Emotional, cognitive and neurochemical alterations in a premotor stage model of Parkinson's disease

Neuroscience 156 (2008) 830 – 840

EMOTIONAL, COGNITIVE AND NEUROCHEMICAL ALTERATIONS IN A PREMOTOR STAGE MODEL OF PARKINSON’S DISEASE M. T. TADAIESKY,a P. A. DOMBROWSKI,b C. P. FIGUEIREDO,c E. CARGNIN-FERREIRA,d C. DA CUNHAb AND R. N. TAKAHASHIa*

have caused the emotional and cognitive deficits observed in this rat model of early phase PD. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

a

Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, Campus Universitário, Trindade, Bloco D/CCB, C. Postal 476, Florianópolis, SC, Brazil

Key words: 6-OHDA, striatum, prefrontal cortex, depression, anxiety, memory.

b

Laboratório de Fisiologia e Farmacologia do Sistema Nervoso Central, Setor de Ciências Biológicas, Universidade Federal do Paraná, UFPR, Centro Politécnico, Curitiba, PR, Brazil

The cardinal biochemical abnormality in Parkinson’s disease (PD) is the profound deficit in brain dopamine (DA) level, primarily, but not exclusively, attributed to the loss of neurons of the nigrostriatal dopaminergic pathway (Dauer and Przedborski, 2003). PD is most often considered a disorder of movement. However, there is growing evidence that a number of symptoms can precede the classical motor features of PD. The period when these symptoms arise can be referred to as the premotor phase of the disease and this notion has been strengthened by recent epidemiological, pathological, and clinical studies which have provided data in favor of the existence of this premotor phase in PD (Tolosa et al., 2007). During this phase, the neuropathological process progresses without concomitant motor manifestations (Spiegel et al., 2006). When cardinal motor signs (bradykinesia, rest tremor and rigidity) required for PD diagnosis appear, as many as 58 – 64% of dopaminergic neurons in the substantia nigra pars compacta (SNc) have been lost and striatal DA content has been reduced by 60 – 80% (Tissingh et al., 1998). Imaging studies of the dopaminergic system and postmortem cell counts of pigmented neurons in the SN have shown that the onset of dopaminergic neuronal loss precede by approximately 4 – 6 years the clinical diagnosis of PD (Marek et al., 2001). Whereas motor impairments result in disability, premotor complications including depression, anxiety and cognitive decline may be of equal or greater significance in some patients’ quality of life (Ziemssen and Reichmann, 2007). Depressive disorders commonly occur in patients with PD (Cummings and Masterman, 1999), affecting approximately 40% of patients at early stages of the disease (Tolosa et al., 2007). Several studies suggest that the pathophysiology underlying mood disorders in PD may be different from the mechanisms that account for the behavioral symptoms observed in the general population (Lieberman, 2006). Similarly, up to 40% of patients with PD suffer from clinically significant anxiety. Although the pathophysiology of psychiatric symptoms in PD is not fully understood, striatal, frontal and limbic dopaminergic, cholinergic, serotonergic, noradrenergic and GABAergic pathways are thought to be involved in their genesis (Schrag, 2004).

c

Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, Florianópolis, Brazil

d

Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, Florianópolis, Brazil

Abstract—In addition to classic motor symptoms, Parkinson’s disease (PD) is characterized by cognitive and emotional deficits, which have been demonstrated to precede motor impairments. The present study addresses the question of whether a partial degeneration of dopaminergic neurons using 6-hydroxydopamine (6-OHDA) in rats is able to induce premotor behavioral signs. The time-course of nigrostriatal damage was evaluated by tyrosine hydroxylase immunohistochemistry and the levels of dopamine, noradrenaline, and 5-HT in various brain regions were analyzed by high performance liquid chromatography (HPLC). Behavioral tests that assessed a variety of psychological functions, including locomotor activity, emotional reactivity and depression, anxiety and memory were conducted on 6-OHDA lesioned rats. Bilateral infusion of 6-OHDA in the striatum of rats caused early (1 week) damage of dopaminergic terminals in striatum and in cell bodies in substantia nigra pars compacta. The nigrostriatal lesion was accompanied by early loss of dopamine in the striatum, which remained stable through a 3-week period of observation. In addition, a late (3 weeks) loss of dopamine in the prefrontal cortex, but not in the hippocampus, was seen. Additional noradrenergic and serotonergic alterations were observed after 6-OHDA administration. The results indicated that 6-OHDA lesioned rats show decreased sucrose consumption and an increased immobility time in the forced swimming test, an anhedonic-depressive-like effect. In addition, an anxiogenic-like activity in the elevated plus maze test and cognitive impairments were observed on the cued version of the Morris water maze and social recognition tests. These findings suggest that partial striatal dopaminergic degeneration and parallel dopaminergic, noradrenergic and serotonergic alterations in striatum and prefrontal cortex may *Corresponding author. Tel: ⫹55-48-37219491; fax: ⫹55-48-33375479. E-mail address: [email protected] (R. N. Takahashi). Abbreviations: ANOVA, analysis of variance; DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; EDTA, ethylenediaminetetraacetic acid; HPLC, high performance liquid chromatography; HVA, homovallinic acid; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NA, noradrenaline; PD, Parkinson’s disease; SNc, substantia nigra pars compacta; TH, tyrosine hydroxylase; 6-OHDA, 6-hydroxydopamine. 0306-4522/08 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.08.035

830

M. T. Tadaiesky et al. / Neuroscience 156 (2008) 830 – 840

Cognitive impairments are also common in PD. Between 15% and 20% of PD patients develop a frank dementia, and less severe cognitive impairment is a wellrecognized feature early in the disease that has been shown to be an important predictor for quality of life (Schrag et al., 2000). Patients with PD are impaired on the acquisition of various kinds of procedural memory that are thought to be produced by striatal alterations (Packard and Knowlton, 2002). In addition, PD patients also present evidence of executive dysfunction and impairments of working memory which resemble that produced by frontallobe damage (Lewis et al., 2003). Patients with executive dysfunction have been reported to exhibit more prominent procedural learning impairment (Jackson et al., 1995), suggesting as well the involvement of the prefrontal cortex– basal ganglia loop in the observed deficit in this type of learning. The idea that a degenerated nigrostriatal system plays a role in cognitive impairments in PD has been further substantiated by studies from our and other groups employing animal models of this disease. Indeed, most work trying to mimic early PD focuses on cognitive impairments. Studies using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin used to mimic PD in primates and rodents have shown both procedural and working memory impairments in rats (Da Cunha et al., 2007; Gevaerd et al., 2001b; Bellissimo et al., 2004; Braga et al., 2005; Prediger et al., 2006a; Reksidler et al., 2007). Moreover, 6-hydroxydopamine (6-OHDA), when injected in SNc, was shown to cause alterations in cued and spatial memory versions of water maze, reflecting memory deficits similar to PD’s early phase (Lindner et al., 1999; Ferro et al., 2005). Few pre-clinical studies, however, have determined whether the full array of premotor parkinsonian symptoms can be detected in rats with incomplete nigrostriatal cell loss and partial striatal DA depletions similar to early stages of the disease. Therefore, we addressed the question of whether DA depletion is able to induce emotional and cognitive signs in a bilateral model of PD by partially destroying dopaminergic neurons with an infusion of 6-OHDA into the rat’s striatum. This procedure, which represents a variant of the original method described by Sauer and Oertel (1994), has been proposed by Lindner (1999) as a reliable tool to induce incomplete progressive nigrostriatal cell loss and partial striatal DA depletions similar to a preclinical stage of the disease. Unlike the direct injection of 6-OHDA into the SNc and/or MFB, which yields a faster lesion of dopaminergic cell bodies, followed by degeneration of striatal terminals, infusion of the toxin into the striatum causes a fast damage of dopaminergic terminals, followed by retrograde loss of tyrosine hydroxylase (TH)–positive cells in the ipsilateral SNc (Blandini et al., 2007). In this case, the SNc cell loss takes place gradually following the striatal injection (Lee et al., 1996), thus mimicking more closely the slowly evolving nature of the nigral lesion of PD. This creates a window, which can be used for the investigation of premotor symptoms of this disease.

