level o f S I A a p p r o a c h i n g that o b s e r v e d in the adult (Kitchen and Pinker, 1990) was o b s e r v e d in 20- and 25-day-old rats and was also sensitive to naloxone reversal.
Discussion Using the swimming harness described in this p a p e r it has p r o v e d possible to o b s e r v e swim S1A as early as day 10, an age w h e n unaided swimming is only possible for short periods ( < I min). T h e sensitivity to reversal by naloxone at this age points to opioid r e c e p t o r mediation and provides a contrasting d e v e l o p m e n t to the nonopioid f o r m of swim S I A which is not evident until postnatal day 25 (Jackson and Kitchen, 1989a). The lack of SIA before day 10 m a y be related to immaturity of the h y p o t h a l a m u s - p i t u i t a r y - a d r e n a l s y s t e m as short swim S I A is h o r m o n e d e p e n d e n t (Jackson and Kitchen, 1989a) and the h y p o t h a l a m u s - p i t u i t a r y - a d r e nal axis does not b e c o m e truly functional until the second postnatal w e e k in the rat (Henning, 1978; Walker et al., 1986). It is unlikely to reflect u n d e r d e v e l o p e d opioid s y s t e m s as in young rats Vt-opioid receptors operate SIA (Jackson and Kitchen, 1989a) and w-mediated behaviors can be clearly d e m o n s t r a t e d in 2- to 5-day-old pups (Pasternak et al., 1980; Z h o n g - Z h a n g and Pasternak, 1981; J a c k s o n and Kitchen, 1989b). Further, in r e s p o n s e to social or sexual isolation, shortterm analgesia is o b s e r v e d in 14-day-old animals and not in 7-day-old rats, again pointing to a lack of stressmediated pain suppression until the second postnatal w e e k (Takahashi et al., 1991).
141
In conclusion, we h a v e s h o w n using a swimming aid device that short, w a r m - w a t e r swims can induce SIA as early as postnatal day 10 and that this response is opioid r e c e p t o r - m e d i a t e d and develops rapidly up to the age of weaning. The authors thank Mr. P. Bishop for his help in developing swimming devices. B. Y. Muhammad is supported by a Kebbi State Scholarship, Nigeria.
References Bodnar RJ (1984) Types of stress which induce analgesia. In Stress Induced Analgesia. Eds., MD Tricklebank and G Cruzon. New York: Wiley & Sons, pp. 19-32. De Cabo C, Kitchen I, Viveros MP (1992) Adaptation of nociceptive procedures to developing animals. Sciences et Techniques de l'Animol de Laboratoire 17:145-148. Henning SJ (1978) Plasma concentrations of total and free corticostetone during development in the rat. A m J Physiol 235: (E)451-456. Jackson HC, Kitchen I (1989a) Swim-stress induced antinociception in young rats. Br J Pharmocol 96:617-622. Jackson HC, Kitchen I (1989b) Behavioural effects of selective mu, kappa and delta opioid agonists in neonatal rats. Psychopharmacology 97:404-409. Kitchen I, McDowell J, Winder C, Wilson JM (1984) Low-level lead exposure alters morphine antinociception in neonatal rats. Toxicol Lett 22:119-123. Kitchen I, Pinker SR (1990) Antagonism of swim-stress induced antinociception by the 8-opioid receptor antagonist naltrindole in neonatal and adult rats. Br J Pharmacol 100:685-688. Pasternak GW, Zhong-Zhang A, Tecott L (1980) Development differences between high and low affinity opiate binding studies; their relationship to analgesia and respiratory depression. Life Sci 27:1185-1190. Salas M (1971) Effects of early malnutrition on the development of swimming ability in the rat. Toxicol Behav 8:119-122. Schapiro S, Salas M, Vukovich K (1970) Hormonal effects on ontogeny of swimming ability in the rat: Assessment of central nervous system development. Science 168:147-151. Takahashi LK, Turner JG, Kalin NH (1991) Development of stressinduced responses in preweanling rats. Dev Psychobiol 24: 341-360. Walker CD, Perrin M, Vale W, Rivier C (1986) Ontogeny of the stress response in the rat: Role of the pituitary and the hypothalamus. Endocrinology 118:1445-1451. Zhong-Zhang A, Pasternak GW (1981) Ontogeny of opioid pharmacology and receptors: High and low affinity site differences. Eur J Pharmacol 73:29-40.
