Differential behavioral effects of partial bilateral lesions of ventral tegmental area or substantia nigra pars compacta in rats

Neuroscience 153 (2008) 1213–1224

DIFFERENTIAL BEHAVIORAL EFFECTS OF PARTIAL BILATERAL LESIONS OF VENTRAL TEGMENTAL AREA OR SUBSTANTIA NIGRA PARS COMPACTA IN RATS E. Y. PIOLI,a W. MEISSNER,a,b R. SOHR,c C. E. GROSS,a E. BEZARDa AND B. H. BIOULACa*

occur across three distinct disorders: parkinsonism, depression and negative symptoms of schizophrenia (De Ajuriaguerra, 1975; Bermanzohn and Siris, 1992). The occurrence of akinetic symptoms in diverse psychiatric disorders supports the notion that akinesia involves not only motor behavior but also cognitive and motivational processes. All these functions are known to be modulated by dopamine (DA). Indeed, DA in the basal ganglia contributes to the regulation of motor and limbic processes and DA at the cortical level is correlated with cognitive functions (Nieoullon, 2002; Nieoullon and Coquerel, 2003). In this view, clinical evidence shows that motor symptoms in neurodegenerative disorders such as PD are accompanied by cognitive deficits (Brown and Marsden, 1990; Brown and Jahanshahi, 1996). Anatomically, the substantia nigra pars compacta (SNc) mainly projects to the caudate nucleus and the putamen. By contrast, the ventral tegmental area (VTA) mainly innervates the frontal cortex, the ventral striatum and the hippocampal formation (Fuxe, 1965; Lindvall and Bjorklund, 1974; Fallon et al., 1978; Fallon and Moore, 1978a,b; Haber and Fudge, 1997). While SNc/VTA projections to the forebrain are segregated, a degree of overlap exists in other cortical terminal fields and some subcortical areas. In particular, neurons projecting to the frontal cortex, the cingulate cortex, the septum rostro-medial caudate and the nucleus accumbens are located in the medial SNc as well as the lateral VTA (Fallon and Moore, 1978b). Bilateral DA-containing neuron degeneration of the SNc is the main characteristic of human PD. Although damage to the SNc primarily causes PD, the VTA is also modestly affected, even in early stages of PD (Damier et al., 1999a,b), rendering it difficult to attribute a specific deficit to a given nucleus. To solve this ambiguity, we induced selective partial bilateral 6-hydroxydopamine (6-OHDA) lesions of either the lateral SNc or the medial VTA in the rat (Pioli et al., 2004). The rats were subjected to various motor, motivational and cognitive behavioral experiments. Motor impairments were estimated by the measure of fine motor control in the stepping test and in the paw reaching test. The motivational behavior was evaluated by the 100 pellets test. Two paradigms were employed to assess cognitive impairments: spontaneous alternation in the Y maze and an object exploration task. Object exploration is associated with an index of cognitive activity, especially in rodents. Both paradigms (in which rats explore without contingent reinforcement) allow the study of various behavioral func-

a

Universite Victor Segalen, Bordeaux 2, Centre National de la Recherche Scienctifique, Bordeaux Institute of Neuroscience, UMR 5227, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France

b

Department of Neurology, CHU de Bordeaux, Hôpital du HautLévêque Bordeaux, France

c

Institute of Pharmacology and Toxicology, Charité Campus Mitte, Humboldt Universitaet, Berlin, Germany

Abstract—Akinesia (or absence of movement) is a prominent feature of Parkinson’s disease. Akinetic symptoms, however, are also observed in depression and schizophrenia, which support the hypothesis that akinesia involves more than only motor behavior. A common feature of these disorders is the disruption of dopamine homeostasis in the CNS. Here we aimed at relating the respective involvement of the nigrostriatal and mesocortical dopaminergic pathways to akinesia. We investigated in the rat the relative effects of selective bilateral partial lesions of substantia nigra pars compacta (SNc) or ventral tegmental area (VTA) which did not affect locomotion, on fine motor, motivational and cognitive behaviors. Motor impairments were measured by the evaluation of fine motor control in the stepping test and in the paw reaching test. Cognitive functions were assessed by various paradigms: spontaneous alternation in the Y maze and object exploration task. Motivational behavior was evaluated by the 100-pellets test. The results suggested that specific behavioral impairments are obtained following selective lesions of either SNc or VTA. SNc-lesioned rats exhibited deficits in fine motor functions as previously described in animal models of Parkinson’s disease, whereas VTA-lesioned rats demonstrated traits of perseveration without significant motor impairments. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: akinesia, dopamine, 6-OHDA.

Akinesia is considered to be a principal feature of Parkinson’s disease (PD) along with rigidity and tremor. It is defined as poverty of initiation and execution of willed and associated movements in the absence of paralysis (Lee, 1989). Akinesia represents a clinical syndrome which can *Corresponding author. Tel: ⫹33-557-571-551; fax: ⫹33-556-901-421. E-mail address: [email protected] (B. H. Bioulac). Abbreviations: ANOVA, analysis of variance; DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; EDTA, ethylenediaminetetraacetic acid; HPLC, high-pressure liquid chromatography; HVA, homovanillic acid; NE, northeast; NW, northwest; PD, Parkinson’s disease; SE, southeast; S.E.M., standard error of the mean; SNc, substantia nigra pars compacta; SW, southwest; TH, tyrosine hydroxylase; TH-IR, tyrosine hydroxylase immunoreactive; VTA, ventral tegmental area; 6-OHDA, 6-hydroxydopamine.