831

Our hypothesis predicts alterations in behavioral tests of depressive-like, anxiety and cognitive tests after striatal dopaminergic degeneration. Behavior was assessed by sucrose consumption, forced swimming test, elevated plus maze, the cued version of Morris water maze and social recognition tests. Additionally, motor system function was analyzed through the open-field test. Nigrostriatal dopaminergic damage was evaluated by TH-immunohistochemistry in both substantia nigra and striatum. Moreover, neurochemical analysis of DA, noradrenaline (NA) and 5-HT were performed in striatum, prefrontal cortex and hippocampus, areas known to be involved in emotional and cognitive processes (Owen, 2004; Levy and Dubois, 2006; Vertes, 2006), in order to understand the neurotransmitter alterations in these areas. To gain information with reference to early changes following the toxic insult we studied the time-course of neuronal damage and metabolic changes in the brain areas along with behavioral modifications following the bilateral injection of 6-OHDA in the dorsal striatum of rats.

EXPERIMENTAL PROCEDURES Animals Forty male Wistar rats from our own colony, weighing 250 –370 g at the beginning of the experiments, were used for this study. Rats were housed in standard cages in groups of a maximum of seven animals in a temperature-controlled room (23⫾1 °C) on a 12-h light/dark cycle with free access to food and water. All procedures used in the present study complied with the guidelines on animal care of the UFSC Ethics Committee on the Use of Animals, which follows the “Principles of laboratory animal care” from NIH. All efforts were made to minimize the number of animals used and their suffering. The rats were divided into two groups, comprising 20 animals each. These two groups were further subdivided into two subgroups: (i) a 6-OHDA group (n⫽10) that received 6-OHDA in the striatum and (ii) a sham group (n⫽10) that received vehicle in the striatum. For evaluation of early behavioral alterations, all tests were performed with the first group (n⫽20) during the first week after surgery and injection of 6-OHDA or vehicle. The tests, during which no behavioral alterations were seen in 6-OHDA-injected animals, were later (3 weeks) repeated with the second group of 20 animals, for comparison with time-course of neurochemical alterations. For the social recognition test, seven additional young, 1-month-old male rats were used.

Surgery and 6-OHDA lesion All surgical procedures were conducted with aseptic technique. To produce a PD model with incomplete destruction of nigrostriatal DA cells, 6-OHDA (12 ␮g per injection, diluted in 0.9% NaCl, supplemented with 0.1% ascorbic acid, injection volume 2.5 ␮l at the rate of 0.5 ␮l/min; Sigma Chemical, St. Louis, MO, USA) was injected over 5 min bilaterally into the ventrolateral area of the dorsal striatum, which is the primate analogous to the putamen, the area that accounts for the greater dopaminergic degeneration in PD. Stereotaxic infusion followed the coordinates of the Paxinos and Watson (2005) atlas: AP: 1.1 mm, ML: ⫾3.2 mm and DV: ⫺7.2 mm from bregma and dura, using a Hamilton 10 ␮l syringe with a 26-gauge needle connected to a 30-gauge cannula. Following injection, the cannula was left in place for 2 min before being retracted, to allow complete diffusion of the drug. All animals were treated with i.p. injection of 20 mg/kg desipramine (Sigma) 30 min before surgery, in order to protect noradrenergic terminals

832

M. T. Tadaiesky et al. / Neuroscience 156 (2008) 830 – 840

from 6-OHDA toxicity. The stereotaxic surgery was performed under ketamine (75 mg/kg)/xylazine (15 mg/kg) anesthesia. Sham-operated rats followed the same protocol except for the fact that vehicle was injected instead of 6-OHDA. The behavioral experiments were carried out 1 and 3 weeks after the surgery. Subgroups of four to eight animals were killed by decapitation following behavioral tests for neurochemical analysis.

Behavioral tests Tests for assessment of emotional parameters. Sucrose consumption. Sucrose consumption is frequently used as a measure of anhedonia in rodents (Craft and DeVries, 2006). After 1 week of recovery, animals were transferred into single housing cages with free access to food. Each rat was provided with two water bottles on the extreme sides of the cage during the 24-h training phase to adapt the rats to drinking from two bottles. After training, one bottle was randomly switched to contain 0.8% sucrose solution, as described previously (Slattery et al., 2007), and 24 h later, the bottles were reversed to avoid perseveration effects. The use of a 48-h testing period allowed us to preclude any effects of neophobia, artifactual bias toward any particular side and perseveration effects. The consumption of water and sucrose solution was estimated simultaneously in sham and 6-OHDA groups by weighing the bottles. Forced-swimming test. The procedure was previously described by Porsolt et al. (1978). Rats were placed in individual glass cylinders (40 cm in height and 17 cm in diameter) containing water (water depth was 30 cm; 25⫾1 °C). Two swimming sessions were conducted (an initial 15-min pretest followed 24 h later by a 5-min test). The total duration of immobility was manually scored continuously for a 5-min period. A rat was regarded as immobile when floating motionless or making only those movements necessary to keep its head above the water. Elevated plus maze. The elevated plus maze consisted of two open arms (50⫻10⫻0.75 cm) and two enclosed arms (50⫻10⫻40 cm), arranged such that two pairs of identical arms were opposite to each other. Arms emerged from a central platform (10⫻10 cm), and the entire apparatus was raised to a height of 50 cm above floor level. Experiments were performed under dim light conditions. At the beginning of the test, the rat was placed on the central platform facing an open arm. After each 5 min test, the maze was carefully cleaned up with a wet towel. Anxiety was evaluated through the following behavioral parameters: percent open arm entries (% open arm entries: open entries/ total entries⫻100) and percent time on open arms (% open time). Both measures are inversely related to the anxiety level in rodents. Since anxiolytic or anxiogenic effects can be confounded by changes in motor activity, the rat’s locomotion was also evaluated on the basis of closed arm entries and total arm entries, parameters which are considered the best indicators of locomotor activity of rodents placed on the elevated plus maze (Dawson and Tricklebank, 1995). Operational criterion for entry was whole body and four paws. Tests for assessment of cognitive performance. Water maze task. To evaluate the occurrence of cognitive deficits after the intrastriatal administration of 6-OHDA in rats, animals were submitted to the cued version of the Morris water maze task. The test was performed in a circular swimming pool similar to that previously used in this laboratory (Prediger et al., 2006a). The pool was made of black painted fiberglass, 1.7 m inside diameter, 0.8 m high, and was filled to a depth of 0.6 m with water maintained at 25 °C. The target platform (10⫻10 cm) was made of transparent Plexiglas and was submerged 1–1.5 cm beneath the surface of the water. The position of the escape platform was cued by a 7-cm diameter white ball attached to the top of the platform and protruding above the water. Visual cues were placed on the walls of the water maze room. The apparatus was located in a room

