Brain Research 960 (2003) 42–47www.elsevier.com/ locate/brainres

Research report

E ffects of maternal separation on behavioural sensitization producedby repeated cocaine administration in adulthood

*YiLin Li, Terry E. Robinson, Seema BhatnagarDepartment 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 repeatedintravenous (i.v.) injections of cocaine in adult female rats. For the first 21 days of life litters were separated from their mother for either15 min (MS-15) or 180 min/day (MS-180). Control animals were subjected only to regular cage changes (animal facility reared; AFR). Inadulthood, these three groups did not differ in their locomotor response to placement in a novel environment or to an acute i.v. injectionof cocaine. After six additional daily injections of either saline or cocaine and following a 7-day drug-free interval, sensitization wasassessed 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 behaviouralsensitization 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 The length of daily maternal separation prior to weaning isimportant in determining the nature of these effects. Rats

Repeated administration of psychomotor stimulant drugs exposed to 15 min of separation daily (MS-15 or neonatalsuch as amphetamine and cocaine produces a progressive handling) exhibit lower hypothalamic–pituitary–adrenalincrease in their psychomotor activating effects, including responses to acute [13,14] and chronic stress [1] inlocomotor behaviour, a phenomenon known as behavioural adulthood as well as less age-related memory impairmentsensitization [20]. The development of behavioural sensiti- and neuronal cell loss [13,14] compared to rats subjectedzation is known to be influenced by a host of biological to longer periods of maternal separation (e.g., 180 min) orand environmental factors [8,18,19,21], but most studies to no manipulation (e.g., non-handled rats; [15,16]). Theseon susceptibility to sensitization have focussed on events neuroendocrine and behavioural differences between ratsand/or conditions in adulthood. Therefore, little is known separated for various periods of time in early postnatal lifeabout how events in the early postnatal environment alter are related to variations in maternal care [4,11]. Thus,behavioural sensitization to psychomotor stimulant drugs variations in maternal care, either naturally occurring orin adulthood. induced by maternal separation, constitute one aspect of

Maternal separation during early postnatal life affects the early environment that has long-term effects on theneuroendocrine responses to stress, anxiety-related behav- physiology and behaviour of the offspring.iour in adulthood and age-related cognitive deficits [15]. Maternal separation has been shown to affect behav-

ioural responses to acute amphetamine administration orself-administration of cocaine in some studies but not*Corresponding author. Tel.:11-734-615-3744; fax:11-734-763-others, depending on the length of separation [10,12]. In7480.

E-mail address: bhatnags@umich.edu(S. Bhatnagar). the present study, we examined the long-lasting effects of

0006-8993/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 02 )03752-6

43Y. Li et al. / Brain Research 960 (2003) 42–47

maternal separation on the susceptibility to behavioural 2 .3. Proceduressensitization produced by repeated injections of cocaine infemale rats. Little is known about the influence of early 2 .3.1. Surgeryenvironmental events on behavioural sensitization in When the animals were approximately 6 months oldfemales even though females generally exhibit more robust they were transferred to transparent plastic cages, wheresensitization than do males [19]. Locomotor responses to they were housed individually, and accustomed to handlingacute and repeated intravenous (i.v.) administrations of for 1 week. They were then prepared with indwellingcocaine were determined in adult female rats that had been intravenous (i.v.) catheters to later allow i.v. drug adminis-maternally separated for either 15 min (MS-15) or 180 min tration. We used i.v. administration because (a) the rapida day (MS-180), or not separated at all (animal facility onset of drug action produced by i.v. administration betterreared-AFR), for the first 21 days of life. approximates how addicts take drugs, and (b) the rapid i.v.

administration of cocaine is especially effective in produc-ing behavioral sensitization [22]. All rats were anesthetizedwith a mix of ketamine, xylazine, and acepromazine

2 . Materials and methods (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

2 .1. Animals anesthesia during surgery. An i.v. catheter was placed intothe right external jugular vein using procedures described

