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Effects of maternal separation on behavioural sensitization produced by repeated cocaine administration in adulthood
Brain Research 960 (2003) 42–47 www.elsevier.com / locate / brainres
Research report
Effects of maternal separation on behavioural sensitization produced by repeated cocaine administration in adulthood YiLin Li, Terry E. Robinson, Seema Bhatnagar* Department of Psychology and Program in Neuroscience, The University of Michigan, 525 East University, Ann Arbor, MI 48109 -1109, USA Accepted 30 August 2002
Abstract We examined whether early maternal separation influenced the susceptibility to behavioural sensitization produced by repeated intravenous (i.v.) injections of cocaine in adult female rats. For the first 21 days of life litters were separated from their mother for either 15 min (MS-15) or 180 min / day (MS-180). Control animals were subjected only to regular cage changes (animal facility reared; AFR). In adulthood, these three groups did not differ in their locomotor response to placement in a novel environment or to an acute i.v. injection of cocaine. After six additional daily injections of either saline or cocaine and following a 7-day drug-free interval, sensitization was assessed by giving all rats a challenge i.v. injection of cocaine. Rats exposed to either period of maternal separation (MS-15 or MS-180) showed significantly less robust sensitization than AFR rats. Therefore, early maternal separation decreased susceptibility to behavioural sensitization produced by cocaine in adulthood and the specific duration of maternal separation had no effect on this outcome. 2002 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Behavioral pharmacology Keywords: Psychomotor stimulant; Maternal separation; Neonatal handling; Plasticity
1. Introduction Repeated administration of psychomotor stimulant drugs such as amphetamine and cocaine produces a progressive increase in their psychomotor activating effects, including locomotor behaviour, a phenomenon known as behavioural sensitization [20]. The development of behavioural sensitization is known to be influenced by a host of biological and environmental factors [8,18,19,21], but most studies on susceptibility to sensitization have focussed on events and / or conditions in adulthood. Therefore, little is known about how events in the early postnatal environment alter behavioural sensitization to psychomotor stimulant drugs in adulthood. Maternal separation during early postnatal life affects neuroendocrine responses to stress, anxiety-related behaviour in adulthood and age-related cognitive deficits [15]. *Corresponding author. Tel.: 11-734-615-3744; fax: 11-734-7637480. E-mail address: [email protected] (S. Bhatnagar).
The length of daily maternal separation prior to weaning is important in determining the nature of these effects. Rats exposed to 15 min of separation daily (MS-15 or neonatal handling) exhibit lower hypothalamic–pituitary–adrenal responses to acute [13,14] and chronic stress [1] in adulthood as well as less age-related memory impairment and neuronal cell loss [13,14] compared to rats subjected to longer periods of maternal separation (e.g., 180 min) or to no manipulation (e.g., non-handled rats; [15,16]). These neuroendocrine and behavioural differences between rats separated for various periods of time in early postnatal life are related to variations in maternal care [4,11]. Thus, variations in maternal care, either naturally occurring or induced by maternal separation, constitute one aspect of the early environment that has long-term effects on the physiology and behaviour of the offspring. Maternal separation has been shown to affect behavioural responses to acute amphetamine administration or self-administration of cocaine in some studies but not others, depending on the length of separation [10,12]. In the present study, we examined the long-lasting effects of
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03752-6
Y. Li et al. / Brain Research 960 (2003) 42–47
maternal separation on the susceptibility to behavioural sensitization produced by repeated injections of cocaine in female rats. Little is known about the influence of early environmental events on behavioural sensitization in females even though females generally exhibit more robust sensitization than do males [19]. Locomotor responses to acute and repeated intravenous (i.v.) administrations of cocaine were determined in adult female rats that had been maternally separated for either 15 min (MS-15) or 180 min a day (MS-180), or not separated at all (animal facility reared-AFR), for the first 21 days of life.
2. Materials and methods
2.1. Animals Litters from two waves (17 and 12 females, respectively) of pregnant Sprague–Dawley female rats (13–15 days gestation; purchased from Harlan Sprague–Dawley, Indianapolis, IN) were used to generate the female subjects used in this study. In each wave, three to six litters were assigned to each of the MS-15, MS-180 and AFR groups. Pups from each litter were eventually randomly assigned to the two drug treatment groups. On the day of birth, litters were randomly assigned to one of three groups: (1) litters subjected to daily maternal separation for 15 min (MS-15); (2) litters subjected to daily maternal separation 180 min (MS-180); (3) litters that were reared in the animal facility without any manipulation other than regular cage changes (animal facility reared; AFR). On day 21, all pups were weaned and housed in same-sex groups of up to five animals, and then housed two to three rats per cage as body weights increased. All rats were housed in a temperature- and humidity-controlled colony maintained on a 14:10-h light:dark cycle (lights on at 07:00 h) throughout the experiment. Food and water were available ad libitum. A total of 64 female rats were then tested at 6 months of age. Only female rats were used in the experiment presented here primarily because female rats show more robust sensitization than do male rats [18].
2.2. Maternal separation Maternal separation consisted of removing the mother and then the pups from the home cage during the early lights on period. All pups from a given litter were placed together in a tub cage lined with bedding material. Pups and then the mothers were returned to their home cage either (1) 15-min later (MS-15) or (2) 180 min later (MS-180). This procedure occurred once a day for the first 21 days of life. Another set of litters was not disturbed during this time except for normal cage changes (AFR). All animals remained undisturbed until catheterization.