0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.01.084

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tions, such as attention, memory, and reactions to change in the environment.

EXPERIMENTAL PROCEDURES Animals Twenty-nine Wistar rats (Depré, St. Doulchard, France) weighing 450 – 480 g were housed under controlled conditions of light (12-h light/dark cycle, i.e. lights on from 07:00 –19:00 h), temperature (22 °C) and humidity. Food and water were available ad libitum. All experiments were carried out in accordance with the European Committee Council Directive of 24 November 1986 (86/609/EEC) and were approved by the local ethical committee. All reasonable efforts were made to minimize animal suffering and to use the minimum number of animals necessary to perform statistically valid analyses.

6-OHDA procedure Rats were anesthetized with 4% chloral hydrate (400 mg/kg i.p.; Sigma, St. Quentin Fallavier, France) and placed in a stereotaxic apparatus as previously described (Pioli et al., 2004). Animals were pre-treated with desipramine (25 mg/kg i.p.; Sigma) 30 min prior to the 6-OHDA injection in order to protect noradrenergic neurons. 6-OHDA (2 ␮g/␮l in 0.9% NaCl containing 0.01% ascorbic acid; Sigma) was injected first into the right and 1 week later into the left hemisphere. The following coordinates were employed for the SNc injection (4 ␮l, 0.5 ␮l/min): 5.2 mm posterior to bregma; ⫾2.2 mm from midline; ⫺7.7 mm from dura; and for the VTA injection (1 ␮l, 0.125 ␮l/min): 5.2 mm posterior to bregma; ⫾0.8 mm from midline; ⫺7.6 mm from dura. After each injection, the needle was left in position for 10 min to allow absorption of the injected solution and to minimize spread of the toxin along the needle tract. Sham-operated controls received an identical procedure with the exception that no toxin was infused but only vehicle (0.9% NaCl containing 0.01% ascorbic acid). This procedure was selected based on numerous pilot studies which reliably demonstrated selective lesions of the target region.

Experimental protocol The animals were allowed to recover for 3 weeks before behavioral testing commenced. This period of recovery has been described to produce stable 6-OHDA lesions (Schwarting and Huston, 1996a,b). All rats were subjected to a sequence of behavioral tests as summarized in Fig. 1. After completion of the behavioral evaluations, the rats were killed with an anesthetic overdose of urethane (Sigma) and the brains were rapidly removed. The selectivity of the 6-OHDA lesions was confirmed by stereological counting of mesencephalic neurons characterized by tyrosine hydroxylase (TH) immunochemistry and by measurement of DA and 5-HT content and their metabolites in cortical and striatal areas. For this purpose, the cortex and striatum were dissected, flash-frozen on dry ice and stored at ⫺80 °C. The caudal part of the brain containing the mesencephalon was postfixed in PFA 4% for 2 days at 4 °C and transferred to 20% sucrose in PBS buffer for cryoprotection. After 1 day, this part of the brain was frozen by immersion in ⫺40 °C isopentane and stored at ⫺80 °C. The entire mesencephalon was then cut at ⫺20 °C in 20 ␮m coronal sections. The sections were collected free floating for further immunohistochemical processing.

Behavioral testing Locomotor activity. Spontaneous motor activity was measured using a photocell apparatus (Panlab, Barcelona, Spain) as described by Diguet et al. (2004). The apparatus consisted of a squared open field (44⫻44⫻32 cm). On each side of the open

Fig. 1. Experimental schedule.

field, a frame placed at 3 cm height with 16 photocell beams per side ensured movement detection. Total motor activity was recorded over a period of 7 min on three consecutive days. Animals from sham and lesioned groups were tested in random order. After each test, the open field was cleaned with a moistened sponge. Results are expressed as mean activity of the 3 days⫾standard error of the mean (S.E.M.) and analyzed using a one-way analysis of variance (ANOVA) with group as independent variable. Stepping adjustments. Stepping adjustments were measured as described by Olsson et al. (1995). Briefly, the experimenter took the rat with one hand holding both hind limbs and the other hand holding one of the forelimbs. The free paw was placed in contact with a flat surface. The experimenter then moved the animal slowly sideways in forehand and backhand directions. The number of adjusting steps was counted for both paws in the backhand and forehand directions. The test was repeated twice a day for three consecutive days. Animals from sham and lesioned groups were tested in random order. Results are expressed as the mean number of adjusting steps during the six sessions⫾S.E.M. The means were subjected to a two-way ANOVA with group as the independent variable and paw as the dependent factor. When appropriate, ANOVAs were followed by post hoc t-tests corrected for multiple comparisons by the method of Bonferroni. Food consumption test. Food consumption was assessed as the time taken to eat 100 pellets as described by Baunez et al. (2002). First, the rats were familiarized with the environment and with the food to prevent neophobia. Animals were fed ad libitum during all the time of the test. The test started with 5 min of exploration in the bowl. Then, 100 sweet pellets (45 mg, Phymep, Paris, France) were introduced and the time taken by each rat to eat the pellets was measured in seconds. The bowl was then cleaned with water and left for a few minutes before the next rat was placed in the bowl with another 100 pellets. Animals from sham and lesioned groups were tested in random order. Results are expressed as the mean time required for eating the 100 pellets⫾S.E.M. The data were submitted to a one-way ANOVA with group as the independent variable. When appropriate,