with indirect incandescent illumination. The scores for latency of escape from the starting point to the platform were measured. Rats were submitted to the cued version of the water maze as previously described by Miyoshi et al. (2002). This consisted of four training days, four consecutive trials per day, during which the animals were left in the tank facing the wall, then being allowed to swim freely to the submerged platform placed in the center of one of the four imaginary quadrants of the tank. The position of the platform was always changed during each trial of the day. The position of the escape platform was cued by a 7-cm diameter white ball attached to the top of the platform and protruding above the water. The initial position in which the animal was left in the tank was one of the four vertices of the imaginary quadrants of the tank, and this was varied among trials in a pseudo-random way. If a rat did not find the platform during a period of 60 s, it was gently guided to it. The animal was allowed to remain on the platform for 10 s and then removed from the tank for 20 s before being placed in the next random initial position. Social recognition test. Short-term social memory was assessed with the social recognition task as previously evaluated in our laboratory (Prediger et al., 2006b). Adult rats were housed individually in plastic cages (42⫻34⫻17 cm) and they were used after 3 days of habituation to their new environment. The test was scored in an observation room, where the rats had been habituated for 30 min before the beginning of the test. All juveniles were isolated in individual cages for 30 min prior to the beginning of the experiment. The social recognition test consisted of two successive presentations (5 min each) separated by a short period of time where a juvenile rat was placed in the home cage of the adult rat and the time spent by the adult in investigating the juvenile (nosing, sniffing, grooming or pawing) was recorded. At the end of the first presentation, the juvenile was removed and kept in an individual cage during the delay period and re-exposed to the adult rat after 30 min. In this kind of task, if the delay period is less than 40 min, the adult male rats usually display recognition of this juvenile rat by significantly reducing the social investigation time during the second presentation (Prediger et al., 2006b). Olfactory discrimination task. With the aim of discarding possible interference of any impairment in the olfactory discrimination ability impairment in the social recognition test, the olfactory discrimination task was performed 3 weeks after surgery. This test, previously evaluated in our laboratory (Prediger et al., 2006a), consisted of placing each rat for 5 min in a cage, which was divided into two identical compartments (30⫻30⫻20 cm) separated by an open door, where it could choose between one compartment with fresh sawdust and another with unchanged sawdust (familiar compartment) that the same rat had occupied for 48 h before the test. Each animal was initially placed in the center of the non-familiar compartment and the time (s) spent by the rat in both compartments (familiar vs. non-familiar) was recorded. Usually, mature male rats are able to discriminate between the familiar and the non-familiar compartments, spending much more time in the familiar compartment since they significantly prefer their own odor to no odor at all.

Assessment of spontaneous locomotor activity In order to assess possible effects of 6-OHDA on locomotor activity, open-field test was performed 1 and 3 weeks after surgery. The apparatus, made of wood covered with impermeable Formica, had a white floor of 100⫻100 cm (divided by black lines into 25 squares of 20⫻20 cm) and white walls, 40 cm high. Each rat was placed in the center of the open field, and the number of squares crossed and of rearings was registered for 5 min.

Neurochemistry TH-immunohistochemistry. One and 3 weeks after 6-OHDA treatment animals were intracardially perfused first with saline,

M. T. Tadaiesky et al. / Neuroscience 156 (2008) 830 – 840 then with 4% of the fixative solution formaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and kept overnight in the same solution. Serial sections (3 ␮M) were selected to include the striatal and midbrain. Immunohistochemistry was assessed using the mouse anti-TH monoclonal antibody (1:200, catalog MAB318) from Millipore, Chemicon International, Technology, Billerica, MA, USA). High temperature antigen retrieval was performed by immersing of the slides in a water bath at 95–98 °C in 10 mM trisodium citrate buffer pH 6.0, for 45 min. The nonspecific binding was blocked by incubating sections for 1 h with goat normal serum diluted in PBS. After overnight incubation at 4 °C with primary antibodies, the slides were washed with PBS and incubated with the secondary antibody Envision plus (Dako Cytomation, Carpinteria, CA, USA), ready-to-use, for 1 h at room temperature. The sections were washed in PBS, and the visualization was completed by use of DAB (3,3=-diaminobenzidine) (Dako Cytomation) in chromogen solution and counterstained lightly with Harris’s hematoxylin. Control and experimental tissues were placed on the same slide and processed under the same conditions. Digital images were acquired by Sight DS-5M-L1 digital camera connected to a light microscope Eclipse-80i (Nikon, Melville, NY, USA). Settings for image acquisition were identical for control and experimental tissues. Sections were analyzed approximately in the levels corresponding to bregma 5.8 mm of the Paxinos and Watson (2005) atlas. We obtained three images of SN and four images of striatum for each rat. Digitized images were transferred to a computer, and the average pixel intensity of immunostaining for TH was calculated using NIH ImageJ 1.36b imaging software (NIH, Bethesda, MD, USA). Values obtained in the images of the regions were averaged for each rat. Results were demonstrated in percentage TH-staining area. Monoamine contents. For determining DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovallinic acid (HVA), NA and 5-HT contents in brain, the rats were killed by decapitation 1 and 3 weeks after 6-OHDA administration. Brains were removed immediately and the structures striatum, prefrontal cortex and hippocampus were dissected from the preparation, frozen in liquid nitrogen and stored at ⫺70 °C. The concentration of DA, DOPAC, HVA, NA and 5-HT in striatum, prefrontal cortex and hippocampus was assayed by reverse-phase high performance liquid chromatography (HPLC) with electrochemical detection (ED). The system consisted of a Synergi Fusion-RP C-18 reverse-phase column (150⫻4.6 mm i.d., 4 ␮m particle size) fitted with a 4⫻3.0 mm pre-column (SecurityGuard Cartridges Fusion-RP); an electrochemical detector (ESA Coulochem III Electrochemical Detector) equipped with a guard cell (ESA 5020) with the electrode set at 350 mV and a dual electrode analytical cell (ESA 5011A); and a LC-20AT pump (Shimadzu) equipped with a manual Rheodyne 7725 injector with a 20 ␮l loop. The column was maintained inside a temperature-controlled oven (25 °C; Shimadzu). The cell has two chambers in series: each chamber includes a porous graphite coulometric electrode, a double counter electrode and a double reference electrode. Oxidizing potentials were set at 100 mV for the first electrode and at 450 mV for the second electrode. DA, DOPAC, HVA, NA and 5-HT were detected at the second electrode. The tissue samples were homogenized with an ultrasonic cell disrupter (Sonics) in 0.1 M perchloric acid containing sodium metabisulfite 0.02% and internal standard. After centrifugation at 10,000⫻g for 30 min, 4 °C, 20 ␮l of the supernatant was injected into the chromatograph. The mobile phase, used at a flow rate of 1 ml/min, had the following composition: 20 g citric acid monohydrated (Merck), 200 mg octane-1-sulfonic acid sodium salt (Merck), 40 mg EDTA (Sigma), 900 ml HPLC-grade water. The pH of the running buffer solution was adjusted to 4.0 then filtered through a 0.45 ␮m filter. Methanol (Merck) was added to give a final composition of 10% methanol (v/v). The peak areas of the external standards were used to quantify the sample peaks.