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2.3. Procedures 2.3.1. Surgery When the animals were approximately 6 months old they were transferred to transparent plastic cages, where they were housed individually, and accustomed to handling for 1 week. They were then prepared with indwelling intravenous (i.v.) catheters to later allow i.v. drug administration. We used i.v. administration because (a) the rapid onset of drug action produced by i.v. administration better approximates how addicts take drugs, and (b) the rapid i.v. administration of cocaine is especially effective in producing behavioral sensitization [22]. All rats were anesthetized with a mix of ketamine, xylazine, and acepromazine (77:1.5:1.5 mg / ml, i.p., at 0.1 ml / 100 g of body weight) and methoxyflurane was used as needed to maintain anesthesia during surgery. An i.v. catheter was placed into the right external jugular vein using procedures described previously [22,23]. Briefly, i.v. catheters were constructed from silicone tubing and a backport guide cannula [3]. Under anesthesia, the silicone end of the catheter was inserted into the right external jugular vein and a backport exited dorsally between the shoulder blades. Following surgery, the catheter was flushed with 0.1 ml of gentamicin (50 mg / ml) and 0.1 ml of heparin solution (30 units / ml heparin in 0.9% sterile bacteriostatic saline, ph 7.4) to prevent occlusions and potential microbial buildup in the catheter. The rats were then returned to the home cage and allowed to recover for 3–6 days before testing began. During this period and throughout the duration of the experiment, catheters were manually flushed once daily with 0.1 ml of heparin solution. The day following the last pretreatment day and the cocaine challenge testing day, catheters were screened for patency by infusing intravenously 0.1 ml of pentothal (thiopental sodium, 20 mg / ml in sterile water). Rats that did not become ataxic within 10 s were excluded from the experiment. 2.3.2. Behavioural testing procedures Three to 6 days after surgery, animals in each of the three groups were further subdivided into either saline or cocaine pretreatment groups. On each treatment day each animal was transferred from its home cage to a test cage located in an adjacent room. The test cages consisted of circular clear plastic buckets with a diameter of 25 cm and a height of 36 cm from the base. The floor was lined with granulated corn cob bedding (Bed-o-cobs). Each rat was tethered to a liquid swivel fixed to a moveable counterbalanced arm suspended above the animal by a stainless steel cable. PE20 tubing connected the liquid swivel to each animal’s catheter and served as the drug / vehicle infusion line that was connected to a syringe mounted on a syringe pump. Thirty minutes after placement into the test environment each animal received an i.v. injection over 10 s of either saline or cocaine (1.0 mg / kg) by activation of the syringe pumps. Thirty minutes after the injection the
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animals were disconnected from the liquid swivel and returned to their home cage in the animal colony room. This procedure was repeated for six consecutive days. Drug treatment was then discontinued for the next 7 days. On the 15th day after the first treatment session, all rats in all groups were transferred to the testing cages, and after a 30-min habituation period they all received a challenge i.v. infusion of 0.5 mg / kg cocaine. Each test chamber was equipped with a photocell-based automated rotometer that recorded every 908 turn in each direction using an XT-based personal computer (McFarlane). The total number of 90 8 turns in either direction was used as an index of locomotor activity. Behavioral data were obtained during the initial 30 min habituation period on the first treatment day, and used as an index of the locomotor response to placement in a novel environment. On the first treatment day, the behavioral response to the first injection of cocaine was used to assess group differences in the response to an acute injection of cocaine. On the challenge test day, sensitization was assessed by comparing the magnitude of the behavioural response to cocaine in saline-pretreated versus cocaine-pretreated groups.
2.4. Statistical analysis The locomotor responses to drug treatment were statistically evaluated by two-way (Maternal Separation condition3Drug treatment) analyses of variance (ANOVA). Fisher’s post-hoc tests were used when significant main effects were observed.
3. Results
3.1. Locomotor response to a novel environment Fig. 1 shows the time course of locomotor activity (908 movements) over the first 30 min after placement into the test cage on the first treatment day, prior to the injection of either cocaine or saline. Locomotor activity decreased over time in all groups, indicating that all groups habituated to the novel environment, and there were no significant group differences.
3.2. Locomotor response to acute saline or cocaine treatment At the end of the 30-min habituation period, the rats received an i.v. injection of either saline or cocaine (1.0 mg / kg). Fig. 2 shows a significant Drug treatment main effect (F(2,732)5100.4; P50.001) over the 30-min period but no significant main effect of Maternal Separation or significant Interaction effect, indicating that, compared to saline, cocaine significantly increased locomotor activity in all groups. In all groups locomotor activity peaked within
Fig. 1. Locomotor activity (shown as quadrant entries per 3 min) is shown during the first 30-min period after the animals were placed in a novel environment. Rats were exposed to either 15 min (MS 15), 180 min (MS 180) of separation or normal cage changes (AFR) daily for the first 21 days of life. All groups showed progressively lower locomotor activity over the 30 min period and there was no significant effect of maternal separation on locomotor responses to the novel environment (MS 15, n528; MS 180, n527; AFR, n527). At 30 min, rats were injected with either saline or cocaine (see Fig. 2).
6 min after the injection and had returned to baseline levels within 18 min (Fig. 2).