ORIGINAL
ARTICLES
A Method for the Study of Swimming Stress and Stress-Induced Antinociception in Preweanling Rats B a l a Y. M u h a m m a d a n d I a n K i t c h e n
Receptors and Cellular Regulation Research Group, School of Biological Sciences, University of Surrey, Guildford, Surrey, England, U.K.
Stress-induced antinociception (SIA) which is well characterized in the adult rat can also be observed in young rats, and, by varying swimming times, it can be dissociated into opioid and nonopioid forms. However, swimming ability in the rat does not fully develop until the third postnatal week and this has precluded the study of swim SIA in neonates. We report here the development of a harness device to aid swimming in young rats which we have successfully employed down to the age of 2 days without distress to the animals. Further we have also shown the development of the opioid form of swim SIA in the rat using this device. Swim SIA is absent at days 2 and 5, but at postnatal day 10 a small level of SIA is evident which is reversed by naloxone (10 mg/kg). Swim SIA develops rapidly thereafter, and the adult profile is observed by day 25.
Keywords: Stress-induced antinociception; Swimming stress; Opioid; Naloxone; Development; Ontogeny
Introduction Stress-induced antinociception (SIA) is an adaptive physiological response which can be induced by a number of different stressors including cold, immobilization, electroshock, and swimming (Bodnar, 1984). The latter stressor has been particularly useful as by varying the duration or temperature of swimming it is possible to dissociate opioid and nonopioid forms of the behavior, on the basis of naloxone reversal or naloxone insensitivity, respectively. We recently reported that swim SIA can be observed in young (20 days old) rats and that the neurochemistry and r e c e p t o r control of this response differs at this preweanling age in comparison with the adult (Jackson and Kitchen, 1989a; Kitchen and Pinker, 1990). Because swimming behavior in the rat does not properly develop until days 12-15
Address reprint requests to Dr. I. Kitchen, Receptors and Cellular Regulation Research Group, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, England, U.K. Received October 1992; revised and accepted December 1992. Journal of Pharmacological and Toxicological Methods 29, 139-141 (1993) © 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010
(Schapiro et al., 1990; Salas, 1972), it has proved difficult to study swim SIA in preweanl~ng animals. Further, in contrast to swim stress, other stressors, such as electroshock and cold exposure, are more severe and for ethical reasons are thus less desirable. We have, therefore, attempted to develop a methodology for using warm-water swimming stress in rat pups and report here the development of a swimming harness that allows the study of swim SIA down to the age of 2 days without distress to the animals and describe how the opioid form of swim SIA develops.
Methods Animals and Nociceptive Testing Wistar albino rats (University of Surrey strain) of mixed sexes were used in all experiments and maintained in litters of eight pups. All rats were maintained at 21 ° _+ I°C in a constant 12 hr light/dark cycle (lights on at 0:7 hr 00 min), and experimental procedures were carried out in a quiet, windowless, air-controlled laboratory. Pups up to the age of 20 days remained with the
1056-8719/93/$6.00
140
mother except during naloxone (10 mg/kg) administration, swimming, and nociceptive testing to minimize stress due to maternal deprivation. All procedures were carried out between 11 hr 00 min and 16 hr 00 min. Nociceptive responses were measured by the warmwater tail-immersion test adapted for use in neonates to give equivalent basal nociceptive measures (Kitchen et al., 1984; De Cabo et al., 1992) using the following temperatures (43.5°C, days 2 and 5; 47.5°C, day 10; 50°C, days 15, 20, and 25). The choice of temperatures were based on previous studies (De Cabo et al., 1992) which have shown that at ages below 10 days, deviation of _+0.5°C from 43.5°C produces either instant removal of the tail or absence of response. Use of 47.5°C at day 10 and 50°C at later ages was aimed at producing an equivalent basal response time (De Cabo et al., 1992), since in animals > 10 days of age consistent nociceptive responses are not achievable at the lower temperature (43.5°C). Nociceptive responses were recorded immediately before i.p. administration of naloxone or saline, 10 min before a 3-min period of swimming stress and at 1, 5, 10, 15, and 30 min following swim stress.