E. Y. Pioli et al. / Neuroscience 153 (2008) 1213–1224 ANOVAs were followed by post hoc t-test corrected for multiple comparisons by the method of Bonferroni. Spontaneous alternation in Y maze. Spontaneous alternation was tested in a Y maze made of gray opaque Perspex® (Syma, Le Haillan, France). It consisted of three identical alleys (named A, B, C). Each was 40 cm long, 15 cm wide and had 20 cm high walls. The three alleys diverged at a 120° angle from a central point. Rats had free access to the three arms of the maze for 10 min, during which the sequence of arm-entrances was recorded as described by Taghzouti et al. (1986). The Y maze was cleaned with water between each run. Alternation was determined from successive entries of the three arms on overlapping triplet sets in which three different arms were entered. For example, a sequence of entries in arms ABC that consisted of ACBABACBAB would yield five alternations, namely, ACB (1), CBA (2), BAC (3), ACB (4) and CBA (5). The number of alternations was then divided by the number of alternation opportunities, i.e. the total arm entries minus two. This test was repeated for three consecutives days. Animals from the sham and lesioned groups were tested in random order. Data recorded during the 3 days were then averaged and expressed as mean⫾S.E.M. The raw data were submitted to a Kruskal-Wallis test with group as the independent factor. When appropriate, this test was followed by a post hoc Dunn’s test for multiple comparisons.

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experimental environment, except for a conspicuous striped pattern (21 cm⫻29.7 cm). A “videotrack” system (EthoVision, Wageningen, The Netherlands) allowed continuous monitoring of the position of the rat and also permitted selection of events occurring in a defined area of the open field. The open field was divided in four areas: northeast (NE), northwest (NW), southeast (SE) and southwest (SW) zones (see Fig. 2A, 2B, 2C). Three objects were used. They differed primarily by shape and texture. Object A was a Perspex tube (8 cm in diameter, 25 cm in height), object B was a wood squared parallelepiped (10⫻10 cm, 23 cm in height) and object C was a Perrier® glass bottle (7 cm in diameter, 28 cm in height). Animals were submitted individually to five successive 6-min sessions, separated by 3-min inter-session intervals, during which

Skilled paw reaching test. Skilled paw reaching was measured using the staircase test apparatus (Montoya et al., 1991) according to the modifications applied by Barneoud et al. (1995, 2000). The apparatus was developed to assess the independent forelimb use in skilled reaching and grasping tasks. Briefly, the apparatus consisted of a clear Perspex® chamber with a hinged lid. A narrow compartment with a central platform running along its length, creating a trough on either side, was connected to the chamber. A removable double staircase was inserted into the end of the box, sliding into the troughs on either side of the central platform. One week before the skilled paw reaching test, the rats were deprived of food and their body weight was stabilized at 85% of pre-test weight. Two 45 mg pellets (Phymep) were placed into each well of the double staircase of the test apparatus. The design is such that the rat can only reach pellets on one side of the staircase with its left paw and on the other side with the right paw thereby providing separate measures of performance for each limb. First, the rats were familiarized with the staircase boxes and the location of the pellets. Thereafter, the paw-reaching task commenced. For 2 weeks, the rats were placed in the test boxes once daily for 15 min. Animals from sham and lesioned groups were tested in random order. Three parameters were taken into account. The number of pellets eaten during the test period indicates the success in grasping and retrieving the pellets. The number of steps from which pellets have been removed (empty stair) provides an index of the attempts to reach the food. The number of missed pellets remaining at the end of the test on the floor of the compartment indicates the clumsiness, i.e. impaired sensori-motor coordination in grasping and retrieving the pellets. For the three parameters, data were subjected to a two-way ANOVA with group as the independent variable and session day as the dependent variable. ANOVAs were followed by post hoc t-test corrected for multiple comparisons by the method of Bonferroni. Object exploration task. The object exploration task consists of three successive phases: i.e. habituation, spatial change and non–spatial-change. We used a modified version of the paradigm used by Buhot and Naili (1995) without the non–spatial change assessment. The apparatus consisted of a gray Perspex®made circular open field (1.20 m in diameter). White curtains surrounded the open field in order to provide a visually uniform

Fig. 2. The object exploration task. Schematic representation of object configuration during successive sessions.