3.3. Cocaine challenge test Fig. 3 shows the time course of the behavioural response to a cocaine challenge in both saline- and cocaine-pretreated groups. It is obvious from inspection of Fig. 3 that the behavioral effect of an i.v. cocaine challenge peaked in the first 3-min interval and had largely returned to baseline within 9 min. Sensitization is typically manifest by both a more rapid onset of a drug effect and by an increase in the peak drug effect. Therefore, to test for group differences in sensitization, we analyzed the peak drug effect (3-min interval). Cocaine increased locomotor behaviour in both saline- and cocaine-pretreated animals at this peak time. However, the cocaine-pretreated rats exhibited significantly higher locomotor responses to the challenge injection of cocaine than did saline-pretreated rats (a significant Drug treatment effect; F(1,58)548.9; P50.001), indicating sensitization in all cocaine-pretreated groups. The main effect of Maternal Separation was not significant (F(2,58)52.14; P50.13) but there was a significant Maternal Separation group3Drug treatment interaction (F(2,58)54.40; P5 0.02). Post-hoc tests (Fisher’s) indicated that both MS-15 and MS-180 rats pretreated with cocaine exhibited a significantly lower locomotor response than did cocainepretreated AFR rats, and there was no significant difference between the MS15 and MS180 cocaine-pretreated
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Fig. 2. Following a 30-min acclimation period to the novel environment (shown in Fig. 1), rats were injected with either saline or cocaine (1.0 mg / kg i.v.) and locomotor activity recorded for 30 min. Locomotor responses (shown as quadrant entries per 3 min) are shown for 6 min before and 18 min following the first injection of saline or cocaine in adult female rats exposed to maternal separation for either 15 min (MS-15), 180 min (MS-180) or subject to regular cage changes (AFR) for the first 21 days of life. No significant differences in locomotor responses to saline or cocaine were observed between the three maternal separation groups (MS-15-saline, n512; MS-15-cocaine, n516; MS-180-saline, n513; MS-180-cocaine, n514; AFR-saline, n514; AFR-cocaine, n513).
groups. To better illustrate group differences in the response to the challenge dose of cocaine in saline- versus cocaine-pretreated groups, the peak drug effect on the challenge test day is shown in the inset in Fig. 3.
4. Discussion We examined whether manipulations of the neonatal environment influenced behavioural sensitization produced
Fig. 3. The time course of locomotor activity (shown as quadrant entries per 3 min) is shown prior to and following a challenge dose of cocaine in adult female rats exposed to maternal separation for either 15 min (MS-15), 180 min (MS-180) or subject to regular cage changes (AFR) for the first 21 days of life. These groups were pretreated for 7 days with either saline or cocaine followed by a 7-day drug-free period prior to challenge with cocaine. Cocaine pretreated groups exhibited increased locomotor activity to the challenge dose of cocaine compared to saline-pretreated groups, providing evidence of sensitization. The MS-15 and MS-180 cocaine-pretreated groups exhibited significantly less robust behavioural responses to a cocaine challenge compared to cocaine-pretreated AFR rats, indicating reduced sensitization (MS-15-saline, n59; MS-15-cocaine, n513; MS-180-saline, n513; MS-180-cocaine, n511; AFR-saline, n59; AFR-cocaine, n59). The peak locomotor response in is shown in the inset. *P50.05; indicates significant differences between the AFR group versus the MS-15 and MS-180 groups.
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by repeated i.v. cocaine administration given in adulthood. Female rats were exposed to daily maternal separation for either 15 (MS-15) or 180 min (MS-180), or no maternal separation (AFR) for the first 3 weeks of life. When they reached adulthood, we examined their locomotor responses to a novel environment and to the first i.v. injection of saline or cocaine. There were no differences amongst the three groups on either of these measures. These animals then received six additional daily injections of saline or cocaine (1.0 mg / kg, i.v.), and the ability of these treatments to produce behavioural sensitization was assessed by a challenge injection of cocaine (0.5 mg / kg, i.v.) following a 7-day drug-free interval. Rats exposed to either period of maternal separation (MS-15 or MS-180) showed a significantly less robust behavioral response to the drug challenge than did control AFR rats. Given that the groups did not differ in their initial response to cocaine (as assessed on the first day of cocaine treatment and by the response of saline pretreated rats challenged with cocaine), but did differ after repeated treatment with cocaine, we conclude that early maternal separation influenced the ability of cocaine to induce behavioural sensitization. Specifically, maternal separation decreased susceptibility to sensitization relative to the AFR control group, but the duration of maternal separation had no effect on this outcome. Maternal separation, of the durations used in the present study, produces long-lasting effects on neuroendocrine responses, anxiety responses and age-related cognitive impairments [15]. These effects of varying durations of maternal separation are related to variations in maternal care that induce plasticity in neural systems, including central corticotropin releasing hormone (CRH) systems. For example, MS-15 rats exhibit lower CRH mRNA in some brainstem and limbic regions compared to MS-180 rats [9]. Central CRH systems also regulate behaviour produced by psychomotor stimulants. Intracerebroventricular administration of CRH increases amphetamine-induced stereotypy [6] and potentiates the development of sensitization to amphetamine produced by repeated exposure to stress [7]. The latter effect can be blocked by central administration of CRH antagonists [7]. In addition, repeated injections of central CRH produce behavioural sensitization to subsequent amphetamine administration [2]. Given the role of CRH in governing maternal separation effects on neuroendocrine responses to stress- and anxiety-related behaviors, as well as sensitization to psychomotor stimulants, we hypothesized that the known changes in central CRH circuits produced by maternal separation would also manifest themselves in differences in behavioural sensitization to cocaine between MS-15 and MS-180 groups. The lack of difference between MS-15 and MS-180 female rats in behavioural sensitization to cocaine in the present study suggests that the mechanisms, including those involving CRH, by which different periods of maternal separation alter neuroendocrine function are not involved in regulating behavioural sensitization to