Litters from two waves (17 and 12 females, respective- previously [22,23]. Briefly, i.v. catheters were constructedly) of pregnant Sprague–Dawley female rats (13–15 days from silicone tubing and a backport guide cannula [3].gestation; purchased from Harlan Sprague–Dawley, In- Under anesthesia, the silicone end of the catheter wasdianapolis, IN) were used to generate the female subjects inserted into the right external jugular vein and a backportused in this study. In each wave, three to six litters were exited dorsally between the shoulder blades. Followingassigned to each of the MS-15, MS-180 and AFR groups. surgery, the catheter was flushed with 0.1 ml of gentamicinPups from each litter were eventually randomly assigned to (50 mg/ml) and 0.1 ml of heparin solution (30 units /mlthe two drug treatment groups. On the day of birth, litters heparin in 0.9% sterile bacteriostatic saline, ph 7.4) towere randomly assigned to one of three groups: (1) litters prevent occlusions and potential microbial buildup in thesubjected to daily maternal separation for 15 min (MS-15); catheter. The rats were then returned to the home cage and(2) litters subjected to daily maternal separation 180 min allowed to recover for 3–6 days before testing began.(MS-180); (3) litters that were reared in the animal facility During this period and throughout the duration of thewithout any manipulation other than regular cage changes experiment, catheters were manually flushed once daily(animal facility reared; AFR). On day 21, all pups were with 0.1 ml of heparin solution. The day following the lastweaned and housed in same-sex groups of up to five pretreatment day and the cocaine challenge testing day,animals, and then housed two to three rats per cage as catheters were screened for patency by infusing intraven-body weights increased. All rats were housed in a tempera- ously 0.1 ml of pentothal (thiopental sodium, 20 mg/ml inture- and humidity-controlled colony maintained on a sterile water). Rats that did not become ataxic within 10 s14:10-h light:dark cycle (lights on at 07:00 h) throughout were excluded from the experiment.the experiment. Food and water were available ad libitum.A total of 64 female rats were then tested at 6 months of 2 .3.2. Behavioural testing proceduresage. Only female rats were used in the experiment Three to 6 days after surgery, animals in each of thepresented here primarily because female rats show more three groups were further subdivided into either saline orrobust sensitization than do male rats [18]. cocaine pretreatment groups. On each treatment day each

animal was transferred from its home cage to a test cagelocated in an adjacent room. The test cages consisted of

2 .2. Maternal separation circular clear plastic buckets with a diameter of 25 cm anda height of 36 cm from the base. The floor was lined with

Maternal separation consisted of removing the mother granulated corn cob bedding (Bed-o-cobs). Each rat wasand then the pups from the home cage during the early tethered to a liquid swivel fixed to a moveable counter-lights on period. All pups from a given litter were placed balanced arm suspended above the animal by a stainlesstogether in a tub cage lined with bedding material. Pups steel cable. PE20 tubing connected the liquid swivel toand then the mothers were returned to their home cage each animal’s catheter and served as the drug/vehicleeither (1) 15-min later (MS-15) or (2) 180 min later infusion line that was connected to a syringe mounted on a(MS-180). This procedure occurred once a day for the first syringe pump. Thirty minutes after placement into the test21 days of life. Another set of litters was not disturbed environment each animal received an i.v. injection over 10during this time except for normal cage changes (AFR). s of either saline or cocaine (1.0 mg/kg) by activation ofAll animals remained undisturbed until catheterization. the syringe pumps. Thirty minutes after the injection the

Y. Li et al. / Brain Research 960 (2003) 42–4744

animals were disconnected from the liquid swivel andreturned 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 inall groups were transferred to the testing cages, and after a30-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-basedautomated rotometer that recorded every 908 turn in eachdirection using an XT-based personal computer (McFar-lane). The total number of 908 turns in either direction wasused as an index of locomotor activity. Behavioral datawere obtained during the initial 30 min habituation periodon the first treatment day, and used as an index of thelocomotor response to placement in a novel environment.On the first treatment day, the behavioral response to thefirst injection of cocaine was used to assess group differ-

Fig. 1. Locomotor activity (shown as quadrant entries per 3 min) isences in the response to an acute injection of cocaine. Onshown during the first 30-min period after the animals were placed in athe challenge test day, sensitization was assessed bynovel environment. Rats were exposed to either 15 min (MS 15), 180 min

comparing the magnitude of the behavioural response to(MS 180) of separation or normal cage changes (AFR) daily for the firstcocaine in saline-pretreated versus cocaine-pretreated21 days of life. All groups showed progressively lower locomotor activity

over the 30 min period and there was no significant effect of maternalgroups.separation on locomotor responses to the novel environment (MS 15,n528; MS 180,n527; AFR, n527). At 30 min, rats were injected with2 .4. Statistical analysiseither saline or cocaine (see Fig. 2).