833

Data analysis and statistics Statistical analyses were performed with the computer software GraphPad Prism 4.0 (GraphPad Software, Inc.). All data are expressed as mean⫾S.E.M. For the social recognition test, statistical evaluation of results was carried out using one-way analysis of variance (ANOVA). Morris water maze escape latencies for the individual trials (averaged by day) and TH-immunostaining were analyzed by two-way ANOVA with repeated measures. Following significant ANOVAs, data were analyzed by the Bonferroni test. Statistical analyses for the rest of the data were carried out by unpaired Student’s t-test. A P value of less than 0.05 was considered significant.

RESULTS Effects of intrastriatal administration of 6-OHDA on emotional parameters in rats Sucrose consumption. During the initial training phase, in which the rats were habituated to drink from two bottles (both filled with water), no significant difference was observed in water intake between sham and 6-OHDA rats. Fig. 1 illustrates the mean 48-h intake of water and sucrose solution in sham and 6-OHDA 1 week after surgery. Both groups clearly consumed more sucrose solution than water. Student’s t-test comparison showed that rats from the sham group consumed significantly more sucrose than those from the 6-OHDA group [F(1,13)⫽1.29, P⬍0.05)]. In addition, no difference in total water intake during this time was observed. Forced-swimming test. The forced-swimming test was used to monitor depressive-like behavior. One week after surgery, Student’s t-test indicated that exposure to 6-OHDA treatment significantly increased immobility time [F(1,35)⫽10.7, P⬍0.001)] (Fig. 2). Elevated plus maze. Three weeks after surgery, 6-OHDA decreased significantly the percentage of entries into the open arms of the elevated plus maze [F(1,12)⫽ 5.28, P⬍0.01)] (Fig. 3B). Moreover, 6-OHDA-treated rats, as compared with sham, tended to spend less time on the open arms (P⫽0.07) (Fig. 3A). Finally, 6-OHDA did not significantly affect the number of total and closed-arm entries, considered as a measure of motor activity parameters (Fig. 3D and C, respectively).

Fig. 1. Effect of 6-OHDA lesion during the 48-h sucrose and water intake and during the 24-h training phase. 6-OHDA-treated animals consumed less sucrose solution than the sham group. Values are shown as mean⫾S.E.M. * P⬍0.05 (in comparison with sham, Student’s t-test).

834

M. T. Tadaiesky et al. / Neuroscience 156 (2008) 830 – 840

Fig. 2. Effect of 6-OHDA lesion on the forced-swimming test. 6-OHDA treatment increased immobility time. Values are shown as mean⫾ S.E.M. * P⬍0.05 (in comparison with sham, Student’s t-test).

Effects of intrastriatal administration of 6-OHDA on cognitive performance in rats Morris water maze. Three weeks after administration of 6-OHDA, rats were tested in the cued version of the Morris water maze. At this time, animals did not present gross motor alterations that would otherwise confound the interpretation of cognitive impairment in memory tasks (Table 2). The effect of striatal lesion on the scores of the rats in the cued version of water maze is presented in Fig. 4. All rats were able to learn the task, since their mean escape latency improved throughout the training days. However, 6-OHDA-lesioned animals spent more time to find the platform. Two-way ANOVA (treatment vs. repeated measures) revealed a significant effect for the main factor [treatment: F(1,13)⫽25.91, P⬍0.0001]; [repeated measures: F(1,13)⫽83, P⬍0.0001], but not for the interaction between these variables: [interaction: F(1,13)⫽0.76,

Fig. 4. Effect of 6-OHDA lesion on the cued version of the water maze task. 6-OHDA-treated animals spent more time to find the platform. Values are shown as mean⫾S.E.M. latency to escape to a submersed platform during four training days, with four consecutive trials per day. Escape latencies for the individual trials were averaged by day. * P⬍0.05 (in comparison with sham, Bonferroni test after two-way ANOVA).

P⬎0.05]. Subsequent Bonferroni tests indicated significant differences (P⬍0.05) between the 6-OHDA and sham groups for the first to third training days of the task. Social recognition test. Fig. 5 summarizes the effect of 6-OHDA injection on the social recognition memory of rats. Three weeks after injection, one-way ANOVA followed by Bonferroni test indicated that sham rats spent less time investigating the juvenile rat than in the first exposure. On the other hand, 6-OHDA-treated rats spent as much time investigating the juvenile rat during the second encounter as they did in the first exposure [F(1,15)⫽ 4.43, P⬍0.05], reflecting a clear impairment of the juvenile’s recognition ability.

Fig. 3. Effect of 6-OHDA lesion on the elevated plus-maze test as measured by spatiotemporal activities parameters. 6-OHDA treatment decreased the percentage entries into the open arms. (A) % Time in open arms. (B) % Open armentries. (C) Closed arm entries. (D) Total arm entries. Data represent mean⫾S.E.M. * P⬍0.05 (in comparison with sham, Student’s t-test).

M. T. Tadaiesky et al. / Neuroscience 156 (2008) 830 – 840

835

Table 2. Effects of 6-OHDA lesion on spontaneous locomotor activity in rats 1 and 3 weeks after surgery Parameters

No. squares crossed No. rearings

1 Week

3 Weeks

Sham

6-OHDA

Sham

6-OHDA

89⫾9.2 20.3⫾2.7

79⫾6 19.7⫾2.5

59.6⫾9.3 12.7⫾2.5

43.7⫾11.4 12⫾2.9

Values are shown as mean⫾S.E.M.