cocaine. Thus, it seems that the affects of maternal separation on neuroendocrine function and anxiety-related behaviours and on behavioural sensitization to cocaine can be dissociated. In a recent study, Meaney et al. [17] reported that maternal separation had no effect on sensitization produced by repeated intraperitoneal injections of amphetamine, although the MS-180 group showed greater sensitization to the repeated stress of saline injection compared to MS-15 and AFR groups. These data differ from ours in that we found maternal separation (MS-15 or MS-180) attenuated sensitization to cocaine but did not alter the effect of repeated treatment with saline. There are a number of methodological differences between the Meaney et al. [17] study and ours that could account for the disparate findings, including the drug (amphetamine versus cocaine), route of administration (i.p. versus i.v.), different pretreatment regimens and perhaps the sex of the animal (we used females and Meaney et al. do not provide this information but presumably used males). Other studies have examined the effects of maternal separation on acute responses to amphetamine and on self-administration of cocaine. In a maternal separation paradigm similar to that used in the present study, no differences in locomotor responses to acute amphetamine were observed between rats exposed to different periods of maternal separation [5], which is consistent with the results reported here. Sixty minutes of separation on days 2–9 after birth is reported to facilitate the acquisition of cocaine self-administration in male rats [10], suggesting increased sensitivity to the reinforcing effects of cocaine. However, 10 periods of 6 h of maternal separation between days 5 and 20 of life did not significantly alter acquisition or maintenance of self-administration of cocaine in male rats [12]. Thus, the effect of maternal separation on self-administration may be related to the duration and frequency of maternal separation. However, we found that duration of separation had no effect on the behavioural sensitization produced by i.v. cocaine in female rats (Figs. 2 and 3). Furthermore, our finding that both MS female groups exhibit decreased sensitization to cocaine compared to AFR rats suggests that maternal separation, for either 15 or 180 min, may reduce the ability of cocaine to produce whatever neuroadaptations that lead to the development of sensitization. The mechanism through which this effect of maternal separation occurs is not known. Nonetheless, this study shows that early maternal separation, regardless of duration, can have long-lasting effects on the susceptibility to psychomotor sensitization in female rats, perhaps by altering the ability of cocaine to induce sensitizationrelated neuroadaptive processes.
Acknowledgements This research was funded by a NARSAD Young Investigator Award to SB and a NIDA grant to TER (R34
Y. Li et al. / Brain Research 960 (2003) 42–47
DA04294). TER was also supported by a Senior Research Scientist Award from NIDA (K05 DA00473).
References [1] S. Bhatnagar, M.J. Meaney, Hypothalamic-pituitary-adrenal function in chronic intermittently cold-stressed neonatally handled and non handled rats, J. Neuroendocrinol. 7 (2) (1995) 97–108. [2] M. Cador, B.J. Cole, G.F. Koob, L. Stinus, M. Le Moal, Central administration of corticotropin releasing factor induces long-term sensitization to D-amphetamine, Brain Res. 606 (1993) 181–186. [3] S.B. Caine, R. Lintz, G.F. Koob, Intravenous drug self-administration techniques in animals, in: A. Sahgal (Ed.), Behavioral Neuroscience, A Practical Approach, Vol. II, IRL Press at Oxford University Press, New York, 1993, pp. 117–143. [4] C. Caldji, B. Tannenbaum, S. Sharma, D. Francis, P.M. Plotsky, M.J. Meaney, Maternal care during infancy regulates the development of neural systems mediating the expression of fearfulness in the rat, Proc Natl Acad Sci USA 95 (9) (1998) 5335–5340. [5] J. Campbell, L.P. Spear, Effects of early handling on amphetamineinduced locomotor activation and conditioned place preference in the adult rat, Psychopharmacology 143 (1999) 183–189. [6] B.J. Cole, G.F. Koob, Low doses of corticotropin-releasing factor potentiate amphetamine-induced stereotyped behavior, Psychopharmacology 99 (1989) 27–33. [7] B.J. Cole, M. Cador, L. Stinus, J. Riviere, W. Vale, G.F. Koob, M. Le Moal, Central administration of a CRF antagonist blocks the development of stress-induced behavioral sensitization, Brain Res. 512 (1990) 343–346. [8] H. Crombag, A. Badiani, T.E. Robinson, Signalled versus unsignalled intravenous amphetamine: large differences in the acute psychomotor response and sensitization, Brain Res. 722 (1996) 227–231. [9] D.D. Francis, C. Caldji, F. Champagne, P.M. Plotsky, M.J. Meaney, The role of corticotropin-releasing factor-norepinephrine systems in mediating the effects of early experience on the development of behavioral and endocrine responses to stress, Biol. Psychiatry 46 (1999) 1153–1166. [10] T.A. Kosten, M.J.D. Miserendino, P. Kehoe, Enhanced acquisition of cocaine self-administration in adult rats with neonatal isolation stress experience, Brain Res. 875 (2000) 44–50. [11] D. Liu, J. Diorio, B. Tannenbaum, C. Caldji, D. Francis, A. Freedman, S. Sharma, D. Pearson, P.M. Plotsky, M.J. Meaney, Maternal care, hippocampal glucocorticoid receptors, and hypothalamic–pituitary–adrenal responses to stress, Science 277 (5332) (1997) 1659–1662.
