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Cross-fostering does not alter the differential sensitivity of Fischer and Lewis rats to central neurotensin-induced locomotion and hypothermia
Behavioural Brain Research 171 (2006) 142–146
Research report
Cross-fostering does not alter the differential sensitivity of Fischer and Lewis rats to central neurotensin-induced locomotion and hypothermia Pat Bauco ∗ , Pierre-Paul Rompr´e Centre de Recherche Fernand-Seguin, Hˆopital Louis-H. Lafontaine et, D´epartment de Psychiatrie, Universit´e de Montr´eal, Montr´eal, Que., Canada H1N 3V2 Received 8 December 2005; received in revised form 18 March 2006; accepted 22 March 2006 Available online 5 May 2006
Abstract A cross-fostering paradigm was used to determine whether the differential locomotor and hypothermic responses to neurotensin (NT) in Fischer (F344) and Lewis (LEW) rats are mediated by the post-natal environment. From post-natal day (PD) 1 to PD 21, male pups from each strain were assigned to a same-strain dam (in-fostered) or were cross-fostered, and at adulthood were implanted with a guide cannula over the lateral ventricle. They were then tested for locomotion and hypothermia following injection of vehicle, 0.18, 1.8 or 18 nmol of NT or D-Tyr[11] NT. In-fostered LEW, but not F344, displayed a strong dose-orderly hypothermic response to NT and to D-Tyr[11] NT while in-fostered F344, but not LEW, rats displayed strong locomotor responses to D-Tyr[11] NT. Cross-fostering had no effect on D-Tyr[11] NT-induced locomotor responses in either strain; it had no effect also on NT- and D-Tyr[11] NT-induced hypothermia in F344 rats while it slightly increased the sensitivity to NT in LEW rats. The results show that these NT-mediated actions are not influenced by cross-fostering or the pre-weaning environment. © 2006 Elsevier B.V. All rights reserved. Keywords: Cross-foster; Exploratory locomotion; Fischer; Hypothermia; Lewis; Neurotensin; Peptide transmitters; Strain differences
1. Introduction Fischer (F344) and Lewis (LEW) are two inbred rat strains that differ in their appetitive and consummatory responses to several drugs of abuse. Lewis rats more readily self-administer opioids [25] and cocaine [23] compared to F344 rats. Compared to F344 rats, LEW rats more readily establish conditioned preferences towards environments associated with opioids, cocaine [16,22] and nicotine [18]; they also sensitize to the locomotorstimulant actions of cocaine [7,22] and amphetamine [7] to a greater degree than F344 rats. A large body of evidence implicates mid-brain dopamine (DA) in the appetitive effects of drugs of abuse [35,36]. Fischer and LEW rats have been shown to differ significantly in dopamine synthesis, transport [5,16], and release [34] as well as in second messenger and immediate early gene expression linked to DA receptor activation [17]. Such
∗ Corresponding author at: Centre de Recherche Fernand-Seguin, Hˆ opital Louis-H. Lafontaine, 7331, rue Hochelga, Montr´eal, Que., Canada H1N 3V2. Tel.: +1 514 251 4015; fax: +1 514 251 2617. E-mail address: [email protected] (P. Bauco).
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differences have been proposed as relevant features of the neurobiological substrate of drug addiction [31]. Neurotensin (NT) is an endogenous tridecapapetide that modulates DA function at pre- and post-synaptic levels in several reward-relevant limbic regions [6], and it has been shown that the endogenous NT circuitry differs in F344 and LEW rats; central NT receptor activation produces differential reward-potentiating [1], locomotor [3] and hypothermic [2] responses. It has been well documented that environmental factors during postnatal development can alter gene expression and have a significant impact on neuroendocrine [28], physiological [10] and behavioral measures [26]. Maternal care, for example, has been shown to regulate and endocrine responses to stress [29] and synaptic development [24]. A question that arises then is whether early environmental factors contribute to differences observed in F344 and LEW rats following central NT receptoractivation. Cross-fostering has been used with other rat strains to show that factors such as maternal and pup behavior, and maternal/pup interactions, play a key role in phenotypic differences between strains [27]. F344 and LEW dams have been also shown to differ in their maternal behavior. For example, LEW dams show a shorter latency to retrieve pups, a different temporal
P. Bauco, P.-P. Rompr´e / Behavioural Brain Research 171 (2006) 142–146
pattern of pup retrieval [15], and more time spent in contact with the litter [37] compared to F344 dams. The aim of the present experiment, therefore, was to use cross-fostering of F344 and LEW rats to determine whether the differential locomotor and hypothermic responses to central NT receptor activation in these two strains of rats is modulated by the pre-weaning environment. 2. Materials and methods Experiments were carried out in accordance with the Canadian Council on Animal Care Guide to the Care and Use of Experimental Animals. An internal animal care committee approved all experiments and all efforts were made to minimize animal discomfort.
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the box. Movements detected within the virtual box were considered as nonambulatory and were quantified as time (in min) during which photocell beam interruptions were detected. Vertical activity was quantified as the total number of photocell beam interruptions produced by rearing (see [11] for validation data on these measures of activity).