Swimming Aid Devices and Swim-Stress Procedures In confirmation of the work of Schapiro et al. (1970), 15-day-old animals were able to swim properly, unaided. However, 10-day-old rats exhibited swimming behavior for < 1 min and were unable to continue to float. Flotation devices consisting of rings of polystyrene (akin to the rubber ring used for children) were initially assessed in 10-day-old rats. These proved unsatisfactory as they prevented freedom of movement causing the pups to struggle and within 1 min the rats became immobile. The alternative approach which proved successful was to attach a harness to the trunk of the pups attached by a hook and a single string to a horizontal bar connected to a retort stand placed centrally over the swimming tank. 5-mm plastic cable ties with self-locking fasteners (R. S. Components, London, England, U.K.) were used for the harness device which allowed infinite adjustment from 1 to 3.5 cm diameter. The harness was adjusted so that both front and hind paws were free and no pressure was exerted on the thorax or abdomen. The degree of immersion of the animals could be finely adjusted using a rack and pinion adjuster on the stand. Neonatal rats were immersed sufficiently to induce a paddling type of response, and the harness allowed appreciable movement around the swimming tank while maintaining a fixed ventral posture. Neonate rats (2-20 days) were made to swim individually in a plastic tank (29 x 22 × 28 cm deep) containing water at 20° _+ 10°C for a period
JPM Vol. 29, No. 3 June 1993:139-141
of 3 min as previously described for 20-day-old animals (Jackson and Kitchen, 1989a). At the end of the swimming period, rats were removed from the harness by cutting the cable ties with a pair of scissors and returned to the home cage before subsequent nociceptive testing.
Results The swimming harness enabled 3-min periods of swimming to be observed in 2-, 5-, and 10-day-old rats without distress. Figure I shows the ontogenetic profile of swim-stress-induced antinociception from days
Figure 1. Effect of 3 min of swimming stress on nociceptive response in (a) 2-, (b) 5-, (c) 10-, (d) 15-, (e) 20-, and (f) 25day-old rats. Values represent means, and vertical bars, SE mean for groups of six animals. (O) Saline-injected, unstressed. (0) Saline-injected, swim-stressed. (D) Naloxone l0 mg/kg, unstressed. (I) Naloxone l0 mg/kg, swimstressed. Pre- Pretest responses 10 min before swim stress. Measures at 30 min were equivalent to those at 15 min at all ages and are omitted for clarity of presentation. Treatment groups were compared using ANOVA and posthoc comparisons made by Duncans New Multiple Range Test. *p < 0.05 swim-stressed versus appropriate unstressed control. +p < 0.05 naloxone-treated, swim-stressed versus saline-treated swim-stressed. a
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B. Y. MUHAMMADAND I. KITCHEN STRESS-INDUCEDANTINOCICEPTIONIN NEONATES
2 to 25. T h e r e was no indication of antinociception in 2- or 5-day-old rats after swimming stress. At day 10, h o w e v e r , swimming p r o d u c e d a small but significant increase in tail-immersion latency 5 min after swim stress; an effect which was c o m p l e t e l y r e v e r s e d by naloxone (10 mg/kg). At day 15, the level of S I A was greater and also significantly attenuated by the opioid antagonist. At this age, a greater level o f S I A could be achieved (5-min r e s p o n s e latency; 6.59 _+ 0.5 sec.) if the immersion t e m p e r a t u r e was reduced to 47.5°C. N a l o x o n e (10 mg/kg) c o m p l e t e l y antagonized SIA using these p a r a m e t e r s (5-min r e s p o n s e latency; 3.05 _+ 0.55 sec.). A level o f S I A a p p r o a c h i n g that o b s e r v e d in the adult (Kitchen and Pinker, 1990) was o b s e r v e d in 20- and 25-day-old rats and was also sensitive to naloxone reversal.