The locomotor responses to drug treatment were statisti-cally evaluated by two-way (Maternal Separation 6 min after the injection and had returned to baseline levelscondition3Drug treatment) analyses of variance within 18 min (Fig. 2).(ANOVA). Fisher’s post-hoc tests were used when signifi-cant main effects were observed. 3 .3. Cocaine challenge test

Fig. 3 shows the time course of the behavioural response3 . Results to a cocaine challenge in both saline- and cocaine-pre-

treated groups. It is obvious from inspection of Fig. 3 that3 .1. Locomotor response to a novel environment the behavioral effect of an i.v. cocaine challenge peaked in

the first 3-min interval and had largely returned to baselineFig. 1 shows the time course of locomotor activity (908 within 9 min. Sensitization is typically manifest by both a

movements) over the first 30 min after placement into the more rapid onset of a drug effect and by an increase in thetest cage on the first treatment day, prior to the injection of peak drug effect. Therefore, to test for group differences ineither cocaine or saline. Locomotor activity decreased over sensitization, we analyzed the peak drug effect (3-mintime in all groups, indicating that all groups habituated to interval). Cocaine increased locomotor behaviour in boththe novel environment, and there were no significant group saline- and cocaine-pretreated animals at this peak time.differences. However, the cocaine-pretreated rats exhibited significant-

ly higher locomotor responses to the challenge injection of3 .2. Locomotor response to acute saline or cocaine cocaine than did saline-pretreated rats (a significant Drugtreatment treatment effect;F(1,58)548.9; P50.001), indicating sen-

sitization in all cocaine-pretreated groups. The main effectAt the end of the 30-min habituation period, the rats of Maternal Separation was not significant (F(2,58)52.14;

received an i.v. injection of either saline or cocaine (1.0 P50.13) but there was a significant Maternal Separationmg/kg). Fig. 2 shows a significant Drug treatment main group3Drug treatment interaction (F(2,58)54.40; P5effect (F(2,732)5100.4;P50.001) over the 30-min period 0.02). Post-hoc tests (Fisher’s) indicated that both MS-15but no significant main effect of Maternal Separation or and MS-180 rats pretreated with cocaine exhibited asignificant Interaction effect, indicating that, compared to significantly lower locomotor response than did cocaine-saline, cocaine significantly increased locomotor activity in pretreated AFR rats, and there was no significant differ-all groups. In all groups locomotor activity peaked within ence between the MS15 and MS180 cocaine-pretreated

45Y. Li et al. / Brain Research 960 (2003) 42–47

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 followingthe 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 toregular cage changes (AFR) for the first 21 days of life. No significant differences in locomotor responses to saline or cocaine were observed between thethree 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 re- 4 . Discussionsponse to the challenge dose of cocaine in saline- versuscocaine-pretreated groups, the peak drug effect on the We examined whether manipulations of the neonatalchallenge test day is shown in the inset in Fig. 3. 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 adultfemale 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 oflife. 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. Cocainepretreated groups exhibited increased locomotor activity to the challenge dose of cocaine compared to saline-pretreated groups, providing evidence ofsensitization. The MS-15 and MS-180 cocaine-pretreated groups exhibited significantly less robust behavioural responses to a cocaine challenge comparedto 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 betweenthe AFR group versus the MS-15 and MS-180 groups.