2.4. Histology At the end of each experiment, animals were deeply anesthetized with urethane (1.4 g/kg, i.p.) and transcardially perfused with 0.9% saline followed by a 10% formalin solution. Brains were removed, stored in 10% formalin and subsequently sliced in serial 40-m sections that were stained with a formal-thionin solution. Location of the injection site was determined under light microscopic examination. Only data from animals with a confirmed ventricular injection site were included in the analyses.
2.1. Subjects and cross-fostering procedure 2.5. Drugs Pregnant Fischer (F344) and Lewis (LEW) rats were obtained at approximately 14 days of gestation (Charles River, St-Constant, Qu´ebec, Canada) and housed individually with free access to food and water in a temperature- and humidity-controlled room with a 12-h light/dark cycle; lights on at 06:30 h. On the day of birth, all female pups were removed from the litters and the remaining F344 and LEW male pups were returned to their respective same strain dam (in-fostered) or to an opposite strain dam (cross-fostered); this resulted in four test groups: (i) F344 pups reared by F344 dams; (ii) F344 pups reared by LEW dams; (iii) LEW pups reared by LEW dams and (iv) LEW pups raised by F344 dams. All testing was performed during the light phase of the day/night cycle (between 06:30 and 16:30), in a room separate from the housing colony.
2.2. Surgery Adult rats (275 and 325 g) were injected with atropine methylnitrate (0.4 mg/kg, i.p.), anesthetized with sodium pentobarbital (65 mg/kg, i.p.) and mounted onto a stereotaxic apparatus. A guide cannula (Plastic One Inc., VA, USA, model C315G) was implanted above the left lateral ventricle (flat-skull coordinates: 0.8 mm posterior to bregma, 1.2 mm lateral and 2.8 mm below the skull surface) according to the atlas of Paxinos and Watson [32] and anchored to stainless-steel screws threaded into the skull with dental cement.
Neurotensin-(1-13); (NT) and (D-Tyr11 )-Neurotensin (D-Tyr[11] NT; Bachem Bioscience Inc., Torrance, CA, USA) were dissolved in sterile 0.9% saline at a concentration of 1.8 nmol/l and stored frozen at −20 ◦ C in aliquots pre-coated with silicone. The peptide solution was thawed just prior to testing and diluted to the required concentration with sterile 0.9% saline when necessary.
2.6. Data analysis Rectal temperature was measured prior to (baseline) and every 30 min for 2 h after microinjection of peptide or vehicle. Data were expressed as the mean of the four post-injection measurements for each respective treatment and were analyzed using a 2 × 2 × 5 (dam strain × pup strain × treatment) MANCOVA (with baselines as the co-variate). Comparisons among means were made with Duncan’s multiple range post-hoc test with level of significance set at 0.05. Parameters of locomotor activity (ambulatory activity, non-ambulatory activity and vertical activity) were computed for the total (2 h) test period, and group means were analyzed with a two-way analysis of variance (ANOVA) with strain (four levels) and treatment (two levels) as the two independent factors.
3. Results 2.3. Testing All animals were tested once weekly in the locomotor activity or hypothermia phases of the experiment; the order of testing was counterbalanced such that for each group, half the animals completed the locomotion tests (D-Tyr[11] NT 18 nmol and vehicle) first and the other half completed the hypothermia tests (NT 0.18, 1.8, 18 nmol, D-Tyr[11] NT 18 nmol and vehicle) first. Concentrations of peptide (and vehicle) were chosen based on our previous studies [2,3] and were tested in a randomized sequence for each animal. 2.3.1. Neurotensin-induced hypothermia Body temperature was measured by inserting a lubricated flexible thermoprobe 6–7 cm into the rectum and kept it in place for approximately 10 s while the rat was hand-restrained. Rectal temperature was assessed prior to and at 30 min intervals for a total of 2 h after injection. Animals were returned to holding cages between trials. Testing was performed in a temperature-controlled room (ambient temperature 22 ◦ C). 2.3.2. Neurotensin-induced locomotion Locomotor activity was measured using an Opto-Varimex Auto Track System (Columbus Instruments, Columbus, OH, USA). This system consists of Plexiglass cages (42 cm × 42 cm × 35 cm) with wire meshed floor equipped with two arrays of 15 infrared photocells located 1.5 and 14.5 cm above the floor to detect horizontal and vertical movements, respectively. Computer software quantified ambulatory activity by calculating the distance traveled beyond a “virtual box” of 9.6 cm × 9.6 cm (3 × 3 photocells) drawn around the animal; the computer assessed (10 times per second) the location of the animal within
3.1. Test for hypothermia Neurotensin induced a dose-orderly hypothermic response in in-fostered LEW rats and the analog produced a significantly larger response at an equimolar concentration than the native peptide (Fig. 1, top panel). Neurotensin also induced a dose-orderly hypothermia in in-fostered F344 rats (Fig. 1, bottom panel) but the magnitude of the response was much weaker than that observed in LEW rats and the NT analog produced a hypothermic response of the same magnitude as NT. LEW rats fostered by F344 dams did not show a blunted hypothermic response like the in-fostered F344 rats. F344 rats fostered by LEW dams were not different than their in-fostered mates; they were isosensitive to the hypothermic effects at each dose of NT and its analog. Mean pre-injection rectal temperatures differed significantly between strains; in-fostered and cross-fostered LEW rats had significantly lower pre-injection rectal temperatures compared to in-fostered and cross-fostered and F344 rats (Fig. 1, top and bottom panels). There were, however, no significant differences in pre-injection temperatures as a function of cross-fostering (Fig. 1, top and bottom panels).