Fig. 5. Effect of 6-OHDA lesion on social investigation time of rats. 6-OHDA-treated animals spent as much time investigating the juvenile rat during the second encounter as they did on the first exposure Values are shown as mean⫾S.E.M. * P⬍0.05 (in comparison with first presentation from the same group, Bonferroni test after one-way ANOVA). # P⬍0.05 (in comparison with second presentation from sham, Bonferroni test after one-way ANOVA).

In order to discard any deleterious effect of 6-OHDA on the olfactory discrimination ability of rats that could otherwise confound cognitive impairments observed in the social recognition test, the olfactory discrimination task was performed 3 weeks after surgery. The results are illustrated in Table 1. Student’s t-test indicated that 3 weeks after surgery both sham and 6-OHDA-treated rats were able to discriminate between the familiar and non-familiar compartments, spending much more time in the familiar compartment. Effects of 6-OHDA on locomotor activity in rats Table 2 summarizes the effects of striatal administration of 6-OHDA on locomotor activity of rats evaluated in the open field test. The exploratory behavior evaluated by number of squares crossed and number of rearing was not affected by 6-OHDA administration 1 and 3 weeks after the lesion. Thus, the effects of 6-OHDA on emotional and cognitive tests are not directly related to their locomotor condition. In addition, both groups displayed a decreased number of squares crossed and rearing on week 3, reflecting habituation. Analysis of neurochemical alterations induced by 6-OHDA administration Nigrostriatal damage. The TH-immunoreactive neurons were detectable in the striatum of sham and 6-OHDATable 1. Effects of 6-OHDA lesion on odor discrimination ability of rats three weeks after surgery Group

Sham 6-OHDA

% Time in compartment Familiar

Non-familiar

59.7⫾3.2 54⫾3

44.2⫾4.9* 46⫾3*

Values are shown as mean⫾S.E.M. P represents statistical analysis. * P⬍0.05 (compared to the percentage of time spent in the familiar compartment, Student’s t-test).

treated rats. The fibers of dopaminergic neurons showed intense TH-immunostaining. There were differences in % TH-stained area between sham and 6-OHDA-treated animals 1 week after 6-OHDA injection; the main effect for lesion was statistically significant [F(3,3)⫽31.42, P⬍0.01]. The magnitude of this difference persisted after 3 weeks; the lesion-time interaction was not statistically significant [F(3,3)⫽0.57, P⬎0.05]. In SNc, cell number declined with 6-OHDA infusions even 1 week after surgery; the main effect for lesion was statistically significant [F(3,4)⫽48.43, P⬍0.001]. The reduced cell number also persisted until the third week, but was diminished at this time point in relation to the first week; the lesion-time interaction was statistically significant [F(3,4)⫽9.19, P⬍0.05]. Fig. 6 shows representative photomicrographs of TH immunohistochemistry in the striatum (Fig. 6A) and SNc (Fig. 6B) at each time point. Fig. 6C and D indicates percentage of stained area in 6-OHDA and sham groups after 1 and 3 weeks in striatum and SNc, respectively. Monoamine contents. With the purpose of determining the relationship between the behavioral alterations induced by 6-OHDA in rats and neurochemical alterations in dopaminergic, noradrenergic and serotonergic neurotransmission, the levels of DA, DOPAC, HVA, NA, and 5-HT in the striatum, prefrontal cortex and hippocampus were measured 1 and 3 weeks after intrastriatal administration of 6-OHDA. The levels of DA [F(1,21)⫽3.23, P⬍0.0001)], its metabolites DOPAC [F(1,21)⫽1.10, P⬍0.005)] and HVA [F(1,21)⫽ 13.79, P⬍0.01)] and 5-HT [F(1,21)⫽2.62, P⬍0.005)] were decreased in the striatum even 1 week after surgery. An average of 59% of dopaminergic depletion was seen in the striatum of rats lesioned with 6-OHDA. Moreover, there was an increase in NA levels in this area [F(1,6)⫽6.96, P⬍0.005)] (Fig. 7). As expected, DA levels in the prefrontal cortex were much lower than in the striatum (121⫾44.7 ng DA/g wet tissue). DA, DOPAC, HVA and 5-HT levels in that area were unaffected by 6-OHDA, while there was a significant NA decrease [F (1,14)⫽3.23, P⬍0.02)] (Table 3). No differences in the neurotransmitter levels were observed in hippocampus. Three weeks after surgery, DA [F(1,21)⫽1.74, P⬍0.005)], DOPAC [F(1,21)⫽1.42, P⬍0.005)] and HVA [F(1,21)⫽ 1.06, P⬍0.005)] were still depleted in the striatum. At this time point, the volume of dopaminergic terminal lesion remained substantially unchanged (51.36%). Additionally, the increase in NA levels [F(1,6)⫽1.32, P⬍0.005)] per-

836

M. T. Tadaiesky et al. / Neuroscience 156 (2008) 830 – 840

Fig. 6. Effects of 6-OHDA lesion on the number of TH-positive neurons in striatum and SNc. 6-OHDA infusions reduced DA cell number in these areas after 1 and 3 weeks. (A) Representative photomicrographs of brain coronal sections containing the sham and 6-OHDA-injected striatum from animals killed 1 and 3 weeks following intrastriatal injection of 6-OHDA (B) Representative photomicrographs of brain coronal sections containing the sham and 6-OHDA-lesioned SNc from animals killed 1 and 3 weeks following intrastriatal injection of 6-OHDA (C) Time-dependent loss of dopaminergic terminals in the striatum. (D) Time-dependent loss of dopaminergic neurons in the SNc. Bars represent the mean (⫾S.E.M.) % TH-stained area. Values are shown as mean⫾S.E.M. P represents statistical analysis. * P⬍0.05 (in comparison with the respective sham group, Bonferroni test after two-way ANOVA).

sisted in this area (Fig. 8A and B). On the other hand, at this point, DA [F(1,6)⫽1.77, P⬍0.05)] and DOPAC [F(1,20)⫽1.83, P⬍0.05)] were decreased in the prefrontal cortex in comparison to the sham group, whereas no alterations were seen in NA and 5-HT levels (Fig. 8C and D). No differences were observed in the hippocampus, except

for a decrease in DOPAC [F(1,6)⫽1.31, P⬍0.05)] levels (Table 4). Table 5 summarizes the time-course of emotional and cognitive deficits found in this study. The results suggest that the occurrence of emotional and cognitive deficits induced by intrastriatal administration of 6-OHDA in rats

Fig. 7. Effects of 6-OHDA lesion on DA, DOPAC, HVA, NA and 5-HT levels in rat striatum 1 week after surgery. (A) DA, DOPAC and HVA levels in sham and 6-OHDA rats. (B) NA and 5-HT levels in sham and 6-OHDA rats. Values are shown as mean⫾S.E.M. P represents statistical analysis. * P⬍0.05 (in comparison with sham, Student’s t-test). Numbers in parentheses indicate percentage depletion calculated by taking sham value as 100%.