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they were returned to their cages. The rats were placed at the same initial position (i.e. coordinates and body orientation) in the open field arena. Animals from sham and lesioned groups were tested in random order. The open field was cleaned only at the end of the series of experiments for each given rat. For a schematic representation of the apparatus and object configuration during successive sessions see Fig. 2 (2A, 2B, 2C). The five individual sessions consisted of: Session 1 (served as a measure of locomotor activity, Fig. 2A). The arena was empty. Session 2 (served as object exploratory activity, Fig. 2B). A striped pattern was added to serve as a landmark within the environment. Three objects were placed in the arena at three corners of a fictitious square: object A in the NW zone, object B in the SE zone and object C in the NE zone. The last corner remained empty (i.e. the “neutral” area). Sessions 3 and 4 (measured exploratory habituation, Fig. 2B). As in session 2. Session 5 (assessed the reaction to spatial change, Fig. 2C). Object C was moved to the “neutral” area. All sessions were videotaped to be analyzed off-line afterward. The distance run during session one reflected locomotor activity. The raw data were submitted to ANOVAs for statistical analysis. Object exploration activity was assessed during session two by comparing the mean exploratory activity in the three zones NW, NE, SE containing the A, B, C objects, respectively [(NW⫹NE⫹ SE)/3] (object zone (OZ) in Fig. 6B) with the exploratory activity in the empty zone (EZ in Fig. 6B). These values were subjected to a two-way ANOVA with group and zone as the main variables. This analysis was followed, when appropriate, by post hoc t-tests corrected for multiple comparisons by the method of Bonferroni. Exploratory habituation was assessed by comparing the mean exploratory activity toward the objects [(NW⫹NE⫹SE)/3] from session 2 to session 4 (Fig. 6C). After repeated expositions of the same environment, object exploration should decrease from the first session with objects (session 2) to the session before the spatial change (session 4). Data were subjected to a two-way ANOVA with group and session as the main variables. When appropriate, the analysis was followed by post hoc t-tests corrected for multiple comparisons by the method of Bonferroni. The reaction to the spatial change was assessed by comparing the mean exploratory activity toward objects during sessions 3 and 4 with that during session 5 for the displaced object C in the SW zone (Fig. 6D) and the non-displaced objects A and B (Fig. 6E) separately. Data were subjected to a two-way ANOVA with group and session status as the main variables. When appropriate, post hoc t-tests corrected for multiple comparisons by the method of Bonferroni were conducted.

Assessment of lesion extent Stereological counting of tyrosine hydroxylase immunoreactive (TH-IR) neurons. Free-floating mesencephalic serial sections (20 ␮m) were processed for TH immunohistochemistry and subsequently counterstained with Cresyl Violet (Nissl staining) as previously described (Pioli et al., 2004). Unbiased stereological techniques (Gundersen et al., 1988; West, 1999) were used to estimate the number of mesencephalic DA-containing neurons. In every eighth section, the boundaries of the SNc and VTA were determined by examining the size and shape of the different TH-IR neuronal groups (German and Manaye, 1993; Oorschot, 1996; Paxinos and Watson, 1997). The volume was calculated by using the formula: V⫽⌺S td; where ⌺S is the sum of the surface areas, t is the average section thickness and d is the number of slices between two consecutives analyzed sections measured (Theoret et al., 1999). A total of 11 sections were counted. The optical dissectors were distributed using a systematic sampling scheme. In the SNc, dissectors (80 ␮m length, 60 ␮m width) were sepa-

rated from each other by 50 ␮m (x) and 40 ␮m (y); and in the VTA dissectors (120 ␮m length, 80 ␮m width) were separated from each other by 75 ␮m (x) and 65 ␮m (y). The following formula was used to estimate the number of TH-IR neurons: N⫽V(SNc) (⌺Q⫺/ ⌺V(dis)); where N is the estimation of cell number, V is the volume of the SNc or VTA, ⌺Q⫺ is the number of cells counted in the dissectors, and ⌺V(dis) is the total volume of all the dissectors (Theoret et al., 1999). Mean estimated number of neurons and S.E.M. were then calculated for each group. Data were subjected to a two-tailed Student’s t-test. Neurochemical analysis. The extent of striatal and cortical DA denervation was assessed by measuring levels of DA, 3,4dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in the dorsal and ventral part of the striatum as well as in the frontal cortex using high-pressure liquid chromatography (HPLC) with electrochemical detection as previously described (Bezard et al., 2001; Pioli et al., 2004). Frontal cortex, anterior and posterior striatum were dissected and homogenized in 1 ml of 0.1 M perchloric acid. Protein concentrations were measured according to Lowry et al. (1951) with human serum albumin as standard. A volume of 20 ␮l was injected with a manual injection valve (RH 7725, Rheodyne, Cotati, USA) and separated by HPLC on a reverse phase column (ProntoSil 120 C18 SH, 5 ␮m, 150⫻3 mm, VDS Optilab, Germany). The mobile phase consisted of 100 mM sodium dihydrogen phosphate, 0.8 mM EDTA (disodium salt), 1.3 mM 1-octanesulfonic acid (sodium salt) and the pH was adjusted to 3.9 with phosphoric acid. The buffer solution was mixed with 40 ml 2-propanol and distilled water was added to a final volume of 1000 ml. DA, DOPAC, HVA, 5-HT and HIAA were quantified electrochemically by a glassy carbon electrode at a potential of 0.8 V (Ag/AgCl electrode). The chromatograms were recorded with a chromatographic data system (CSW 1.7, Data Apex Ltd., Czech Republic) and quantified by determination of peak areas in relation to the known concentration of respective standards. Mean and S.E.M. values were calculated for both striatal and cortical levels. Neurochemical data were submitted to a two-tailed Student’s t-test.