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Fig. 1. Mean ± S.E.M. pre-injection rectal temperatures in in-fostered and cross-fostered Lewis (top panel) and in-fostered and cross-fostered Fischer (bottom panel) rats. In-fostered and cross-fostered Lewis rats had respectively significantly lower pre-injection rectal temperatures compared to in-fostered and to cross-fostered Fischer rats as determined by a Student’s t-test for independent samples († p < 0.05). There were no significant within-strain differences in preinjection rectal temperature. Figure also presents the mean ± S.E.M. changes in rectal temperature (◦ C) over 2 h following injection with vehicle or one of three concentrations of neurotensin (NT) and its analog D-Tyr[11] NT (D-NT) in in-fostered and cross-fostered Lewis (top panel, n = 13 and 9, respectively) and Fischer (bottom panel, n = 9 and 8, respectively) rats. A MANCOVA yielded a significant main effect of strain (dam; F(1,33) = 50.5, p < 0.0001), a significant main effect of treatment (F(4,136) = 124.91, p < 0.0001) as well as a signficant strain (dam) by treatment interaction (F(4,136) = 54.86, p < 0.0001). Post-hoc tests confirmed significant differences between NT and vehicle (* p < 0.05), 18 nmol of NT and of D-Tyr[11] NT (# p < 0.05), and Fischer and Lewis rats (the corresponding treatment and fostering condition, + p < 0.05).
3.2. Test for locomotion The NT analog which mimicks the effect of NT on core body temperature induced in in-fostered F344 rats strong ambulatory and non-ambulatory activity and suppressed vertical activity
Fig. 2. Total (2 h) ambulatory activity expressed as distance in meters travelled within the activity box (top panels), non-ambulatory activity expressed as time in minutes making movements within the “virtual box” (see Section 2.3.2 for details, middle panels), and vertical activity expressed as total number of photocell counts (bottom panels) measured in in-fostered and cross-fostered Fischer (n = 9 and 8, respectively) and Lewis (n = 13 and 8, respectively) rats. Data are presented as group means ± S.E.M. The ANOVA yielded a significant effect of strain by treatment interaction: ambulatory activity F(3,68) = 24.8, p < 0.001, non-ambulatory activity F(3,68) = 22.8, p < 0.001, vertical activity F(3,68) = 4.14, p < 0.01. Post-hoc tests confirmed the significant differences between NT and vehicle (* p < 0.05; ** p < 0.1) and between Fischer and Lewis rats (for the corresponding treatment and fostering condition, + p < 0.05; ++ p < 0.01).
compared to the respective vehicle control (Fig. 2). The NT analog had no effect on ambulatory and non-ambulatory activity in in-fostered LEW rats; it produced a significant suppression of vertical activity compared to vehicle, an effect that tended to be stronger than in F344. Cross-fostered F344 and LEW rats responded almost identically to the NT analog; F344, but not LEW, displayed an increase in ambulatory and non-ambulatory activity while both strains showed a suppression of vertical movements. The locomotor response to vehicle did not differ between in-fostered and cross-fostered rats of both strains and the time course of the locomotor responses (not shown) vehicle
P. Bauco, P.-P. Rompr´e / Behavioural Brain Research 171 (2006) 142–146
and peptide were very similar to that previously reported (see Fig. 2 in [3]). 4. Discussion The results of the hypothermia experiment show that NT produced a dose-orderly decrease in core body temperature in in-fostered rats of both strains but with a much lower magnitude in F344 rats. As reported previously [2], it is the response of F344 rats that is atypical because the hypothermic response of LEW rats to NT is similar to what has previously been found for other strains [8]. In order to determine whether the lower potency of NT in F344 rats was due to a more effective and rapid enzymatic degradation of the peptide, we tested the effectiveness of an equimolar concentration of D-Tyr[11] NT. The NT analog which is more resistant to degradation [9] was slightly more potent in LEW rats but as potent as NT in F344 rats; this excludes then the possibility that in F344 rats the blunted hypothermic response to NT is due pharmacokinetic properties. This conclusion is reinforced by the fact that both strains are iso-sensitive to NT-induced analgesia in the hot-plate test [2]. Previous studies have shown that NT plays a role in ethanol-induced hypothermia, and it has been suggested that these substances act in a synergistic manner on specific neuronal processes to induce a hypothermic response [13,14]. It can be inferred that the atypical hypothermic response of F344 rats is due to an atypical functional characteristic of the NT relevant circuitry, rather than an anomaly of the hypothermic response per se, since F344 rats show a normal hypothermic response to ethanol [21]. There is a large body of evidence showing that NT acts preand post-synaptically to modulate central DA neurotransmission. The pre-synaptic action of NT results in an increase in DA neurotransmission (DA agonist-like effect) and the postsynaptic action of NT results in a functional decrease (DA antagonist-like effect) in DA neurotransmission [6]. Because selective DA depletion and blockade of DA receptors increase NT-induced hypothermia [30], one possibility is that the presynaptic action of NT on DA contributes, at least in part, to reduce or attenuate its hypothermic effect. This would suggest that the blunted hypothermic response is due to a stronger presynaptic action of NT on DA neurotransmission in F344 than LEW rats. This hypothesis is indirectly supported by the results obtained in the locomotor activity test. In effect, central injection of the NT analog, D-Tyr[11] NT (which mimics the effect of NT), produced strong ambulatory and non-ambulatory movements in in-fostered F344 but not LEW rats, results that replicate our previous findings [3]. Although the mechanism by which NT alters locomotor activity remains to be fully characterized, evidence from other rat strains and from F344 and LEW rats [3] suggest that it involves NT receptors in the nucleus accumbens (N. Acc.) and in the ventral tegmental area (VTA). Microinjection of NT or D-Tyr[11] NT into the VTA stimulates locomotion [19,33]. When injected into the N. Acc. NT has no effect on spontaneous locomotion but inhibits DA-dependent increases in locomotion [12]. Furthermore, the locomotor-stimulant effect of NT in the VTA is attenuated by simultaneous injection of NT in the N. Acc. [20]. In F344 but not in LEW rats, we found that D-Tyr[11] NT acts
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at both the VTA and the N. Acc. to stimulate locomotion [3]. Locomotor activity following ICV injection of NT likely reflects the net effect of the peptide in the VTA (pre-synaptically) and the N. Acc. (post-synaptically). However, neurotensin receptor binding, as measured by quantitative autoradiography, shows no significant differences in neurotensin receptor binding in the VTA or N. Acc. in F344 and LEW rats [4]. The major aim of the present study was to determine whether the differential response of F344 and LEW rats to NT-induced locomotion and hypothermia is under genetic control or is mediated by strain differences in the pre-weaning environment. Rats from each strain were either in-fostered or cross-fostered by PD 1 until weaning at PD 21 and tested later at adulthood. The hypothesis was that cross-fostering LEW pups with F344 dams would render adult LEW rats less sensitive to NT-induced hypothermia and more sensitive to NT-induced locomotion (and/or the opposite for F344 pups cross-fostered with LEW dams). The results show that cross-fostering had no effect on the responses of each strain to NT. LEW rats fostered by F344 dams displayed responses to NT that were not different than that of their infostered mates. In fact, the opposite was observed in the test of hypothermia; cross-fostered LEW rats showed higher sensitivity to NT than their in-fostered mates. Maternal care has been shown to significantly impact development of progeny on neuroendocrine [29], synaptic development [24], physiological [10] and behavioral measures [26]. With respect to F344 and LEW rats, LEW dams show a shorter latency to retrieve pups, a different temporal pattern of pup retrieval [15], and more time spent in contact with the litter [37] compared to F344 dams. These latter strain differences in maternal care have been shown to mediate strain differences in animal models of schizophrenia [37] whereas other measures, such as the differential response of F344 and LEW rats to proinflammatory stimuli, are under tight genetic control and are not readily influenced by strain differences in maternal care [15]. In the present experiment cross-fostering did not significantly alter the differential locomotor and hypothermic responses of F344 and LEW rats to central NT-receptor activation, hence showing that the differences are genetically determined and not readily changed by the pre-weaning environment. In view of the modulation of central DA neurotransmission by NT and of the role of DA in these NT-mediated effects, further analyses of central NT circuitry of these two strains likely constitute a promising strategy to better characterize the neural and genetic bases of DA-dependent disorders. Acknowledgement Supported by a grant from the Canadian Institutes of Health Research (CIHR) to PPR. References [1] Bauco P, Rompr´e P-P. Effects of neurotensin receptor activation on brain stimulation reward in Fischer 344 and Lewis rats. Eur J Pharmacol 2001;432:57–61.
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[20] Kalivas PW, Nemeroff CB, Prange AJ. Neuroanatomical site specific modulation of spontaneous motor activity by neurotensin. Eur J Pharmacol 1982;78:471–4. [21] Khanna JM, Kalant H, Shah G, Sharma H. Comparison of sensitivity and alcohol consumption in four outbred strains of rats. Alcohol 1990;7:429–34. [22] Kosten TA, Miserendino MJD, Chi S, Nestler EJ. Fischer and Lewis rat strains show differential cocaine effects in conditioned place preference and behavioral sensitization but not in locomotor activity or conditioned taste aversion. J Pharmacol Exp Ther 1994;269:137–44. [23] Kosten TA, Miserendino MJ, Haile CN, DeCaprio JL, Jatlow PI, Nestler EJ. Acquisition and maintenance of intravenous cocaine selfadministration in Lewis and Fischer inbred rat strains. Brain Res 1997;778:418–29. [24] Liu D, Diorio J, Day JC, Francis DD, Meaney MJ. Maternal care, hippocampal synaptogenesis and cognitive development