Psychopharmacology (2002) 161:434–441 DOI 10.1007/s00213-002-1092-4
O R I G I N A L I N V E S T I G AT I O N
Gwenaëlle Le Pen · Laurent Gaudet · Patrick Mortas Roland Mory · Jean-Luc Moreau
Deficits in reward sensitivity in a neurodevelopmental rat model of schizophrenia Received: 25 November 2001 / Accepted: 2 March 2002 / Published online: 20 April 2002 © Springer-Verlag 2002
Abstract Rationale: Neonatal ventral hippocampal lesions in rats have been shown to result in behavioral abnormalities at adulthood thought to simulate some aspects of positive and cognitive deficits classically observed in schizophrenic patients. Objectives: We investigated whether such lesions can also induce deficits in reward sensitivity that are related to the negative symptoms of psychotic disorders. Methods: To investigate the effects of neonatal and adult lesions of the ventral hippocampus on reward-related behaviors we used the conditioned place preference (CPP) test and the saccharin consumption model. Results: In contrast to adult-lesioned animals, neonatally lesioned rats exhibited a deficit in amphetamine-induced CPP and a significant reduction in saccharin preference. These deficits are unlikely due to lesion-induced motor impairments as both neonatal- and adult-lesioned rats exhibited a similar hyperlocomotor response to amphetamine. Conclusions: Taken together, these results show that neonatal ventral hippocampal lesions induce a reduction in reward-seeking behaviors in adulthood that mimic some aspects of the negative symptoms (anhedonia) in psychotic patients. Keywords Schizophrenia · Conditioned place preference · Saccharin intake · Neonatal lesion · Ventral hippocampus · Reward
Introduction Schizophrenia is one of the most disabling and emotionally devastating illnesses of the brain known to man. Schizophrenia is characterized by a constellation of distinctive symptoms. Relevant animal models of schizophrenia must be developed to better understand schizophrenia and to develop effective pharmacotherapies G. Le Pen · L. Gaudet · P. Mortas · R. Mory · J.-L. Moreau (✉) Pharma Division, Preclinical CNS Research, F-Hoffmann-La Roche Ltd, 4070 Basel, Switzerland e-mail:
[email protected] Tel.: +41-61-6886951, Fax: +41-61-6881895
against this illness. The most common models rely on the use of psychotogenic substances to produce schizophrenic-like symptoms in animals. Recently new nonpharmacological models have emerged based on the neurodevelopmental hypothesis of schizophrenia, which suggests that the disorder has its origin in an abnormality of brain development (Duncan et al. 1999; Weinberger 1986). Among these new models long-term consequences induced by neonatal ventral hippocampal lesions in the rat have been proposed by Lipska and coworkers (Lipska and Weinberger 1993, 2000; Lipska et al. 1993) as offering a valid simulation of psychotic disorders. Indeed, neonatal ventral hippocampal lesions in rats have been shown to induce behavioral abnormalities thought to simulate some aspects of positive symptoms and cognitive deficits classically observed in schizophrenic patients (Al-Amin et al. 2000; Chambers et al. 1996; Le Pen et al. 2000; Lipska et al. 1993, 1995a 1995b). In addition, neonatal lesioned rats exhibit a postpubertal emergence of abnormal behaviors such as hyperresponsiveness to stress, novelty, dopamine agonists, and glutamate antagonists (Al-Amin et al. 2000; Black et al. 1998; Lipska et al. 1993, 1995a). These postpubertal anomalies are reminiscent of the classically described postpubertal onset of psychotic symptoms in schizophrenic patients. Indeed, the onset of schizophrenia is frequently triggered by a stressful event (Duncan et al. 1999), and psychological stress (Duncan et al. 1999), dopamine agonists (Angrist and Van Kammen 1984; Lieberman et al. 1987), and glutamate antagonists (Meador-Wooddruff et al. 2000) are well documented to precipitate or exacerbate psychotic symptoms in humans. Regarding cognitive processes, neonatal ventral hippocampal lesions induce a postpubertal emergence of deficits in sensorimotor gating (Le Pen et al. 2000; Lipska et al. 1995b) and in spatial learning and working memory, as investigated using the radial-arm maze model (Chambers et al. 1996). More recently we have extended these results by showing lesion-induced deficits in spatial and associative learning using avoidance tasks and the Morris water maze procedure (Le Pen et al. 2000).