M. T. Tadaiesky et al. / Neuroscience 156 (2008) 830 – 840

837

Table 3. Effect of 6-OHDA lesion on DA, DOPAC, HVA, NA and 5-HT levels in rat prefrontal cortex and hippocampus one week after surgery Region

Group DA

PFC HIP

DOPAC

HVA

NA

5-HT

Sham

6-OHDA

Sham

6-OHDA

Sham

6-OHDA

Sham

6-OHDA

Sham

6-OHDA

121⫾44.7 33.3⫾0.2

97⫾73.9 30.9⫾1.9

97⫾40.5 24.9⫾1.3

73.9⫾2.8 21⫾3.2

125⫾19.1 65.6⫾27.3

81.9⫾1.4 66.8⫾44.6

238.1⫾21.1 405.8⫾137

*177.4⫾17.9 237.8⫾34.3

415.4⫾53.7 87.6⫾35.8

327.5⫾51.8 104⫾34

Values are shown as mean⫾S.E.M. P represents statistical analysis. * P⬍0.05 (in comparison with sham, Student’s t-test). Abbreviations: HIP, hippocampus; PFC, prefrontal cortex.

may be related to time-dependent alterations in dopaminergic, noradrenergic and serotonergic neurotransmission in the striatum and prefrontal cortex.

DISCUSSION The primary findings of this study are that specific striatal lesion was shown to induce anhedonia and behavioral despair in rats, in addition to an increase in anxiety-like responses. Moreover, we support previous data that show impaired procedural memory after nigrostriatal lesion and provide new information on alterations on the social recognition test in a PD model. These behavioral deficits in 6-OHDA-lesioned rats are significant because they correlate with neurochemical alterations in SNc, striatum and prefrontal cortex evaluated at different time intervals. Importantly, gross impairments in spontaneous motor activity were not apparent suggesting that the behavioral deficits detected were specific.

Consistent with our initial hypothesis, intrastriatal administration of 6-OHDA promoted an early decrease in sucrose consumption measured 1 week after 6-OHDA treatment. The sucrose consumption test has been widely utilized as an animal model of depression, suggesting an anhedonic state in these animals (Craft and DeVries, 2006). Anhedonia in 6-OHDA-treated rats was accompanied by another feature of depressive-like behavior, behavioral despair, reflected by an increased floating time in the forced swimming test, which indicates a negative emotional state. These anhedonic-depressive-like effects were correlated with neurochemical alterations at the striatal level. We found that a clearly detectable loss of TH immunostaining was already present at 1 week after 6-OHDA injection. The cell loss in the present study was partial: 55.75% of the dopaminergic neurons in striatum survived. Similarly, the loss of TH-positive cells in the SNc became evident at the same time point and also resulted in incom-

Fig. 8. Effects of 6-OHDA lesion on DA, DOPAC, HVA, NA and 5-HT levels in rat striatum and prefrontal cortex 3 weeks after surgery. (A, B) Striatum. (C, D) Prefrontal cortex. Values are shown as mean⫾S.E.M. P represents statistical analysis. * P⬍0.05 (in comparison with sham, Student’s t-test). Numbers in parentheses indicate percentage depletion calculated by taking sham value as 100%.

838

M. T. Tadaiesky et al. / Neuroscience 156 (2008) 830 – 840

Table 4. Effect of 6-OHDA lesion on DA, DOPAC, HVA, NA and 5-HT levels in rat hippocampus three weeks after surgery Region

Group DA

HIP

DOPAC

HVA

NA

5-HT

Sham

6-OHDA

Sham

6-OHDA

Sham

6-OHDA

Sham

6-OHDA

Sham

6-OHDA

35.8⫾2.8

23.4⫾7.8

28.2⫾3.2

17.6⫾2.8*

36.7⫾8.2

53.9⫾14.2

274.7⫾23.1

195.9⫾44.4

150.4⫾33.6

116.4⫾59.6

Values are shown as mean⫾S.E.M. P represents statistical analysis. * P⬍0.05 (in comparison with sham, Student’s t-test). Abbreviation: HIP, hippocampus.

plete reductions in the number of DA cell bodies. These results are in accordance with previous studies (Blandini et al., 2007; Lindner, 1999; Sauer and Oertel, 1994), who reported that injection of 6-OHDA into the terminal field of nigral dopaminergic neurons causes a retrograde degeneration of these cells. The infusion of 6-OHDA in the striatum also resulted in a partial depletion of DA, DOPAC, HVA and 5-HT, which is similar to previous reports (Henze et al., 2005; Chen et al., 2007; Aguiar et al., 2008), in addition to an increase in NA levels, possibly due to a compensatory response to DA reduction. Concerning 5-HT striatal depletion consecutive to 6-OHDA administration, one must consider that in our experiments 5-HT terminals were not protected from 6-OHDA damage, which could explain the reduced 5-HT striatal levels. Therefore, consistent with the known role of the serotonergic system in emotional processes, it is conceivable that alterations found on sucrose consumption and forced swimming tests could be related, at least partially, to 5-HT changes. In fact, there are pathophysiological evidences of 5-HT alterations in patients with PDassociated depression (Schrag, 2004), and a “5-HT hypothesis” has even been proposed for depression in PD (Mayeux, 1990). Therefore, we cannot rule out the serotonergic participation in the emotional alterations found in the behavioral tests implemented in this study. The abovementioned alterations in dopaminergic, noradrenergic and serotonergic striatal systems suggest, therefore, that one or more of these neurotransmitter systems play an important role in depressive-like behavior in this PD model, further supporting the involvement of these neurotransmitter systems in PD-related depression (Schrag, 2004). Additionally, an anxiogenic response, quantified as a decrease in open arm entries in the elevated plus maze, was evident after 6-OHDA administration at the latest time point (3 weeks post-lesion). At this interval, the reduction in dopaminergic neurons persisted in both striatum and SNc. The decreased striatal DA, DOPAC and HVA levels as well Table 5. Summary of time-course of behavioral deficits found in 6-OHDA-injected rats Behavioral tests 1 Week