in rats. Nat Neurosci 2000;3:799–806. [25] Martin S, Manzanares J, Corchero J, Garcia-Lecumberri C, Crespo JA, Fuentes JA, et al. Differential basal proenkephalin gene expression in dorsal striatum and nucleus accumbens, and vulnerability to morphine self-administration in Fischer 344 and Lewis rats. Brain Res 1999;821:350–5. [26] Matthews K, Wilkinson LS, Robbins TW. Repeated maternal separation of preweanling rats attenuates behavioral responses to primary and conditioned incentives in adulthood. Physiol Behav 1996;59:99–107. [27] McCarty R, Cierpial MA, Murphy CA, Lee JH, Fields-Okotcha C. Maternal involvement in the development of cardiovascular phenotype. Experientia 1992;48:315–22. [28] Meaney MJ, Mitchell JB, Aitken DH, Bhatnagar S, Bodnoff SR, Iny LJ, et al. The effects of neonatal handling on the development of the adrenocortical response to stress: implications for neuropathology and cognitive deficits in later life. Psychoneuroendocrinology 1991;16:85–103. [29] Meaney MJ. Maternal care, gene expression, and the transmission of individual differences in stress reactivity across generations. Annu Rev Neurosci 2001;24:1161–92. [30] Nemeroff CB, Bissette G, Manberg PJ, Osbahr AJ, Breese GR, Prange AJ. Neurotensin-induced hypothermia: evidence for an interaction with dopaminergic systems and the hypothalamic–pituitary–thyroid axis. Brain Res 1980;195:69–84. [31] Nestler EJ. Molecular basis of long-term plasticity underlying addiction. Nat Rev Neurosci 2001;2:119–28. [32] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. San Diego: Academic Press; 1998. [33] Steinberg R, Brun P, Souilhac J, Bougault I, Leyris R, Le Fur G, et al. Neurochemical and behavioural effects of neurotensin vs [DTyr11 ]neurotensin on mesolimbic dopaminergic function. Neuropeptides 1995;28:43–50. [34] Sutanto W, Rosenfeld P, De Kloet ER, Levine S. Long-term effects of neonatal maternal deprivation and ACTH on hippocampal mineralocorticoid and glucocorticoid receptors. Brain Res Dev Brain Res 1996;92:156–63. [35] Wise RA, Rompr´e P-P. Brain dopamine and reward. Annu Rev Psychol 1989;40:191–225. [36] Wise RA. Dopamine, learning and motivation. Nat Rev Neurosci 2004;5:483–94. [37] Wood GK, Marcotte ER, Quirion R, Srivastava LK. Strain differences in the behavioural outcome of neonatal ventral hippocampal lesions are determined by the postnatal environment and not genetic factors. Eur J Neurosci 2001;14:1030–4.
Research report
Cross-fostering does not alter the differential sensitivity of Fischer and Lewis rats to central neurotensin-induced locomotion and hypothermia Pat Bauco ∗ , Pierre-Paul Rompr´e Centre de Recherche Fernand-Seguin, Hˆopital Louis-H. Lafontaine et, D´epartment de Psychiatrie, Universit´e de Montr´eal, Montr´eal, Que., Canada H1N 3V2 Received 8 December 2005; received in revised form 18 March 2006; accepted 22 March 2006 Available online 5 May 2006
Abstract A cross-fostering paradigm was used to determine whether the differential locomotor and hypothermic responses to neurotensin (NT) in Fischer (F344) and Lewis (LEW) rats are mediated by the post-natal environment. From post-natal day (PD) 1 to PD 21, male pups from each strain were assigned to a same-strain dam (in-fostered) or were cross-fostered, and at adulthood were implanted with a guide cannula over the lateral ventricle. They were then tested for locomotion and hypothermia following injection of vehicle, 0.18, 1.8 or 18 nmol of NT or D-Tyr[11] NT. In-fostered LEW, but not F344, displayed a strong dose-orderly hypothermic response to NT and to D-Tyr[11] NT while in-fostered F344, but not LEW, rats displayed strong locomotor responses to D-Tyr[11] NT. Cross-fostering had no effect on D-Tyr[11] NT-induced locomotor responses in either strain; it had no effect also on NT- and D-Tyr[11] NT-induced hypothermia in F344 rats while it slightly increased the sensitivity to NT in LEW rats. The results show that these NT-mediated actions are not influenced by cross-fostering or the pre-weaning environment. © 2006 Elsevier B.V. All rights reserved. Keywords: Cross-foster; Exploratory locomotion; Fischer; Hypothermia; Lewis; Neurotensin; Peptide transmitters; Strain differences
1. Introduction Fischer (F344) and Lewis (LEW) are two inbred rat strains that differ in their appetitive and consummatory responses to several drugs of abuse. Lewis rats more readily self-administer opioids [25] and cocaine [23] compared to F344 rats. Compared to F344 rats, LEW rats more readily establish conditioned preferences towards environments associated with opioids, cocaine [16,22] and nicotine [18]; they also sensitize to the locomotorstimulant actions of cocaine [7,22] and amphetamine [7] to a greater degree than F344 rats. A large body of evidence implicates mid-brain dopamine (DA) in the appetitive effects of drugs of abuse [35,36]. Fischer and LEW rats have been shown to differ significantly in dopamine synthesis, transport [5,16], and release [34] as well as in second messenger and immediate early gene expression linked to DA receptor activation [17]. Such
∗ Corresponding author at: Centre de Recherche Fernand-Seguin, Hˆ opital Louis-H. Lafontaine, 7331, rue Hochelga, Montr´eal, Que., Canada H1N 3V2. Tel.: +1 514 251 4015; fax: +1 514 251 2617. E-mail address: [email protected] (P. Bauco).