RESULTS Extent of lesion Given the aim of the present study, it was imperative that our lesions be large enough to cover the target area, but strictly restricted to either the SNc or the VTA. We have previously shown with the same model that (i) bilateral lesions were highly comparable on both hemispheres and that (ii) partial lesions of the SNc and VTA were restricted to the targeted nucleus (Pioli et al., 2004). The extent of 6-OHDA-induced lesion in SNc and VTA was quantified by unbiased stereological counting of the total number of TH-IR cell bodies in SNc and in VTA and by the neurochemical assessment of the content of DA, 5-HT and theirs metabolites in the dorsal striatum, ventral striatum and frontal cortex (see Table 1). Bilateral 6-OHDA injections in the SNc induced a significant decrease (i) in the number of TH-IR neurons in the SNc (t⫽6.28, P⬍0.05), but not in the VTA (t⫽0.35, P⬎0.05) and (ii) in DA (t⫽6.34, P⬍0.05), DOPAC (t⫽5.03, P⬍0.05) and HVA content (t⫽3.00, P⬍0.05) along with an increase in 5-HT (t⫽4.70, P⬍0.05) and HIAA content in dorsal striatum (t⫽3.72, P⬍0.05; Table 1). Bilateral 6-OHDA injections in the VTA induced a significant decrease in the number of TH-IR neurons in the

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Table 1. Effect of bilateral partial 6-OHDA-induced lesions in the SNc or the VTA (i) on the estimated number of TH-IR neurons in the SNc and the VTA, (ii) on DA, DOPAC, HVA, 5-HT and 5-HIAA content in the frontal cortex, in the ventral striatum and in the dorsal striatum and (iii) on global locomotion Group TH-IR neurons SNC VTA HPLC (nmol/g of protein) Frontal cortex DA DOPAC HVA 5-HT HIAA Ventral striatum DA DOPAC HVA 5-HT HIAA Dorsal striatum DA DOPAC HVA 5-HT HIAA Actimetry (Mean beam break)

SNc Sham

SNc

VTA Sham

VTA

8516⫾1037 8282⫾933

2938⫾458* 7971⫾718

8525⫾1099 8264⫾550

8045⫾635 6165⫾737*

11.6⫾1.1 5.5⫾0.7 8.1⫾4.3 2.4⫾0.2 2.3⫾0.1

5.4⫾0.9* 1.8⫾1.9* 2.4⫾0.2 9.8⫾1.3* 10.3⫾1.3*

13.1⫾1.5 7.7⫾0.7 3.4⫾0.4 1.7⫾0.1 2.8⫾0.2

10.8⫾2.1 3.7⫾0.6* 3.2⫾0.4 2.6⫾0.8 3.2⫾0.56

157.7⫾21.9 53.5⫾10.3 20.4⫾2.9 4.8⫾0.5 9.9⫾1.6

93.9⫾14.6* 31.3⫾4.9 15.2⫾2.1 16.9⫾2.6* 21.7⫾2.3*

108.2⫾22.4 58⫾7.8 18.6⫾3 4.2⫾0.9 10⫾0.6

141.4⫾17.5 46.1⫾3 19.2⫾2.2 4.9⫾0.7 10.8⫾1.2

340⫾11.4 77.2⫾2.6 29.7⫾1.4 3.1⫾0.2 10.1⫾0.4 2379⫾371

143.2⫾40.7* 33.3⫾6.5* 16.9⫾3.2* 11.1⫾1.3* 20.8⫾2.2* 2357⫾200

313.3⫾18.3 89⫾7.7 29.5⫾1.9 3.4⫾0.4 13.4⫾1 2606⫾418

379.9⫾31.9 82.8⫾6.7 33.2⫾3.3 4.6⫾1 14.1⫾1.7 2386⫾407

Data are expressed as mean⫾S.E.M. * P⬍0.05, a significant difference with the respective sham-operated animals.

VTA (t⫽2.75, P⬍0.05), but not in SNc (t⫽0.67, P⬎0.05; Table 1). The VTA lesions had no significant effect on DA (frontal cortex: t⫽0.86, P⬎0.05; ventral striatum: t⫽1.82, P⬎0.05; dorsal striatum: t⫽0.24, P⬎0.05), HVA (frontal cortex: t⫽0.44, P⬎0.05; ventral striatum: t⫽0.16, P⬎0.05; dorsal striatum: t⫽0.93, P⬎0.05), 5-HT (frontal cortex: t⫽1.06, P⬎0.05; ventral striatum: t⫽0.85, P⬎0.05; dorsal striatum: t⫽1.13, P⬎0.05) and HIAA levels (frontal cortex: t⫽0.64, P⬎0.05; ventral striatum: t⫽0.58, P⬎0.05; dorsal striatum: t⫽0.36, P⬎0.05; Table 1). However, VTA lesion reduced DOPAC content in the frontal cortex (t⫽4.28, P⬍0.05), suggesting that DA homeostasis was disrupted in these animals. Behavioral assessment Locomotor activity. To ensure that our partial 6-OHDA lesions were not associated with changes in locomotion, horizontal activity was measured in an open field. As expected, there was no significant difference in horizontal activity between groups (F(3,28)⫽0.19, P⬎0.05, Table 1), validating the partial lesion approach, i.e. partial lesion of either the SNc or the VTA did not affect locomotion. Stepping adjustments. The stepping test assessed fine motor control. An overall estimation showed differences between treatment groups on stepping adjustment in all conditions (forehand: F(3,57)⫽35.8; P⬍0.05; backhand: F(3,57)⫽63.5; P⬍0.05). Further post hoc testing revealed a significant decrease in the performance in the