Y. Li et al. / Brain Research 960 (2003) 42–4746

by repeated i.v. cocaine administration given in adulthood. cocaine. Thus, it seems that the affects of maternalFemale rats were exposed to daily maternal separation for separation on neuroendocrine function and anxiety-relatedeither 15 (MS-15) or 180 min (MS-180), or no maternal behaviours and on behavioural sensitization to cocaine canseparation (AFR) for the first 3 weeks of life. When they be dissociated.reached adulthood, we examined their locomotor responses In a recent study, Meaney et al. [17] reported thatto a novel environment and to the first i.v. injection of maternal separation had no effect on sensitization producedsaline or cocaine. There were no differences amongst the by repeated intraperitoneal injections of amphetamine,three groups on either of these measures. These animals although the MS-180 group showed greater sensitization tothen received six additional daily injections of saline or the repeated stress of saline injection compared to MS-15cocaine (1.0 mg/kg, i.v.), and the ability of these treat- and AFR groups. These data differ from ours in that wements to produce behavioural sensitization was assessed by found maternal separation (MS-15 or MS-180) attenuateda challenge injection of cocaine (0.5 mg/kg, i.v.) following sensitization to cocaine but did not alter the effect ofa 7-day drug-free interval. Rats exposed to either period of repeated treatment with saline. There are a number ofmaternal separation (MS-15 or MS-180) showed a sig- methodological differences between the Meaney et al. [17]nificantly less robust behavioral response to the drug study and ours that could account for the disparatechallenge than did control AFR rats. Given that the groups findings, including the drug (amphetamine versus cocaine),did not differ in their initial response to cocaine (as route of administration (i.p. versus i.v.), different pretreat-assessed on the first day of cocaine treatment and by the ment regimens and perhaps the sex of the animal (we usedresponse of saline pretreated rats challenged with cocaine), females and Meaney et al. do not provide this informationbut did differ after repeated treatment with cocaine, we but presumably used males). Other studies have examinedconclude that early maternal separation influenced the the effects of maternal separation on acute responses toability of cocaine to induce behavioural sensitization. amphetamine and on self-administration of cocaine. In aSpecifically, maternal separation decreased susceptibility maternal separation paradigm similar to that used in theto sensitization relative to the AFR control group, but the present study, no differences in locomotor responses toduration of maternal separation had no effect on this acute amphetamine were observed between rats exposed tooutcome. different periods of maternal separation [5], which is

Maternal separation, of the durations used in the present consistent with the results reported here. Sixty minutes ofstudy, produces long-lasting effects on neuroendocrine separation on days 2–9 after birth is reported to facilitateresponses, anxiety responses and age-related cognitive the acquisition of cocaine self-administration in male ratsimpairments [15]. These effects of varying durations of [10], suggesting increased sensitivity to the reinforcingmaternal separation are related to variations in maternal effects of cocaine. However, 10 periods of 6 h of maternalcare that induce plasticity in neural systems, including separation between days 5 and 20 of life did not sig-central corticotropin releasing hormone (CRH) systems. nificantly alter acquisition or maintenance of self-adminis-For example, MS-15 rats exhibit lower CRH mRNA in tration of cocaine in male rats [12]. Thus, the effect ofsome brainstem and limbic regions compared to MS-180 maternal separation on self-administration may be relatedrats [9]. Central CRH systems also regulate behaviour to the duration and frequency of maternal separation.produced by psychomotor stimulants. Intracerebroventricu- However, we found that duration of separation had nolar administration of CRH increases amphetamine-induced effect on the behavioural sensitization produced by i.v.stereotypy [6] and potentiates the development of sensiti- cocaine in female rats (Figs. 2 and 3). Furthermore, ourzation to amphetamine produced by repeated exposure to finding that both MS female groups exhibit decreasedstress [7]. The latter effect can be blocked by central sensitization to cocaine compared to AFR rats suggestsadministration of CRH antagonists [7]. In addition, re- that maternal separation, for either 15 or 180 min, maypeated injections of central CRH produce behavioural reduce the ability of cocaine to produce whatever neuro-sensitization to subsequent amphetamine administration adaptations that lead to the development of sensitization.[2]. Given the role of CRH in governing maternal sepa- The mechanism through which this effect of maternalration effects on neuroendocrine responses to stress- and separation occurs is not known. Nonetheless, this studyanxiety-related behaviors, as well as sensitization to psy- shows that early maternal separation, regardless of dura-chomotor stimulants, we hypothesized that the known tion, can have long-lasting effects on the susceptibility tochanges in central CRH circuits produced by maternal psychomotor sensitization in female rats, perhaps byseparation would also manifest themselves in differences altering the ability of cocaine to induce sensitization-in behavioural sensitization to cocaine between MS-15 and related neuroadaptive processes.MS-180 groups. The lack of difference between MS-15and MS-180 female rats in behavioural sensitization tococaine in the present study suggests that the mechanisms,A cknowledgementsincluding those involving CRH, by which different periodsof maternal separation alter neuroendocrine function are This research was funded by a NARSAD Young Inves-not involved in regulating behavioural sensitization to tigator Award to SB and a NIDA grant to TER (R34

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