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[12] K. Matthews, T.W. Robbins, B.J. Everitt, S.B. Caine, Repeated neonatal maternal separation alters intravenous cocaine self-administration in adult rats, Psychopharmacology 141 (1999) 123–134. [13] M.J. Meaney, D.H. Aitken, S. Bhatnagar, D. Van Berkel, R.M. Sapolsky, Postnatal handling attenuates neuroendocrine, anatomical and cognitive impairments related to the aged hippocampus, Science 239 (1988) 766–768. [14] M.J. Meaney, D.H. Aitken, S. Bhatnagar, R.M. Sapolsky, Postnatal handling attenuates certain neuroendocrine, anatomical and cognitive dysfunctions associated with aging in female rats, Neurobiol. Aging 12 (1991) 31–38. [15] M.J. Meaney, Maternal care, gene expression, and the transmission of individual differences in stress reactivity across generations, Annu. Rev. Neurosci. 24 (2001) 1161–1192. [16] M.J. Meaney, P.M. Plotsky, Early, postnatal experience alters hypothalamic corticotropin-releasing factor (CRF) mRNA, median eminence CRF content stress-induced release in adult rats, Mol. Brain Res. 18 (1993) 195–200. [17] M.J. Meaney, W. Brake, A. Gratton, Environmental regulation of the development of mesolimbic dopamine systems: a neurobiological mechanism for vulnerability to drug abuse?, Psychoneuroendocrinology 27 (1 / 2) (2002) 127–138. ` S. Maccari, M. Le Moal, P. Mormede, ` [18] P.V. Piazza, J.-M. Deminiere, H. Simon, Individual vulnerability to drug self-administration: action of corticosterone on dopaminergic systems as a possible ¨ pathophysiological mechanism, in: P. Willner, J. Scheel-Kruger (Eds.), The Mesolimbic Dopamine System: From Motivation to Action, Wiley, New York, 1991, pp. 473–495. [19] T.E. Robinson, Behavioral sensitization: characterization of enduring changes in rotational behavior produced by intermittent injections of amphetamine in male and female rats, Psychopharmacology 84 (1984) 466–475. [20] T.E. Robinson, J.B. Becker, Enduring changes in brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal models of amphetamine psychosis, Brain Res. Rev. 396 (1986) 157–198. [21] T.E. Robinson, Stimulant drugs and stress: factors influencing individual differences in the susceptibility to sensitization, in: P.W. Kalivas, C. Barnes (Eds.), Sensitization of the Nervous System, Telford Press, Caldwell, NJ, 1988, pp. 145–173. [22] A.N. Samaha, Y. Li, T.E. Robinson, The rate of intravenous cocaine administration determines susceptibility to sensitization, J. Neurosci. 22 (8) (2002) 3244–3250. [23] J.R. Weeks, Long-term intravenous infusions, in: Myers (Ed.), Methods in Psychobiology, Academic Press, London, 1972, pp. 155–168.
Research report
Effects of maternal separation on behavioural sensitization produced by repeated cocaine administration in adulthood YiLin Li, Terry E. Robinson, Seema Bhatnagar* Department of Psychology and Program in Neuroscience, The University of Michigan, 525 East University, Ann Arbor, MI 48109 -1109, USA Accepted 30 August 2002
Abstract We examined whether early maternal separation influenced the susceptibility to behavioural sensitization produced by repeated intravenous (i.v.) injections of cocaine in adult female rats. For the first 21 days of life litters were separated from their mother for either 15 min (MS-15) or 180 min / day (MS-180). Control animals were subjected only to regular cage changes (animal facility reared; AFR). In adulthood, these three groups did not differ in their locomotor response to placement in a novel environment or to an acute i.v. injection of cocaine. After six additional daily injections of either saline or cocaine and following a 7-day drug-free interval, sensitization was assessed by giving all rats a challenge i.v. injection of cocaine. Rats exposed to either period of maternal separation (MS-15 or MS-180) showed significantly less robust sensitization than AFR rats. Therefore, early maternal separation decreased susceptibility to behavioural sensitization produced by cocaine in adulthood and the specific duration of maternal separation had no effect on this outcome. 2002 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Behavioral pharmacology Keywords: Psychomotor stimulant; Maternal separation; Neonatal handling; Plasticity
1. Introduction Repeated administration of psychomotor stimulant drugs such as amphetamine and cocaine produces a progressive increase in their psychomotor activating effects, including locomotor behaviour, a phenomenon known as behavioural sensitization [20]. The development of behavioural sensitization is known to be influenced by a host of biological and environmental factors [8,18,19,21], but most studies on susceptibility to sensitization have focussed on events and / or conditions in adulthood. Therefore, little is known about how events in the early postnatal environment alter behavioural sensitization to psychomotor stimulant drugs in adulthood. Maternal separation during early postnatal life affects neuroendocrine responses to stress, anxiety-related behaviour in adulthood and age-related cognitive deficits [15]. *Corresponding author. Tel.: 11-734-615-3744; fax: 11-734-7637480. E-mail address: [email protected] (S. Bhatnagar).