stepping test for the forehand (see Fig. 3A) and for the backhand direction (see Fig. 3B) in animals with partial bilateral SNc lesion. By contrast, partial bilateral VTA lesion had no effect on the number of stepping adjustments in either condition (see Fig. 3A and 3B). Food consumption test. An overall estimation indicated differences between experimental groups on the time needed to eat the 100 pellets (F(3,28)⫽4.4, P⬍0.05). Post hoc comparisons revealed that rats with partial bilateral SNc lesion were significantly slower to eat 100 food pellets than shams (see Fig. 4A). In contrast, rats with partial bilateral VTA lesions consumed food reward pellets as fast as shams (see Fig. 4A). Spontaneous alternation in Y maze. As expected, rats alternated at levels significantly above chance indicating their readiness to explore novel environmental stimuli (see for review Dember and Fowler, 1958; Lalonde, 2002; Hughes, 2004). An overall effect was found on the spontaneous alternation in the Y maze (F(3,28)⫽5.4, P⬍0.05). A post hoc comparison revealed that partial bilateral VTA lesioned rats made significantly less spontaneous alternations that their sham controls (see Fig. 4B). In contrast, partial bilateral SNc-lesioned rats alternated in the Y maze at a comparable level as their sham controls (see Fig. 4B). Skilled paw reaching test. The skilled paw-reaching test in the rat is a complex sensorimotor task analogous to skilled reaching in humans (Whishaw and Pellis, 1990). The criteria of success, attempts and sensorimotor coor-

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indicates that each group exhibited increased exploratory activity when facing objects during session 2. The results of the object exploration phase (session 2; Fig. 2B) revealed that the four groups displayed an increase in the distance moved in the objects zone compared with the empty zone (see Fig. 6B). These observations were confirmed by the two-way ANOVA with repeated measures (group⫻zone), which revealed a significant effect of zone (F(1,57)⫽94.1, P⬍0.05), but not of group (F(3,28)⫽1.51, P⬎0.05). The ANOVA also revealed a significant group⫻ zone interaction (F(6,83)⫽3.98, P⬍0.05). Every group showed an object-directed exploration, i.e. an increase of the exploration activity in the object zone in comparison to the empty zone (P⫽0.0001). From sessions 2– 4, rats were exposed to the initial configuration of objects as shown in Fig. 2B, which allowed evaluation of the exploration habituation. Fig. 6C represents the time-course of the average mean exploratory activity in areas containing objects for sessions 2– 4 ([NW⫹NE⫹SE]/3). As can be seen in Fig. 6C, each group

Fig. 3. The stepping test. Effect of bilateral partial 6-OHDA-induced lesions in the SNc or the VTA on adjusting steps in forehand (A) and backhand (B) direction. Black bar, hatched bar, gray bar and open bar correspond to SNc sham group, SNc lesioned group, VTA sham group and VTA lesioned group, respectively. Data are expressed as mean⫾S.E.M. * Denotes a significant difference with the respective sham-operated animals, P⬍0.05.

dination for the different phases of the paw reaching were evaluated. An overall estimation between groups displayed significant differences in the success (F(3,57)⫽9.1, P⬍0.05) and the attempt (F(3,57)⫽10.0, P⬍0.05), but not in the sensorimotor coordination (F(3,57)⫽0.5, P⬎0.05) of paw reaching. Post hoc testing revealed increased success (Fig. 5A) and attempts (Fig. 5B) in rats with partial bilateral VTA lesion, while no difference in sensorimotor coordination (Fig. 5C) was detected in comparison to shams. Rats with partial bilateral SNc lesions did not exhibit significant deficits in success (Fig. 5A), attempts (Fig. 5B) or sensorimotor coordination (Fig. 5C). Object exploration task. During the session 1, the rat was placed into the empty open field (Fig. 2A) to allow the evaluation of locomotor activity. Fig. 6A depicts the mean exploratory activity across the arena during sessions 1 and 2. The two-way ANOVA analysis revealed a significant effect of session (F(1,57)⫽12.14, P⬍0.05) but not of lesion (F(3,28)⫽1.53, P⬎0.05), nor a significant interaction between the two (F(3,57)⫽1.8, P⬎0.05). The session effect

Fig. 4. (A) The food consumption test. (B) The spontaneous alternation. Effect of bilateral partial 6-OHDA-induced lesions in the SNc or in the VTA on the 100 pellets test (A) and on the percentage of spontaneous alternation (B). Black bar, hatched bar, gray bar and open bar correspond to SNc sham group, SNc lesioned group, VTA sham group and VTA lesioned group, respectively. Data are expressed as mean⫾S.E.M. * Denotes a significant difference with the respective sham-operated animals, P⬍0.05.