3 Weeks

Forced swimming test Sucrose preference test

Elevated plus maze Morris water maze Social recognition test

as the increased NA levels were still present, and were found not to change significantly from those of the first week. On the other hand, the loss of DA and DOPAC in the prefrontal cortex became evident only at this point, suggesting a progressive effect of 6-OHDA lesion. This result suggests that the anxiogenic response elicited by 6-OHDA could be related to dopaminergic depletion on the prefrontal cortex, since no alteration in open arm entries was evident at 1 week post-surgery. Indeed, this notion is supported by previous evidence that 6-OHDA injected directly in the prefrontal cortex induces a significant anxiogenic effect evaluated in the elevated plus maze (Espejo, 1997). Even though most of the dopaminergic efferents from the prefrontal cortex arise from the ventral tegmental area, some of them come from the central area of the SNc (Albanese and Bentivoglio, 1982). For this reason, the depletion of DA observed in the prefrontal cortex consecutive to the intrastriatal administration of 6-OHDA in rats could explain their increase in anxiety levels. Therefore, this result suggests that prefrontal DA depletion may play an important role in modulating anxiety in PD. Besides emotional deficits presented in PD patients, PD seems to produce cognitive deficits, especially in procedural memory. The striatum has been the main area implicated in procedural learning dysfunctions (Saint-Cyr et al., 1988). However, the frontal cortex has also been implicated as a component of the neural network that supports procedural learning (Torriero et al., 2007). Consistent with earlier studies reporting that bilateral 6-OHDA or MPTP injections in SNc impair rats’ performance in the cued version of the Morris water maze (Miyoshi et al., 2002; Ferro et al., 2005), intrastriatal 6-OHDA injection performed in this study induced similar impairment in this task. Importantly, the percent of striatal DA depletion observed in this study was similar to previous studies showing deficits in memory tasks (Lindner et al., 1999; Gevaerd et al., 2001a; Miyoshi et al., 2002; Ferro et al., 2005). As for the cognitive deficits found in this test, our findings support the hypothesis of a combined involvement of striatal and prefrontal cortical DA deficits in procedural memory, since no significant deficits were observed in the absence of prefrontal cortex DA alterations, 1 week after 6-OHDA administration. Additionally to cued water maze deficits, our findings demonstrated a deleterious effect of this neurotoxin on the social recognition test in rats. The 6-OHDA-treated rats spent as much time investigating the juvenile rat during the

M. T. Tadaiesky et al. / Neuroscience 156 (2008) 830 – 840

second presentation as they did on the first encounter, demonstrating impairment in the ability to recognize the juvenile after a short period of time (30 min). Since this response occurred in association with reduced prefrontal cortex DA, the behavioral impairment could be interpreted as related to DA depletion in this area. However, our findings contrast with those of a previous study (Fernandez Espejo, 2003). A possible reason for the discrepancy between these results could be that, in our work, the prefrontal DA depletion that occurred concomitantly to social recognition memory impairment was accompanied by alterations in striatal DA levels. Therefore, it is possible that DA depletion in both structures would be necessary for the deficits observed in our study. Importantly, dopaminergic lesion is known to induce anosmia in rats (Prediger et al., 2006a), which could lead to misinterpretation of the results found in the social recognition test. Therefore, it seemed important to evaluate whether 6-OHDA injection alters this function. The present findings demonstrated an absence in odor recognition impairment in 6-OHDA-treated rats, evaluated 3 weeks after surgery. The present olfactory discrimination task, previously standardized in our laboratory (Prediger et al., 2006a), was based on some initial observations of Carr et al. (1976) who had shown that mature male rats significantly prefer their own odor to no odor at all. The ability of both sham and 6-OHDA groups to discriminate between the familiar and the non-familiar compartments suggests that alterations in the social recognition test really reflect a cognitive deficit, and not merely an olfactory impairment. Consistent with previous evidence that lesions of the nigrostrital pathway failed to alter DA hippocampal levels (Miyoshi et al., 2002), in the present study hippocampal DA levels of 6-OHDA-treated animals were not altered after striatal lesion, and may suggest that this area is not involved on the behavioral impairments found herein. However, one may consider that DA hippocampal levels are very low; therefore, we cannot exclude the possibility that the HPLC analysis was unable to detect small differences in DA levels in this area.

CONCLUSION In conclusion, our data indicate that denervation of dopaminergic nigrostriatal pathway by 6-OHDA and accompanying dopaminergic, noradrenergic and serotonergic alterations correlate with emotional and cognitive impairments in rats, symptoms characteristic of the premotor phase, therefore providing a good model of PD presymptomatic period. Certainly, more studies are needed to elucidate the role of these different neurotransmitter systems in the behavioral alterations found in this study. Acknowledgments—This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Brazil.

REFERENCES Aguiar LM, Macedo DS, Vasconcelos SM, Oliveira AA, de Sousa FC, Viana GS (2008) CSC, an adenosine A(2A) receptor antagonist and

839

MAO B inhibitor, reverses behavior, monoamine neurotransmission, and amino acid alterations in the 6-OHDA-lesioned rats. Brain Res 1191:192–199. Albanese A, Bentivoglio M (1982) The organization of dopaminergic and non-dopaminergic mesencephalo-cortical neurons in the rat. Brain Res 238:421– 425. Bellissimo MI, Kouzmine I, Ferro MM, Oliveira BH, Canteras NS, Da Cunha C (2004) Is the unilateral lesion of the left substantia nigra pars compacta sufficient to induce working memory impairment in rats? Neurobiol Learn Mem 82:150 –158. Blandini F, Levandis G, Bazzini E, Nappi G, Armentero MT (2007) Time-course of nigrostriatal damage, basal ganglia metabolic changes and behavioural alterations following intrastriatal injection of 6-hydroxydopamine in the rat: new clues from an old model. Eur J Neurosci 25:397– 405. Braga R, Kouzmine I, Canteras NS, Da Cunha C (2005) Lesion of the substantia nigra, pars compacta impairs delayed alternation in a Y-maze in rats. Exp Neurol 192:134 –141. Carr WJ, Yee L, Gable D, Marasco E (1976) Olfactory recognition of conspecifics by domestic Norway rats. J Comp Physiol Psychol 90:821– 828. Chen H, Jing FC, Li CL, Tu PF, Zheng QS, Wang ZH (2007) Echinacoside prevents the striatal extracellular levels of monoamine neurotransmitters from diminution in 6-hydroxydopamine lesion rats. J Ethnopharmacol 114:285–289. Craft TK, DeVries AC (2006) Role of IL-1 in poststroke depressive-like behavior in mice. Biol Psychiatry 60:812– 818. Cummings JL, Masterman DL (1999) Depression in patients with Parkinson’s disease. Int J Geriatr Psychiatry 14:711–718. Da Cunha C, Wietzikoski S, Wietzikoski E, Silva MHC, Chandler J, Ferro MM, Andreatini R, Canteras NS (2007) Pre-training to find a hidden platform in the Morris water maze can compensate for deficit to find a cued platform in a rat model of Parkinson’s disease. Neurobiol Learn Mem 87:451– 463. Dauer W, Przedborski S (2003) Parkinson’s disease: mechanisms and models. Neuron 39:889 –909. Dawson GR, Tricklebank MD (1995) Use of the elevated plus maze in the search for novel anxiolytic agents. Trends Pharmacol Sci 16:33–36. Espejo EF (1997) Selective dopamine depletion within the medial prefrontal cortex induces anxiogenic-like effects in rats placed on the elevated plus maze. Brain Res 762:281–284. Fernandez Espejo E (2003) Prefrontocortical dopamine loss in rats delays long-term extinction of contextual conditioned fear, and reduces social interaction without affecting short-term social interaction memory. Neuropsychopharmacology 28:490 – 498. Ferro MM, Bellissimo MI, Anselmo-Franci JA, Angellucci ME, Canteras NS, Da Cunha C (2005) Comparison of bilaterally 6-OHDAand MPTP-lesioned rats as models of the early phase of Parkinson’s disease: histological, neurochemical, motor and memory alterations. J Neurosci Methods 148:78 – 87. Gevaerd MS, Miyoshi E, Silveira R, Canteras NS, Takahashi RN, Da Cunha C (2001a) L-Dopa restores striatal dopamine level but fails to reverse MPTP-induced memory deficits in rats. Int J Neuropsychopharmacol 4:361–370. Gevaerd MS, Takahashi RN, Silveira R, Da Cunha C (2001b) Caffeine reverses the memory disruption induced by intra-nigral MPTPinjection in rats. Brain Res Bull 101:6. Henze C, Earl C, Sautter J, Schmidt N, Themann C, Hartmann A, Oertel WH (2005) Reactive oxidative and nitrogen species in the nigrostriatal system following striatal 6-hydroxydopamine lesion in rats. Brain Res 1052:97–104. Jackson GM, Jackson SR, Harrison J, Henderson L, Kennard C (1995) Serial reaction time learning and Parkinson’s disease: evidence for a procedural learning deficit. Neuropsychologia 33:577–593. Lee CS, Sauer H, Bjorklund A (1996) Dopaminergic neuronal degeneration and motor impairments following axon terminal lesion by