The length of daily maternal separation prior to weaning is important in determining the nature of these effects. Rats exposed to 15 min of separation daily (MS-15 or neonatal handling) exhibit lower hypothalamic–pituitary–adrenal responses to acute [13,14] and chronic stress [1] in adulthood as well as less age-related memory impairment and neuronal cell loss [13,14] compared to rats subjected to longer periods of maternal separation (e.g., 180 min) or to no manipulation (e.g., non-handled rats; [15,16]). These neuroendocrine and behavioural differences between rats separated for various periods of time in early postnatal life are related to variations in maternal care [4,11]. Thus, variations in maternal care, either naturally occurring or induced by maternal separation, constitute one aspect of the early environment that has long-term effects on the physiology and behaviour of the offspring. Maternal separation has been shown to affect behavioural responses to acute amphetamine administration or self-administration of cocaine in some studies but not others, depending on the length of separation [10,12]. In the present study, we examined the long-lasting effects of
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03752-6
Y. Li et al. / Brain Research 960 (2003) 42–47
maternal separation on the susceptibility to behavioural sensitization produced by repeated injections of cocaine in female rats. Little is known about the influence of early environmental events on behavioural sensitization in females even though females generally exhibit more robust sensitization than do males [19]. Locomotor responses to acute and repeated intravenous (i.v.) administrations of cocaine were determined in adult female rats that had been maternally separated for either 15 min (MS-15) or 180 min a day (MS-180), or not separated at all (animal facility reared-AFR), for the first 21 days of life.
2. Materials and methods
2.1. Animals Litters from two waves (17 and 12 females, respectively) of pregnant Sprague–Dawley female rats (13–15 days gestation; purchased from Harlan Sprague–Dawley, Indianapolis, IN) were used to generate the female subjects used in this study. In each wave, three to six litters were assigned to each of the MS-15, MS-180 and AFR groups. Pups from each litter were eventually randomly assigned to the two drug treatment groups. On the day of birth, litters were randomly assigned to one of three groups: (1) litters subjected to daily maternal separation for 15 min (MS-15); (2) litters subjected to daily maternal separation 180 min (MS-180); (3) litters that were reared in the animal facility without any manipulation other than regular cage changes (animal facility reared; AFR). On day 21, all pups were weaned and housed in same-sex groups of up to five animals, and then housed two to three rats per cage as body weights increased. All rats were housed in a temperature- and humidity-controlled colony maintained on a 14:10-h light:dark cycle (lights on at 07:00 h) throughout the experiment. Food and water were available ad libitum. A total of 64 female rats were then tested at 6 months of age. Only female rats were used in the experiment presented here primarily because female rats show more robust sensitization than do male rats [18].
2.2. Maternal separation Maternal separation consisted of removing the mother and then the pups from the home cage during the early lights on period. All pups from a given litter were placed together in a tub cage lined with bedding material. Pups and then the mothers were returned to their home cage either (1) 15-min later (MS-15) or (2) 180 min later (MS-180). This procedure occurred once a day for the first 21 days of life. Another set of litters was not disturbed during this time except for normal cage changes (AFR). All animals remained undisturbed until catheterization.
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2.3. Procedures 2.3.1. Surgery When the animals were approximately 6 months old they were transferred to transparent plastic cages, where they were housed individually, and accustomed to handling for 1 week. They were then prepared with indwelling intravenous (i.v.) catheters to later allow i.v. drug administration. We used i.v. administration because (a) the rapid onset of drug action produced by i.v. administration better approximates how addicts take drugs, and (b) the rapid i.v. administration of cocaine is especially effective in producing behavioral sensitization [22]. All rats were anesthetized with a mix of ketamine, xylazine, and acepromazine (77:1.5:1.5 mg / ml, i.p., at 0.1 ml / 100 g of body weight) and methoxyflurane was used as needed to maintain anesthesia during surgery. An i.v. catheter was placed into the right external jugular vein using procedures described previously [22,23]. Briefly, i.v. catheters were constructed from silicone tubing and a backport guide cannula [3]. Under anesthesia, the silicone end of the catheter was inserted into the right external jugular vein and a backport exited dorsally between the shoulder blades. Following surgery, the catheter was flushed with 0.1 ml of gentamicin (50 mg / ml) and 0.1 ml of heparin solution (30 units / ml heparin in 0.9% sterile bacteriostatic saline, ph 7.4) to prevent occlusions and potential microbial buildup in the catheter. The rats were then returned to the home cage and allowed to recover for 3–6 days before testing began. During this period and throughout the duration of the experiment, catheters were manually flushed once daily with 0.1 ml of heparin solution. The day following the last pretreatment day and the cocaine challenge testing day, catheters were screened for patency by infusing intravenously 0.1 ml of pentothal (thiopental sodium, 20 mg / ml in sterile water). Rats that did not become ataxic within 10 s were excluded from the experiment. 2.3.2. Behavioural testing procedures Three to 6 days after surgery, animals in each of the three groups were further subdivided into either saline or cocaine pretreatment groups. On each treatment day each animal was transferred from its home cage to a test cage located in an adjacent room. The test cages consisted of circular clear plastic buckets with a diameter of 25 cm and a height of 36 cm from the base. The floor was lined with granulated corn cob bedding (Bed-o-cobs). Each rat was tethered to a liquid swivel fixed to a moveable counterbalanced arm suspended above the animal by a stainless steel cable. PE20 tubing connected the liquid swivel to each animal’s catheter and served as the drug / vehicle infusion line that was connected to a syringe mounted on a syringe pump. Thirty minutes after placement into the test environment each animal received an i.v. injection over 10 s of either saline or cocaine (1.0 mg / kg) by activation of the syringe pumps. Thirty minutes after the injection the
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animals were disconnected from the liquid swivel and returned to their home cage in the animal colony room. This procedure was repeated for six consecutive days. Drug treatment was then discontinued for the next 7 days. On the 15th day after the first treatment session, all rats in all groups were transferred to the testing cages, and after a 30-min habituation period they all received a challenge i.v. infusion of 0.5 mg / kg cocaine. Each test chamber was equipped with a photocell-based automated rotometer that recorded every 908 turn in each direction using an XT-based personal computer (McFarlane). The total number of 90 8 turns in either direction was used as an index of locomotor activity. Behavioral data were obtained during the initial 30 min habituation period on the first treatment day, and used as an index of the locomotor response to placement in a novel environment. On the first treatment day, the behavioral response to the first injection of cocaine was used to assess group differences in the response to an acute injection of cocaine. On the challenge test day, sensitization was assessed by comparing the magnitude of the behavioural response to cocaine in saline-pretreated versus cocaine-pretreated groups.