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moved from its initial position (NE, Fig. 2B) to the empty location (SW, Fig. 2C). The other objects were not manipulated. In this session, two categories of objects are considered separately for analysis: the displaced object (C) and the nondisplaced objects (A and B). Reaction to spatial change was assessed by the mean exploratory activity zone for both sessions 3 and 4 compared with session 5 in the SW zone (displaced object, Fig. 6D) or in the NW and SE zone (nondisplaced objects, Fig. 6E). The results demonstrated that all groups showed an increase in exploratory activity of the moved object (Fig. 6D), while they explored the nondisplaced object at a constant level throughout the session (Fig. 6E). These observation were confirmed by a two-way ANOVA with repeated measures on the displaced objects (group⫻session) which revealed an effect of session (F(3,28)⫽20.9, P⬍0.05), but no significant effect of group (F(1,57)⫽1.74, P⬎0.05), nor a significant interaction between those variables (F(6,83)⫽0.25, P⬎0.05). A two-way ANOVA with repeated measures on the non-displaced familiar objects (group⫻session) revealed no significant effect of session (F(3,28)⫽2.64, P⬎0.05), no significant effect of group (F(1,57)⫽1.86, P⬎0.05), and no significant interaction between both variables (F(6,83)⫽0.25, P⬎0.05). In summary, during the first session of the object exploration task no group differences were observed in global activity in the empty area (similar as shown in the square open field). During session 2, rats from each group exhibited an object-directed increase of their activities in presence of the object configuration. All groups showed habituation toward the objects after a number of exposures (session 2– 4) and they were able to react to a spatial change during session 5, which cannot be ascribed to a non-specific increase in arousal.

DISCUSSION

Fig. 5. The skilled paw reaching test. Effect of bilateral partial 6-OHDA lesions in the SNc or in the VTA on success (A), attempts (B) and clumsiness (C) of paw reaching performed during the sixteenth testing day. Performances of the rats in the paw reaching test are expressed as mean⫾S.E.M. number of eaten pellets (A), number of steps without pellets (B), and number of missed pellets on the floor (C). Black bar, hatched bar, gray bar and open bar correspond to SNc sham group, SNc-lesioned group, VTA sham group and VTA-lesioned group, respectively. * Denotes a significant difference with the respective sham-operated animals, P⬍0.05.

showed a habituation response, i.e. decrease of exploration activity across experimental sessions. A two-way ANOVA with repeated measures revealed a significant effect of group (F(3,28)⫽3.14, P⬍0.05) and a significant effect of session (F(2,83)⫽9.91, P⬍0.05), but no significant interaction between both variables (F(6,83)⫽1.34, P⬎0.05). Finally, in session 5, the rats are exposed to a new spatial configuration of the objects. A familiar object was

The present study shows that distinctive patterns of behavioral impairment are obtained following selective lesion of either the SNc or VTA. Our results imply that the SNc might be primarily involved in motor-related akinesia behavior, while the VTA is preferentially associated with a perseverative behavior. Methodological considerations The aim of this study was to compare the effects of restricted lesions of two major dopaminergic mesencephalic pathways on different behavioral tasks. To examine the extent of cognitive impairment in 6-OHDA-lesioned rats, we employed a bilateral lesion model acting on either the SNc or the VTA, rather than the different dopaminergic target structures (e.g. striatum, nucleus accumbens, or prefrontal cortex). The SNc and VTA projections are topographically organized (Haber et al., 2000). However, an extensive overlap exists in theirs projections (Fallon and Moore, 1978b; Loughlin and Fallon, 1984). Therefore, to identify distinct effects of SNc and VTA projections on motor and cognitive processes in overlapping target areas, the extent of SNc and VTA lesion had to be restricted to the

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Fig. 6. The object exploration task. Overview of the performances in object exploration task for the different group. (A) Locomotor activity during the first two sessions, data are expressed as mean (⫾S.E.M.) distance run across the arena by each group. (B) Object exploration activity during session 2 was assessed by comparing the mean exploratory activity in the three zones NW, NE, SE containing the A, B, C objects, respectively ([NW⫹NE⫹SE]/3) with the exploratory activity in the empty zone SW. Data represent mean (⫾S.E.M.) distance run. (C) Object habituation from session 2 to session 4 represents the time-course of the average mean exploratory activity in areas containing objects ([NW⫹NE⫹SE]/3) for sessions 2– 4. Data are expressed as mean (⫾S.E.M.) distance run. (D) Reaction to spatial change. Data are expressed as mean (⫾S.E.M.) distance run of SW area for session 3– 4 mean compared with session 5. (E) Non-specific activity around the non-displaced familiar object A and B. Data are expressed as mean (⫾S.E.M.) distance run of NW and SE area for session 3– 4 mean compared with session 5. Black line, black broken line, gray line and gray broken line correspond to SNc sham group, SNc-lesioned group, VTA sham group and VTA-lesioned group, respectively.

injected nucleus. Furthermore, partial lesion of the SNc or the VTA should not impair locomotor activity since it is impossible to investigate cognitive functions in animals

with severe motor deficits. Indeed, extensive SNc lesion (⬎80%) induce locomotor hypo-activity (Sakai and Gash, 1994), while extensive VTA lesion induce hyper-activity

