Introduction

1. ARTIFICIALLY REARED RATS SHOW INCREASED SENSITIVITY TO AMPHETAMINE REVERSAL OF EFFECTS WITH TACTILE STIMULATION V. Lovic1, A.S. Fleming1, M.L. Smith1 & P.J. Fletcher1,2 *.1Department of Psychology, University of Toronto at Mississauga2Centre for Addiction and Mental Health, Toronto, Canada

2. Introduction Mammalian brain and behaviour plasticity is particularly evident during early stages of development while the young organism is in the care of its mother. Previously we have found that female rats that are raised without mothers, through artificial rearing (AR), grow up to show deficits in the attentional-set shifting task (Lovic & Fleming, 2004). These deficits were reversed in AR animals that were provided with maternal-licking like stimulation during early postnatal period. Rat attentional set-shifting task is an analogue of the Wisconsin Card Sorting Task, a test of behavioural flexibility that is sensitive to changes in the DA system and damage of the prefrontal cortex. It has been recently demonstrated in rats that alteration of the DA system, through amphetamine sensitization, produces deficits in the attentional set shifting task (Rizos, Lovic, Tann, Kapur & Fletcher, 2003). We were interested in investigating whether artificial rearing produces changes in the DA system. We decided to assess these changes indirectly by observing changes in DA dependent behaviour (locomotor activity) in response to treatment with two different DA agonists, amphetamine (dopamine releaser) and methylphenidate (Ritalin – dopamine reuptake inhibitor). Furthermore, we were interested in seeing whether maternal-licking like stimulation provided to some of the AR pups would reverse alterations in the DA system associated with AR.

3. Methods Subjects Fifty-four, male Spraque-Dawley rats were used. Animals were reared at the University of Toronto at Mississauga. Once they reached adulthood, they were transported to Centre for Addiction and Mental Health where they were tested. Artificial Rearing On the day of birth (postnatal day – PND 0) litters were culled to 5 females and 8 males. On PND 3, three males from each litter received a cheek cannulea implant. Two of the pups from each litter, that were implanted with cannulea were artificially reared (AR) and the third one was returned to its mother and was mother reared (MR-SHAM). The fourth male sibling was not manipulated and was designated as control (MR-CON). Two AR pups were placed in two different conditions: AR-MIN (AR with minimal amount of tactile stimulation – 2 x 30-sec/day) and AR-MAX (AR with ‘maximal’ amount of stimulation 8 x 2-min/day).

4. Methods

5. Methods Drugs Amphetamine (AMPH) and Methylphenidate (METH) were dissolved in 0.9% saline and administered intraperitoneally, in a volume of 1 ml/kg. Each rat’s activity was assessed after an injection of AMPH (0, 0.25, 0.5 and 1.0 mg/kg) or METH (0, 2, 5 and 10 mg/kg). Order of injections was counterbalanced between groups. Locomotor Activity Locomotor activity was assessed in four large, clear Plexiglas activity chambers (Med Associates Inc.). The apparatus was a square box (WL 43cm x H 30cm) equipped with an array of 16x16 photoreceptors that were used to detect locomotor activity. The boxes were connected to a computer capable of recording distance traveled based on interruptions of photoreceptors. Distance traveled was used as index of ambulatory activity and a dependent measure.All rats were first habituated to the apparatus by placing them in the activity chambers for 1-hour on 3 occasions, approximately a day apart. On the test days, rats were placed in the activity chamber for a 30-min habituation period followed immediately with an injection of one of four doses of AMPH or METH. Following injection, locomotor activity was assessed during a 90-min period.

6. Results AR Rats Showed Increased Activity in a Novel Environment b a a - MR vs. AR-MIN b – MR vs. AR-MAX

7. Results AR-MIN Rats Showed a Dose Dependent Increase in Amphetamine Sensitivity a a a a - MR vs. AR-MIN

8. Results There Were No Significant Group Differences in Methylphenidate Sensitivity

9. Conclusions AR rats showed hyperactivity in a novel environment. This effect dissipated by the last habituation session, indicating that AR are not hyperactive in general, but are rather hyperactive in response to novelty. Locomotor hyperactivity in response to novelty is associated with alterations in the DA and excitatory amino acid neurotransmission (Hooks & Kalivas, 1994). AR rats also showed increased locomotor activity after injections of amphetamine. This increased sensitivity was reversed by providing AR pups with maternal-licking like stimulation as exemplified by the AR-MAX group. There were no significant group differences in groups’ METH induced locomotion. These differences between AMPH and METH effects could be explained by the mechanisms of action of these two drugs (see figure). This would suggest that AR does not produce rats that have chronic overactivity of the DA system, but rather rats that have the potential of having a significantly more active DA system.

10. Conclusions The mechanism of these effects is unknow, however, previous research has suggested that tactile stimulation early in life changes the levels of perinatal hormones, such as thyroid hormone, that in turn produce changes in the brain. We plan to investigate the mechanism of action underlying the changes in the dopamine system associated with early life treatments Furthermore, we are planning to investigate dopamine-associated behaviors of artificially and mother reared animals, specifically their reward system (addiction) , as well as behavioral flexibility and impulsiveness.

11. References Hooks, M.S. & Kalivas, P.W. (1994). Involvement of dopamine and excitatory amino acid transmission in novelty-induced motor activity. Journal of Experimental Therapeutics, 269(3): 976-88. Lovic, V. & Fleming, A.S. (in press). Artificially reared female rats show reduced prepulse inhibition and deficits in the attentional set shifting task – reversal of effects with maternal-like licking stimulation. Behavioural Brain Research. Rizos, Z., Lovic, V., Tenn, C., Kapur, S. & Fletcher, P.J. (2003). Rats sensitized to amphetamine, but not PCP, show deficits in an attentional set-shifting task. Society for Neurscience Abstracts, New Oreleans, USA.

12. Abstract Mammalian brain and behaviour plasticity is particularly evident during early stages of development while the young organism is in the care of its mother. Previously we have found that total maternal deprivation of rat pups, achieved through artificial rearing (AR), leads to a number of behavioural and neurophysiological changes, suggesting a change in the dopamine system. The purpose of this study was to further investigate changes in the dopamine system, associated with AR, using two indirect dopamine agonists, d-amphetamine (dopamine releaser) and methylphenidate (Ritalin – dopamine reuptake inhibitor). Rats were raised with or without mothers (AR, ‘pup-in-a-cup’ paradigm) and some of those raised without mothers were provided with tactile, maternal-licking like stimulation. Rats’ locomotion was measured after an injection of d-amphetamine (0, 0.25, 0.5 and 1.0mg/kg) or methylphenidate (0, 2, 5, 10 mg/kg). While the AR produced increased sensitivity to d-amphetamine in a dose-response fashion, as exemplified by increased locomotion, there were no group differences in sensitivity to methylphenidate. Furthermore, AR animals that were provided with maternal-licking like stimulation were not different from the control, mother-reared animals. These data show that motherless rearing can produce increased sensitivity to amphetamine and that this increase in sensitivity can be reversed by providing motherless pups with tactile stimulation.