2.4. Statistical analysis The locomotor responses to drug treatment were statistically evaluated by two-way (Maternal Separation condition3Drug treatment) analyses of variance (ANOVA). Fisher’s post-hoc tests were used when significant main effects were observed.
3. Results
3.1. Locomotor response to a novel environment Fig. 1 shows the time course of locomotor activity (908 movements) over the first 30 min after placement into the test cage on the first treatment day, prior to the injection of either cocaine or saline. Locomotor activity decreased over time in all groups, indicating that all groups habituated to the novel environment, and there were no significant group differences.
3.2. Locomotor response to acute saline or cocaine treatment At the end of the 30-min habituation period, the rats received an i.v. injection of either saline or cocaine (1.0 mg / kg). Fig. 2 shows a significant Drug treatment main effect (F(2,732)5100.4; P50.001) over the 30-min period but no significant main effect of Maternal Separation or significant Interaction effect, indicating that, compared to saline, cocaine significantly increased locomotor activity in all groups. In all groups locomotor activity peaked within
Fig. 1. Locomotor activity (shown as quadrant entries per 3 min) is shown during the first 30-min period after the animals were placed in a novel environment. Rats were exposed to either 15 min (MS 15), 180 min (MS 180) of separation or normal cage changes (AFR) daily for the first 21 days of life. All groups showed progressively lower locomotor activity over the 30 min period and there was no significant effect of maternal separation on locomotor responses to the novel environment (MS 15, n528; MS 180, n527; AFR, n527). At 30 min, rats were injected with either saline or cocaine (see Fig. 2).
6 min after the injection and had returned to baseline levels within 18 min (Fig. 2).
3.3. Cocaine challenge test Fig. 3 shows the time course of the behavioural response to a cocaine challenge in both saline- and cocaine-pretreated groups. It is obvious from inspection of Fig. 3 that the behavioral effect of an i.v. cocaine challenge peaked in the first 3-min interval and had largely returned to baseline within 9 min. Sensitization is typically manifest by both a more rapid onset of a drug effect and by an increase in the peak drug effect. Therefore, to test for group differences in sensitization, we analyzed the peak drug effect (3-min interval). Cocaine increased locomotor behaviour in both saline- and cocaine-pretreated animals at this peak time. However, the cocaine-pretreated rats exhibited significantly higher locomotor responses to the challenge injection of cocaine than did saline-pretreated rats (a significant Drug treatment effect; F(1,58)548.9; P50.001), indicating sensitization in all cocaine-pretreated groups. The main effect of Maternal Separation was not significant (F(2,58)52.14; P50.13) but there was a significant Maternal Separation group3Drug treatment interaction (F(2,58)54.40; P5 0.02). Post-hoc tests (Fisher’s) indicated that both MS-15 and MS-180 rats pretreated with cocaine exhibited a significantly lower locomotor response than did cocainepretreated AFR rats, and there was no significant difference between the MS15 and MS180 cocaine-pretreated
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Fig. 2. Following a 30-min acclimation period to the novel environment (shown in Fig. 1), rats were injected with either saline or cocaine (1.0 mg / kg i.v.) and locomotor activity recorded for 30 min. Locomotor responses (shown as quadrant entries per 3 min) are shown for 6 min before and 18 min following the first injection of saline or cocaine in adult female rats exposed to maternal separation for either 15 min (MS-15), 180 min (MS-180) or subject to regular cage changes (AFR) for the first 21 days of life. No significant differences in locomotor responses to saline or cocaine were observed between the three maternal separation groups (MS-15-saline, n512; MS-15-cocaine, n516; MS-180-saline, n513; MS-180-cocaine, n514; AFR-saline, n514; AFR-cocaine, n513).
groups. To better illustrate group differences in the response to the challenge dose of cocaine in saline- versus cocaine-pretreated groups, the peak drug effect on the challenge test day is shown in the inset in Fig. 3.
4. Discussion We examined whether manipulations of the neonatal environment influenced behavioural sensitization produced
Fig. 3. The time course of locomotor activity (shown as quadrant entries per 3 min) is shown prior to and following a challenge dose of cocaine in adult female rats exposed to maternal separation for either 15 min (MS-15), 180 min (MS-180) or subject to regular cage changes (AFR) for the first 21 days of life. These groups were pretreated for 7 days with either saline or cocaine followed by a 7-day drug-free period prior to challenge with cocaine. Cocaine pretreated groups exhibited increased locomotor activity to the challenge dose of cocaine compared to saline-pretreated groups, providing evidence of sensitization. The MS-15 and MS-180 cocaine-pretreated groups exhibited significantly less robust behavioural responses to a cocaine challenge compared to cocaine-pretreated AFR rats, indicating reduced sensitization (MS-15-saline, n59; MS-15-cocaine, n513; MS-180-saline, n513; MS-180-cocaine, n511; AFR-saline, n59; AFR-cocaine, n59). The peak locomotor response in is shown in the inset. *P50.05; indicates significant differences between the AFR group versus the MS-15 and MS-180 groups.