840

M. T. Tadaiesky et al. / Neuroscience 156 (2008) 830 – 840

instrastriatal 6-hydroxydopamine in the rat. Neuroscience 72:641– 653. Levy R, Dubois B (2006) Apathy and the functional anatomy of the prefrontal cortex-basal ganglia circuits. Cereb Cortex 16:916 –928. Lewis SJ, Cools R, Robbins TW, Dove A, Barker RA, Owen AM (2003) Using executive heterogeneity to explore the nature of working memory deficits in Parkinson’s disease. Neuropsychologia 41: 645– 654. Lieberman A (2006) Depression in Parkinson’s disease: a review. Acta Neurol Scand 113:1– 8. Lindner MD, Cain CK, Plone MA, Frydel BR, Blaney TJ, Emerich DF, Hoane MR (1999) Incomplete nigrostriatal dopaminergic cell loss and partial reductions in striatal dopamine produce akinesia, rigidity, tremor and cognitive deficits in middle-aged rats. Behav Brain Res 102:1–16. Marek K, Innis R, van Dyck C, Fussell B, Early M, Eberly S, Oakes D, Seibyl J (2001) [I-123]beta-CIT SPECT imaging assessment of the rate of Parkinson’s disease progression. Neurology 57:2089–2094. Mayeux R (1990) The “serotonin hypothesis” for depression in Parkinson’s disease. Adv Neurol 53:163–166. Miyoshi E, Wietzikoski S, Camplessei M, Silveira R, Takahashi RN, Da Cunha C (2002) Impaired learning in a spatial working memory version and in a cued version of the water maze in rats with MPTP-induced mesencephalic dopaminergic lesions. Brain Res Bull 58:41– 47. Owen AM (2004) Cognitive dysfunction in Parkinson’s disease: the role of frontostriatal circuitry. Neuroscientist 10:525–537. Packard MG, Knowlton BJ (2002) Learning and memory functions of the basal ganglia. Annu Rev Neurosci 25:563–593. Paxinos G, Watson C (2005) The rat brain in stereotaxic coordinates, 5th ed, p 205. San Diego: Academic Press. Porsolt RD, Anton G, Blavet N, Jalfre M (1978) Behavioural despair in rats: a new model sensitive to antidepressant treatments. Eur J Pharmacol 47:379 –391. Prediger RD, Batista LC, Medeiros R, Pandolfo P, Florio JC, Takahashi RN (2006a) The risk is in the air: Intranasal administration of MPTP to rats reproducing clinical features of Parkinson’s disease. Exp Neurol 202:391– 403.

Prediger RD, De-Mello N, Takahashi RN (2006b) Pilocarpine improves olfactory discrimination and social recognition memory deficits in 24 month-old rats. Eur J Pharmacol 531:176 –182. Reksidler AB, Lima MM, Zanata SM, Machado HB, da Cunha C, Andreatini R, Tufik S, Vital MA (2007) The COX-2 inhibitor parecoxib produces neuroprotective effects in MPTP-lesioned rats. Eur J Pharmacol 560:163–175. Saint-Cyr JA, Taylor AE, Lang AE (1988) Procedural learning and neostriatal dysfunction in man. Brain 111 (Pt 4):941–959. Sauer H, Oertel WH (1994) Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-hydroxydopamine: a combined retrograde tracing and immunocytochemical study in the rat. Neuroscience 59:401– 415. Schrag A (2004) Psychiatric aspects of Parkinson’s disease: an update. J Neurol 251:795– 804. Schrag A, Jahanshahi M, Quinn N (2000) What contributes to quality of life in patients with Parkinson’s disease? J Neurol Neurosurg Psychiatry 69:308 –312. Slattery DA, Markou A, Cryan JF (2007) Evaluation of reward processes in an animal model of depression. Psychopharmacology (Berl) 190:555–568. Spiegel J, Storch A, Jost WH (2006) Early diagnosis of Parkinson’s disease. J Neurol 253:2–7. Tissingh G, Bergmans P, Booij J, Winogrodzka A, van Royen EA, Stoof JC, Wolters EC (1998) Drug-naive patients with Parkinson’s disease in Hoehn and Yahr stages I and II show a bilateral decrease in striatal dopamine transporters as revealed by [123I]betaCIT SPECT. J Neurol 245:14 –20. Tolosa E, Compta Y, Gaig C (2007) The premotor phase of Parkinson’s disease. Parkinsonism Relat Disord 13 (Suppl):S2–S7. Torriero S, Oliveri M, Koch G, Lo Gerfo E, Salerno S, Petrosini L, Caltagirone C (2007) Cortical networks of procedural learning: evidence from cerebellar damage. Neuropsychologia 45:1208 – 1214. Vertes RP (2006) Interactions among the medial prefrontal cortex, hippocampus and midline thalamus in emotional and cognitive processing in the rat. Neuroscience 142:1–20. Ziemssen T, Reichmann H (2007) Non-motor dysfunction in Parkinson’s disease. Parkinsonism Relat Disord 13:323–332.

(Accepted 18 August 2008) (Available online 9 September 2008)