0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.03.027
differences have been proposed as relevant features of the neurobiological substrate of drug addiction [31]. Neurotensin (NT) is an endogenous tridecapapetide that modulates DA function at pre- and post-synaptic levels in several reward-relevant limbic regions [6], and it has been shown that the endogenous NT circuitry differs in F344 and LEW rats; central NT receptor activation produces differential reward-potentiating [1], locomotor [3] and hypothermic [2] responses. It has been well documented that environmental factors during postnatal development can alter gene expression and have a significant impact on neuroendocrine [28], physiological [10] and behavioral measures [26]. Maternal care, for example, has been shown to regulate and endocrine responses to stress [29] and synaptic development [24]. A question that arises then is whether early environmental factors contribute to differences observed in F344 and LEW rats following central NT receptoractivation. Cross-fostering has been used with other rat strains to show that factors such as maternal and pup behavior, and maternal/pup interactions, play a key role in phenotypic differences between strains [27]. F344 and LEW dams have been also shown to differ in their maternal behavior. For example, LEW dams show a shorter latency to retrieve pups, a different temporal
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pattern of pup retrieval [15], and more time spent in contact with the litter [37] compared to F344 dams. The aim of the present experiment, therefore, was to use cross-fostering of F344 and LEW rats to determine whether the differential locomotor and hypothermic responses to central NT receptor activation in these two strains of rats is modulated by the pre-weaning environment. 2. Materials and methods Experiments were carried out in accordance with the Canadian Council on Animal Care Guide to the Care and Use of Experimental Animals. An internal animal care committee approved all experiments and all efforts were made to minimize animal discomfort.
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the box. Movements detected within the virtual box were considered as nonambulatory and were quantified as time (in min) during which photocell beam interruptions were detected. Vertical activity was quantified as the total number of photocell beam interruptions produced by rearing (see [11] for validation data on these measures of activity).
2.4. Histology At the end of each experiment, animals were deeply anesthetized with urethane (1.4 g/kg, i.p.) and transcardially perfused with 0.9% saline followed by a 10% formalin solution. Brains were removed, stored in 10% formalin and subsequently sliced in serial 40-m sections that were stained with a formal-thionin solution. Location of the injection site was determined under light microscopic examination. Only data from animals with a confirmed ventricular injection site were included in the analyses.
2.1. Subjects and cross-fostering procedure 2.5. Drugs Pregnant Fischer (F344) and Lewis (LEW) rats were obtained at approximately 14 days of gestation (Charles River, St-Constant, Qu´ebec, Canada) and housed individually with free access to food and water in a temperature- and humidity-controlled room with a 12-h light/dark cycle; lights on at 06:30 h. On the day of birth, all female pups were removed from the litters and the remaining F344 and LEW male pups were returned to their respective same strain dam (in-fostered) or to an opposite strain dam (cross-fostered); this resulted in four test groups: (i) F344 pups reared by F344 dams; (ii) F344 pups reared by LEW dams; (iii) LEW pups reared by LEW dams and (iv) LEW pups raised by F344 dams. All testing was performed during the light phase of the day/night cycle (between 06:30 and 16:30), in a room separate from the housing colony.
2.2. Surgery Adult rats (275 and 325 g) were injected with atropine methylnitrate (0.4 mg/kg, i.p.), anesthetized with sodium pentobarbital (65 mg/kg, i.p.) and mounted onto a stereotaxic apparatus. A guide cannula (Plastic One Inc., VA, USA, model C315G) was implanted above the left lateral ventricle (flat-skull coordinates: 0.8 mm posterior to bregma, 1.2 mm lateral and 2.8 mm below the skull surface) according to the atlas of Paxinos and Watson [32] and anchored to stainless-steel screws threaded into the skull with dental cement.
Neurotensin-(1-13); (NT) and (D-Tyr11 )-Neurotensin (D-Tyr[11] NT; Bachem Bioscience Inc., Torrance, CA, USA) were dissolved in sterile 0.9% saline at a concentration of 1.8 nmol/l and stored frozen at −20 ◦ C in aliquots pre-coated with silicone. The peptide solution was thawed just prior to testing and diluted to the required concentration with sterile 0.9% saline when necessary.
2.6. Data analysis Rectal temperature was measured prior to (baseline) and every 30 min for 2 h after microinjection of peptide or vehicle. Data were expressed as the mean of the four post-injection measurements for each respective treatment and were analyzed using a 2 × 2 × 5 (dam strain × pup strain × treatment) MANCOVA (with baselines as the co-variate). Comparisons among means were made with Duncan’s multiple range post-hoc test with level of significance set at 0.05. Parameters of locomotor activity (ambulatory activity, non-ambulatory activity and vertical activity) were computed for the total (2 h) test period, and group means were analyzed with a two-way analysis of variance (ANOVA) with strain (four levels) and treatment (two levels) as the two independent factors.