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by repeated i.v. cocaine administration given in adulthood. Female rats were exposed to daily maternal separation for either 15 (MS-15) or 180 min (MS-180), or no maternal separation (AFR) for the first 3 weeks of life. When they reached adulthood, we examined their locomotor responses to a novel environment and to the first i.v. injection of saline or cocaine. There were no differences amongst the three groups on either of these measures. These animals then received six additional daily injections of saline or cocaine (1.0 mg / kg, i.v.), and the ability of these treatments to produce behavioural sensitization was assessed by a challenge injection of cocaine (0.5 mg / kg, i.v.) following a 7-day drug-free interval. Rats exposed to either period of maternal separation (MS-15 or MS-180) showed a significantly less robust behavioral response to the drug challenge than did control AFR rats. Given that the groups did not differ in their initial response to cocaine (as assessed on the first day of cocaine treatment and by the response of saline pretreated rats challenged with cocaine), but did differ after repeated treatment with cocaine, we conclude that early maternal separation influenced the ability of cocaine to induce behavioural sensitization. Specifically, maternal separation decreased susceptibility to sensitization relative to the AFR control group, but the duration of maternal separation had no effect on this outcome. Maternal separation, of the durations used in the present study, produces long-lasting effects on neuroendocrine responses, anxiety responses and age-related cognitive impairments [15]. These effects of varying durations of maternal separation are related to variations in maternal care that induce plasticity in neural systems, including central corticotropin releasing hormone (CRH) systems. For example, MS-15 rats exhibit lower CRH mRNA in some brainstem and limbic regions compared to MS-180 rats [9]. Central CRH systems also regulate behaviour produced by psychomotor stimulants. Intracerebroventricular administration of CRH increases amphetamine-induced stereotypy [6] and potentiates the development of sensitization to amphetamine produced by repeated exposure to stress [7]. The latter effect can be blocked by central administration of CRH antagonists [7]. In addition, repeated injections of central CRH produce behavioural sensitization to subsequent amphetamine administration [2]. Given the role of CRH in governing maternal separation effects on neuroendocrine responses to stress- and anxiety-related behaviors, as well as sensitization to psychomotor stimulants, we hypothesized that the known changes in central CRH circuits produced by maternal separation would also manifest themselves in differences in behavioural sensitization to cocaine between MS-15 and MS-180 groups. The lack of difference between MS-15 and MS-180 female rats in behavioural sensitization to cocaine in the present study suggests that the mechanisms, including those involving CRH, by which different periods of maternal separation alter neuroendocrine function are not involved in regulating behavioural sensitization to
cocaine. Thus, it seems that the affects of maternal separation on neuroendocrine function and anxiety-related behaviours and on behavioural sensitization to cocaine can be dissociated. In a recent study, Meaney et al. [17] reported that maternal separation had no effect on sensitization produced by repeated intraperitoneal injections of amphetamine, although the MS-180 group showed greater sensitization to the repeated stress of saline injection compared to MS-15 and AFR groups. These data differ from ours in that we found maternal separation (MS-15 or MS-180) attenuated sensitization to cocaine but did not alter the effect of repeated treatment with saline. There are a number of methodological differences between the Meaney et al. [17] study and ours that could account for the disparate findings, including the drug (amphetamine versus cocaine), route of administration (i.p. versus i.v.), different pretreatment regimens and perhaps the sex of the animal (we used females and Meaney et al. do not provide this information but presumably used males). Other studies have examined the effects of maternal separation on acute responses to amphetamine and on self-administration of cocaine. In a maternal separation paradigm similar to that used in the present study, no differences in locomotor responses to acute amphetamine were observed between rats exposed to different periods of maternal separation [5], which is consistent with the results reported here. Sixty minutes of separation on days 2–9 after birth is reported to facilitate the acquisition of cocaine self-administration in male rats [10], suggesting increased sensitivity to the reinforcing effects of cocaine. However, 10 periods of 6 h of maternal separation between days 5 and 20 of life did not significantly alter acquisition or maintenance of self-administration of cocaine in male rats [12]. Thus, the effect of maternal separation on self-administration may be related to the duration and frequency of maternal separation. However, we found that duration of separation had no effect on the behavioural sensitization produced by i.v. cocaine in female rats (Figs. 2 and 3). Furthermore, our finding that both MS female groups exhibit decreased sensitization to cocaine compared to AFR rats suggests that maternal separation, for either 15 or 180 min, may reduce the ability of cocaine to produce whatever neuroadaptations that lead to the development of sensitization. The mechanism through which this effect of maternal separation occurs is not known. Nonetheless, this study shows that early maternal separation, regardless of duration, can have long-lasting effects on the susceptibility to psychomotor sensitization in female rats, perhaps by altering the ability of cocaine to induce sensitizationrelated neuroadaptive processes.
Acknowledgements This research was funded by a NARSAD Young Investigator Award to SB and a NIDA grant to TER (R34
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DA04294). TER was also supported by a Senior Research Scientist Award from NIDA (K05 DA00473).
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