Discussion Using the swimming harness described in this p a p e r it has p r o v e d possible to o b s e r v e swim S1A as early as day 10, an age w h e n unaided swimming is only possible for short periods ( < I min). T h e sensitivity to reversal by naloxone at this age points to opioid r e c e p t o r mediation and provides a contrasting d e v e l o p m e n t to the nonopioid f o r m of swim S I A which is not evident until postnatal day 25 (Jackson and Kitchen, 1989a). The lack of SIA before day 10 m a y be related to immaturity of the h y p o t h a l a m u s - p i t u i t a r y - a d r e n a l s y s t e m as short swim S I A is h o r m o n e d e p e n d e n t (Jackson and Kitchen, 1989a) and the h y p o t h a l a m u s - p i t u i t a r y - a d r e nal axis does not b e c o m e truly functional until the second postnatal w e e k in the rat (Henning, 1978; Walker et al., 1986). It is unlikely to reflect u n d e r d e v e l o p e d opioid s y s t e m s as in young rats Vt-opioid receptors operate SIA (Jackson and Kitchen, 1989a) and w-mediated behaviors can be clearly d e m o n s t r a t e d in 2- to 5-day-old pups (Pasternak et al., 1980; Z h o n g - Z h a n g and Pasternak, 1981; J a c k s o n and Kitchen, 1989b). Further, in r e s p o n s e to social or sexual isolation, shortterm analgesia is o b s e r v e d in 14-day-old animals and not in 7-day-old rats, again pointing to a lack of stressmediated pain suppression until the second postnatal w e e k (Takahashi et al., 1991).
141
In conclusion, we h a v e s h o w n using a swimming aid device that short, w a r m - w a t e r swims can induce SIA as early as postnatal day 10 and that this response is opioid r e c e p t o r - m e d i a t e d and develops rapidly up to the age of weaning. The authors thank Mr. P. Bishop for his help in developing swimming devices. B. Y. Muhammad is supported by a Kebbi State Scholarship, Nigeria.
References Bodnar RJ (1984) Types of stress which induce analgesia. In Stress Induced Analgesia. Eds., MD Tricklebank and G Cruzon. New York: Wiley & Sons, pp. 19-32. De Cabo C, Kitchen I, Viveros MP (1992) Adaptation of nociceptive procedures to developing animals. Sciences et Techniques de l'Animol de Laboratoire 17:145-148. Henning SJ (1978) Plasma concentrations of total and free corticostetone during development in the rat. A m J Physiol 235: (E)451-456. Jackson HC, Kitchen I (1989a) Swim-stress induced antinociception in young rats. Br J Pharmocol 96:617-622. Jackson HC, Kitchen I (1989b) Behavioural effects of selective mu, kappa and delta opioid agonists in neonatal rats. Psychopharmacology 97:404-409. Kitchen I, McDowell J, Winder C, Wilson JM (1984) Low-level lead exposure alters morphine antinociception in neonatal rats. Toxicol Lett 22:119-123. Kitchen I, Pinker SR (1990) Antagonism of swim-stress induced antinociception by the 8-opioid receptor antagonist naltrindole in neonatal and adult rats. Br J Pharmacol 100:685-688. Pasternak GW, Zhong-Zhang A, Tecott L (1980) Development differences between high and low affinity opiate binding studies; their relationship to analgesia and respiratory depression. Life Sci 27:1185-1190. Salas M (1971) Effects of early malnutrition on the development of swimming ability in the rat. Toxicol Behav 8:119-122. Schapiro S, Salas M, Vukovich K (1970) Hormonal effects on ontogeny of swimming ability in the rat: Assessment of central nervous system development. Science 168:147-151. Takahashi LK, Turner JG, Kalin NH (1991) Development of stressinduced responses in preweanling rats. Dev Psychobiol 24: 341-360. Walker CD, Perrin M, Vale W, Rivier C (1986) Ontogeny of the stress response in the rat: Role of the pituitary and the hypothalamus. Endocrinology 118:1445-1451. Zhong-Zhang A, Pasternak GW (1981) Ontogeny of opioid pharmacology and receptors: High and low affinity site differences. Eur J Pharmacol 73:29-40.