3. Results 2.3. Testing All animals were tested once weekly in the locomotor activity or hypothermia phases of the experiment; the order of testing was counterbalanced such that for each group, half the animals completed the locomotion tests (D-Tyr[11] NT 18 nmol and vehicle) first and the other half completed the hypothermia tests (NT 0.18, 1.8, 18 nmol, D-Tyr[11] NT 18 nmol and vehicle) first. Concentrations of peptide (and vehicle) were chosen based on our previous studies [2,3] and were tested in a randomized sequence for each animal. 2.3.1. Neurotensin-induced hypothermia Body temperature was measured by inserting a lubricated flexible thermoprobe 6–7 cm into the rectum and kept it in place for approximately 10 s while the rat was hand-restrained. Rectal temperature was assessed prior to and at 30 min intervals for a total of 2 h after injection. Animals were returned to holding cages between trials. Testing was performed in a temperature-controlled room (ambient temperature 22 ◦ C). 2.3.2. Neurotensin-induced locomotion Locomotor activity was measured using an Opto-Varimex Auto Track System (Columbus Instruments, Columbus, OH, USA). This system consists of Plexiglass cages (42 cm × 42 cm × 35 cm) with wire meshed floor equipped with two arrays of 15 infrared photocells located 1.5 and 14.5 cm above the floor to detect horizontal and vertical movements, respectively. Computer software quantified ambulatory activity by calculating the distance traveled beyond a “virtual box” of 9.6 cm × 9.6 cm (3 × 3 photocells) drawn around the animal; the computer assessed (10 times per second) the location of the animal within
3.1. Test for hypothermia Neurotensin induced a dose-orderly hypothermic response in in-fostered LEW rats and the analog produced a significantly larger response at an equimolar concentration than the native peptide (Fig. 1, top panel). Neurotensin also induced a dose-orderly hypothermia in in-fostered F344 rats (Fig. 1, bottom panel) but the magnitude of the response was much weaker than that observed in LEW rats and the NT analog produced a hypothermic response of the same magnitude as NT. LEW rats fostered by F344 dams did not show a blunted hypothermic response like the in-fostered F344 rats. F344 rats fostered by LEW dams were not different than their in-fostered mates; they were isosensitive to the hypothermic effects at each dose of NT and its analog. Mean pre-injection rectal temperatures differed significantly between strains; in-fostered and cross-fostered LEW rats had significantly lower pre-injection rectal temperatures compared to in-fostered and cross-fostered and F344 rats (Fig. 1, top and bottom panels). There were, however, no significant differences in pre-injection temperatures as a function of cross-fostering (Fig. 1, top and bottom panels).
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Fig. 1. Mean ± S.E.M. pre-injection rectal temperatures in in-fostered and cross-fostered Lewis (top panel) and in-fostered and cross-fostered Fischer (bottom panel) rats. In-fostered and cross-fostered Lewis rats had respectively significantly lower pre-injection rectal temperatures compared to in-fostered and to cross-fostered Fischer rats as determined by a Student’s t-test for independent samples († p < 0.05). There were no significant within-strain differences in preinjection rectal temperature. Figure also presents the mean ± S.E.M. changes in rectal temperature (◦ C) over 2 h following injection with vehicle or one of three concentrations of neurotensin (NT) and its analog D-Tyr[11] NT (D-NT) in in-fostered and cross-fostered Lewis (top panel, n = 13 and 9, respectively) and Fischer (bottom panel, n = 9 and 8, respectively) rats. A MANCOVA yielded a significant main effect of strain (dam; F(1,33) = 50.5, p < 0.0001), a significant main effect of treatment (F(4,136) = 124.91, p < 0.0001) as well as a signficant strain (dam) by treatment interaction (F(4,136) = 54.86, p < 0.0001). Post-hoc tests confirmed significant differences between NT and vehicle (* p < 0.05), 18 nmol of NT and of D-Tyr[11] NT (# p < 0.05), and Fischer and Lewis rats (the corresponding treatment and fostering condition, + p < 0.05).
3.2. Test for locomotion The NT analog which mimicks the effect of NT on core body temperature induced in in-fostered F344 rats strong ambulatory and non-ambulatory activity and suppressed vertical activity
Fig. 2. Total (2 h) ambulatory activity expressed as distance in meters travelled within the activity box (top panels), non-ambulatory activity expressed as time in minutes making movements within the “virtual box” (see Section 2.3.2 for details, middle panels), and vertical activity expressed as total number of photocell counts (bottom panels) measured in in-fostered and cross-fostered Fischer (n = 9 and 8, respectively) and Lewis (n = 13 and 8, respectively) rats. Data are presented as group means ± S.E.M. The ANOVA yielded a significant effect of strain by treatment interaction: ambulatory activity F(3,68) = 24.8, p < 0.001, non-ambulatory activity F(3,68) = 22.8, p < 0.001, vertical activity F(3,68) = 4.14, p < 0.01. Post-hoc tests confirmed the significant differences between NT and vehicle (* p < 0.05; ** p < 0.1) and between Fischer and Lewis rats (for the corresponding treatment and fostering condition, + p < 0.05; ++ p < 0.01).