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Although the ability of this model to mimic some aspects of the positive and cognitive symptoms of schizophrenia has been extensively investigated, its ability to simulate negative symptoms has not. In schizophrenic patients the typical negative symptoms consist of alogia, flat affect, avolition, a loss of capacity to experience pleasure (anhedonia), social isolation, and emotional withdrawal. The only two animal studies addressing negative symptoms in this model of schizophrenia have shown that rats with neonatal lesions of the ventral hippocampus exhibit deficits in social behavior (Becker et al. 1999; Sams-Dodd et al. 1997). Given (a) the considerable body of evidence for alterations in mesolimbic dopamine functions in rats with neonatal lesions of the ventral hippocampus (Lipska and Weinberger 1993, 1994; Lipska et al. 1993, 1995b) and (b) the particular implication of this system in reward processes (Kalivas and Stewart 1991; Robinson and Berridge 1993), we decided to evaluate the capacity of lesioned rats to experience pleasure. Some authors have suggested that the mesolimbic dopamine system mediates the rewarding effects of natural rewards (Mark et al. 1991; Royall and Klemm 1981; Spanagel and Weiss 1999) as well as those of many psychomimetic and/or self-administered drugs (Spanagel and Weiss 1999; Wise and Bozarth 1987). Thus in this study we tested adult male Sprague-Dawley rats with postnatal day 7 ibotenic acid lesions of the ventral hippocampus both in an unbiased amphetamineinduced conditioned place preference (CPP) model and in a saccharin consumption test. For comparison, adult 8-week-old rats were lesioned in the ventral hippocampus and tested in the same models.
Methods All animal experiments were conducted according to the principles of laboratory animal care and were approved by the local animal protection authorities. Animals Sprague-Dawley rat pups were obtained at 3–4 days of age as whole litters together with their mother, and adult male SpragueDawley rats were obtained at 50 days (BRL, Füllinsdorf, Switzerland). They were kept on a 12- h light/12- h dark cycle (on: 6 a.m., off: 6 p.m.) and fed ad libitum. Surgical procedure Neonatal rats On the 7th day of age and at a body weight of 15–20 g male pups in each litter were randomized to sham or lesion status, anesthetized by isoflurane inhalation (4% for induction and 1.5–3% for maintenance) through a mask mounted on a stereotaxic Kopf instrument with an adapter for small animals (Harvard Biosciences) and additionally taped on a heating pad placed on the platform of the stereotaxic frame. The skin overlying the skull was incised and 0.3 µl of either ibotenic acid (Sigma, St. Louis, Mo., USA; 10 µg/µl) or artificial cerebrospinal fluid was bilaterally infused over a 2-min period by a microinfusion pump (PHD Programma-
ble, Harvard Biosciences) using an injection cannula (0.3 mm diameter) aimed at the ventral hippocampal formation (AP –3.0 mm, ML ±3.5 mm, and DV –5.0 mm relative to bregma). After the completion of the infusion the cannula was left in place for an additional 3 min before being slowly removed. Then the skin overlying the skull was sutured and the animals allowed to recover on a heating pad before being returned to their mother. Eighteen days after surgery (i.e., postnatal day 25) rats were weaned and housed four per cage until further testing. Behavioral testing occurred during light cycle between 9 a.m. and 4 p.m. Adult rats We anesthetized 56-day-old rats intraperitoneally with ketamine 80 mg/kg (Ketalar, Parke-Davis) and xylazine 10 mg/kg (Rompun, Bayer). The animals were mounted on a stereotaxic Kopf instrument, and the skin overlying the skull was incised. Then 2×0.2 µl of either ibotenic acid (Sigma; 15 µg/µl) or artificial cerebrospinal fluid was bilaterally infused over a 2-min period by a microinfusion pump (PHD Programmable, Harvard Biosciences) using an injection cannula (0.3 mm diameter) aimed at the ventral hippocampal formation (AP –4.8 mm, ML ±5.2 mm, and DV –7.0 and –5.0 mm relative to bregma). This procedure was adapted from Lipska and colleagues (1992) in order to induce lesions similar in volume and location to those obtained after neonatal injections of ibotenic acid. Following infusion the skin overlying the skull was sutured and the animals allowed to recover on a heating pad before being returned to their home cage per group of four rats. In both neonatal and adult lesion groups the mortality rate following surgery was less than 5%.