compared to the respective vehicle control (Fig. 2). The NT analog had no effect on ambulatory and non-ambulatory activity in in-fostered LEW rats; it produced a significant suppression of vertical activity compared to vehicle, an effect that tended to be stronger than in F344. Cross-fostered F344 and LEW rats responded almost identically to the NT analog; F344, but not LEW, displayed an increase in ambulatory and non-ambulatory activity while both strains showed a suppression of vertical movements. The locomotor response to vehicle did not differ between in-fostered and cross-fostered rats of both strains and the time course of the locomotor responses (not shown) vehicle
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and peptide were very similar to that previously reported (see Fig. 2 in [3]). 4. Discussion The results of the hypothermia experiment show that NT produced a dose-orderly decrease in core body temperature in in-fostered rats of both strains but with a much lower magnitude in F344 rats. As reported previously [2], it is the response of F344 rats that is atypical because the hypothermic response of LEW rats to NT is similar to what has previously been found for other strains [8]. In order to determine whether the lower potency of NT in F344 rats was due to a more effective and rapid enzymatic degradation of the peptide, we tested the effectiveness of an equimolar concentration of D-Tyr[11] NT. The NT analog which is more resistant to degradation [9] was slightly more potent in LEW rats but as potent as NT in F344 rats; this excludes then the possibility that in F344 rats the blunted hypothermic response to NT is due pharmacokinetic properties. This conclusion is reinforced by the fact that both strains are iso-sensitive to NT-induced analgesia in the hot-plate test [2]. Previous studies have shown that NT plays a role in ethanol-induced hypothermia, and it has been suggested that these substances act in a synergistic manner on specific neuronal processes to induce a hypothermic response [13,14]. It can be inferred that the atypical hypothermic response of F344 rats is due to an atypical functional characteristic of the NT relevant circuitry, rather than an anomaly of the hypothermic response per se, since F344 rats show a normal hypothermic response to ethanol [21]. There is a large body of evidence showing that NT acts preand post-synaptically to modulate central DA neurotransmission. The pre-synaptic action of NT results in an increase in DA neurotransmission (DA agonist-like effect) and the postsynaptic action of NT results in a functional decrease (DA antagonist-like effect) in DA neurotransmission [6]. Because selective DA depletion and blockade of DA receptors increase NT-induced hypothermia [30], one possibility is that the presynaptic action of NT on DA contributes, at least in part, to reduce or attenuate its hypothermic effect. This would suggest that the blunted hypothermic response is due to a stronger presynaptic action of NT on DA neurotransmission in F344 than LEW rats. This hypothesis is indirectly supported by the results obtained in the locomotor activity test. In effect, central injection of the NT analog, D-Tyr[11] NT (which mimics the effect of NT), produced strong ambulatory and non-ambulatory movements in in-fostered F344 but not LEW rats, results that replicate our previous findings [3]. Although the mechanism by which NT alters locomotor activity remains to be fully characterized, evidence from other rat strains and from F344 and LEW rats [3] suggest that it involves NT receptors in the nucleus accumbens (N. Acc.) and in the ventral tegmental area (VTA). Microinjection of NT or D-Tyr[11] NT into the VTA stimulates locomotion [19,33]. When injected into the N. Acc. NT has no effect on spontaneous locomotion but inhibits DA-dependent increases in locomotion [12]. Furthermore, the locomotor-stimulant effect of NT in the VTA is attenuated by simultaneous injection of NT in the N. Acc. [20]. In F344 but not in LEW rats, we found that D-Tyr[11] NT acts
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at both the VTA and the N. Acc. to stimulate locomotion [3]. Locomotor activity following ICV injection of NT likely reflects the net effect of the peptide in the VTA (pre-synaptically) and the N. Acc. (post-synaptically). However, neurotensin receptor binding, as measured by quantitative autoradiography, shows no significant differences in neurotensin receptor binding in the VTA or N. Acc. in F344 and LEW rats [4]. The major aim of the present study was to determine whether the differential response of F344 and LEW rats to NT-induced locomotion and hypothermia is under genetic control or is mediated by strain differences in the pre-weaning environment. Rats from each strain were either in-fostered or cross-fostered by PD 1 until weaning at PD 21 and tested later at adulthood. The hypothesis was that cross-fostering LEW pups with F344 dams would render adult LEW rats less sensitive to NT-induced hypothermia and more sensitive to NT-induced locomotion (and/or the opposite for F344 pups cross-fostered with LEW dams). The results show that cross-fostering had no effect on the responses of each strain to NT. LEW rats fostered by F344 dams displayed responses to NT that were not different than that of their infostered mates. In fact, the opposite was observed in the test of hypothermia; cross-fostered LEW rats showed higher sensitivity to NT than their in-fostered mates. Maternal care has been shown to significantly impact development of progeny on neuroendocrine [29], synaptic development [24], physiological [10] and behavioral measures [26]. With respect to F344 and LEW rats, LEW dams show a shorter latency to retrieve pups, a different temporal pattern of pup retrieval [15], and more time spent in contact with the litter [37] compared to F344 dams. These latter strain differences in maternal care have been shown to mediate strain differences in animal models of schizophrenia [37] whereas other measures, such as the differential response of F344 and LEW rats to proinflammatory stimuli, are under tight genetic control and are not readily influenced by strain differences in maternal care [15]. In the present experiment cross-fostering did not significantly alter the differential locomotor and hypothermic responses of F344 and LEW rats to central NT-receptor activation, hence showing that the differences are genetically determined and not readily changed by the pre-weaning environment. In view of the modulation of central DA neurotransmission by NT and of the role of DA in these NT-mediated effects, further analyses of central NT circuitry of these two strains likely constitute a promising strategy to better characterize the neural and genetic bases of DA-dependent disorders. Acknowledgement Supported by a grant from the Canadian Institutes of Health Research (CIHR) to PPR. References [1] Bauco P, Rompr´e P-P. Effects of neurotensin receptor activation on brain stimulation reward in Fischer 344 and Lewis rats. Eur J Pharmacol 2001;432:57–61.
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