Conditioned place preference The apparatus consisted of rectangular Plexiglas chambers divided into two 30×30×50 cm compartments. One compartment had black walls with a black Plexiglas floor and the other one had black and white vertically stripped walls and a black grid floor made of a wire net with square openings of 0.5×0.5 cm. A removable partition separated the two compartments. On the CPP test day this partition was replaced by a similar partition containing an opening in the center (8×11 cm) which allowed the animals free access to both compartments. All sessions were conducted in a sound-attenuated room under conditions of dim illumination (70 lux). Previous experiments have shown that under our conditions (a) Sprague Dawley rats do not exhibit any significant spontaneous preference for either of the two compartments, and (b) a low dose of amphetamine (0.1 mg/kg) does not induce a significant place preference either in neonatal lesioned or in sham-operated animals. The experiment performed in 3-month-old rats consisted of two distinct phases: conditioning and postconditioning. For the conditioning phase on day 1 immediately after intraperitonal D-amphetamine injection (1 mg/kg) the animals were confined to the appropriate compartment for 30 min. Place preference conditioning was conducted by using a counterbalanced procedure so that in each treatment group one-half of the rats received the drug in the black compartment and one-half in the black and white stripped compartment. On the next day they were injected with vehicle and confined to the other compartment. Each rat received two D-amphetamine pairings (on days 1 and 3) and two vehicle pairings (on days 2 and 4). During the conditioning sessions locomotor activity of the rats was quantified by a videotracking system (Noldus, The Netherlands). The postconditioning test was conducted 1 day after the last conditioning session. Uninjected rats were allowed free access to both compartments of the apparatus for 15 min, and the amount of time spent in each compartment was monitored. The image analysis system (Noldus, The Netherlands) consisted of one video camera positioned above the apparatus, a video interface, and a microcomputer. It converted the video input signals into binary images so that each animal corresponded to a white
436 Fig. 1A–D Lesion boundaries in the ventral hippocampus of rats infused bilaterally with ibotenic acid. A A schematic drawing of the ventral hippocampus with boundaries of the largest (stripes) and smallest (black) lesions. B–D Representative photomicrographs of cresyl violet-stained coronal section through the brains of rats that had received sham (B), neonatal (C), or adult (D) lesions of the ventral hippocampus
spot against a black background. Virtual windows on the computer screen corresponded to the different areas of the experimental apparatus. During experimentation the movements of these spots were recorded and translated into the time (s) spent in each compartment. As the time spent in the amphetamine-paired side and that spent in the vehicle-paired side are dependent on each other (one is the mirror image of the other), time spent in amphetaminepaired side was considered as the index of CPP. Two-way analysis of variance (ANOVA) (lesion status × age at lesion) was used to compare the effects of ventral hippocampal (VH) lesions on (a) the time spent on amphetamine-paired side, and (b) the horizontal locomotor activity. Then to determine whether amphetamine produced a significant place conditioning in each population of rats, a one-group t test was used in which time spent in the drug-paired side was compared to the value of 450 s (which signifies no preference). Two-way ANOVAs were followed when appropriate by post hoc analyses (Fisher's LSD). Saccharin-water choice All the saccharin-water choice experiments were conducted in the animals' home cage (adaptation from Touzani et al. 1997). In two
successive experiments at 3.5 months of age neonatal and adult operated animals were placed on a schedule of restricted water access. Over a 5-day period rats were allowed only two periods of drinking, one beginning at 9:30 a.m. and lasting for 1 h and one beginning at 3:30 p.m. and lasting for 2 h. After habituation to the deprivation schedule rats were presented daily with two bottles equipped with stainless steel drinking spouts, one containing tap water and the other containing one of the following four concentrations of saccharin: 0.1, 1.5, 7.5, and 34 mM. An ascending series of saccharin concentrations was used to verify whether the preference and/or aversion thresholds were modified by the lesion. Fresh solutions of the sweetener (Benzoic sulfimide natrium salt, Fluka) were prepared daily by dissolution in tap water. The test session took place each day at 9:30 h and lasted 1 h. The bottles were weighed at the beginning and at the end of the test to the nearest 0.01 g. The different concentrations of saccharin were tested once per day in ascending order. The bottle containing saccharin solution was placed on a different side of the home-cage every day. During the 4 days of testing the deprivation schedule was maintained as indicated above. Thus the choice test took place after 16 h of water deprivation. Two-way ANOVAs (lesion status × saccharin concentrations) with repeated measures on factor saccharin concentration were used to test for significant differences between lesioned rats and sham-operated controls.
437 Rating of lesion size At the completion of behavioral tests rats were killed by decapitation. Brains were rapidly removed, and after fixation in formalin solution (10% in NaCl) 40-µm sections were sliced with a freezing cryostat. The sections through the lesioned area were mounted and stained with cresyl violet. The extent of the lesion on each side of the brain was rated as follows (according to Sams-Dodd et al. 1997): 0, no discernible cell loss in the hippocampal formation; 1, small; 2, medium; 3, large area of cell loss within the ventral hippocampal formation. Scores for both sides were added to yield a total score ranging from 0 to 6.
Results Histology Subjects with only unilateral or extrahippocampal damage were discarded [place preference experiment: four neonatal VH (NVH)- and two adult VH-lesioned rats; saccharine intake experiment: ten NVH- and two adult VH-lesioned animals). Neonatal- and adult-lesioned rats evaluated in CPP and saccharin consumption models exhibited mean lesion score of 3.6±0.4, 3.8±0.3, 3.9±0.1, and 4±0.1, respectively, indicating (a) that the cell loss was restricted to the ventral part of the hippocampus and (b) a similar extent and location of the lesion in adult and neonatal lesioned rats (Fig. 1). Conditioned place preference CPP induced at adulthood by D-amphetamine in neonatal and adult ventral hippocampal lesioned rats is depicted on Fig. 2A. The two-way ANOVA revealed no overall effect of lesion or age (at lesion) on time spent on amphetamine-paired side (F1,66=1.1, NS; F1,66=2, NS, respectively). However, a significant age × lesion interaction was observed (F1,66=4.4, P