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(Le Moal and Simon, 1991). Moreover, extensive bilateral lesions of the SNc and VTA require intensive nursing of the rats as they become aphagic and adipsic (Ungerstedt, 1971; Le Moal and Simon, 1991; Sakai and Gash, 1994). Thus, the majority of studies in rat models of parkinsonism are based on unilateral lesion and only few studies have used bilateral lesion without systematically assessing the effects on non-motor functions (see for review Schwarting and Huston, 1996a,b). In the present study, rats with partial bilateral 6-OHDA lesion of the SNc or VTA did not demonstrate any deficits in locomotion, providing a valuable model for studying cognitive deficits (Schneider and Kovelowski, 1990). Effect of SNc partial bilateral lesions Rats with partial bilateral 6-OHDA lesion of the SNc had an impaired performance in the stepping test and in the 100 pellets test, but displayed normal locomotion and spontaneous alternation, normal performance in skilled paw reaching, and a level of object exploration similar to that observed in the sham group (Table 2). Motivation behaviors have been traditionally divided into preparatory and consummatory acts. Preparatory behavior consists of the approach, exploration and instrumental responding for a reward, whereas consummatory behavior includes eating and drinking. The 100 pellets task only allows the evaluation of consummatory behavior, which mainly involves the sensorimotor system and DA transmission. Rats with partial bilateral SNc lesion had an impaired performance in the motivational task, the 100 pellets test. Our results are in accordance with previous studies showing that DA depletion of the dorsolateral striatum, but not of the dorsomedial part, disrupts the ability to retrieve small food pellets by using the forepaw (Sabol et al., 1985) and that extensive DA depletion of the forebrain produces aphagia and adipsia (Ungerstedt, 1971; Zigmond and Stricker, 1972). Although subtle deficits in skilled grasping and eating (an automated skill) could account for the deficits in this task, the dorsolateral part of striatum is known as well to underpin the development of habit response (White, 1997; Jog et al., 1999; Yin et al., 2004; Faure et al., 2005; Tang et al., 2007). Even if PD was seen so far as primarily affecting motor function, there has Table 2. Summary of the differential behavioral effects induced by partial bilateral lesions of VTA or SNc Behavioral tasks

Locomotor activity Stepping adjustments Food consumption Spontaneous alternation Skilled paw reaching Object exploration

Lesioned groups SNc

VTA

ns 2 2 ns ns ns

ns ns ns 2 1 ns

“1” Denotes a significant increase of performance, whereas “2” symbolizes a significant decrease of performance compared to sham animals; ns signifies that the performances are non-significantly different from the sham animals.

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been an increasing awareness of the associated nonmotor symptoms such ad cognitive deficits (Owen et al., 1997; Marinus et al., 2003; Mollion et al., 2003). Those cognitive deficits would even be present before the motor manifestations. For example, loss of DA in the striatum impairs habit formation (Salmon and Butters, 1995; Knowlton et al., 1996; White, 1997; Hay et al., 2002) and implicit memory (Saint-Cyr et al., 1988; Roncacci et al., 1996; Koenig et al., 1999) in PD patients. Our results further ground the possibility that those symptoms occur earlier in the disease process than previously thought. A recent study showed that the stepping test appears to be a reliable tool to assess the persisting subtle motor dysfunction consequent to dopaminergic deficiency: the degree of altered performance was well correlated with the severity of the underlying dopaminergic lesion and this motor abnormality was improved under L-DOPA treatment (Paille et al., 2007). In our study, the performance of the partial bilateral SNc-lesioned rats on the stepping test altered in comparison with that of the sham rats. This result is in line with the behavioral characterization of the 6-OHDA unilateral lesioned (Lee et al., 1996; Kirik et al., 1998; Roedter et al., 2001; Ogura et al., 2005) and more importantly of the 6-OHDA bilateral lesioned rats as experimental models of PD (Paille et al., 2007). Effect of VTA partial bilateral lesions In the present study, rats with partial bilateral VTA lesion displayed no motor deficits, but behavioral abnormalities in spontaneous alternation and in skilled paw reaching (Table 2). During free exploration of the Y maze, a normal rat alternates arm visits to acquire or process novel information. Spontaneous alternation quantifies an unlearned response in rats, which involves different processes such as perception, attention, memory and motivation (see for review Dember and Fowler, 1958; Lalonde, 2002; Hughes, 2004). Here, rats with partial bilateral VTA lesions lost this innate behavior; their alternation performance remained around chance level. A similar decrease in spontaneous alternation has been described in the Y maze and in the eight-arm radial maze for rats with mesocorticolimbic pathway lesions (Taghzouti et al., 1986), but also for amphetamine-treated rats (Y maze (Kokkinidis, 1987); eight-arm radial maze (Loh et al., 1993; Holter et al., 1996)) and for rats sensitized to the DA agonist quinpirole (T maze (Einat and Szechtman, 1995)). A deficit in spontaneous alternation is seen as an increased tendency to repeat a choice between two arms, which could be interpreted as impairment in spatial memory. However, rats with partial bilateral VTA lesion showed normal performance in the object exploration task, reaching the sham group exploration level of the objects presentation and of the spatial change. This result suggested that VTA-lesioned rats are able to remember a spatial object configuration and to properly orient their behavior to a spatial change. As object exploration like the spontaneous alternation task is an ethologically based test without involving reward delivery and taking advantage of animal’s willingness to explore novel

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environmental stimuli we can compare both results. Because VTA-lesioned rats were not impaired in the object exploration task, their deficit in the spontaneous alternation task cannot be ascribed to some spatial memory impairments. Yadin et al. (1991) proposed that the reduction in spontaneous alternation in a T-maze could serve as an animal model of obsessive-compulsive behavior. They interpreted the finding that rats keep returning to the same arm as a form of compulsive “checking.” Furthermore, bilaterally partially VTA-lesioned rats displayed a higher paw reaching performance compared with sham animals, indicating an abnormal response. This may be interpreted as the result of increased motivation. However, VTA-lesioned rats did not exhibit any performance differences in the 100 pellets test compared with sham animals which is in agreement with no effect of VTA-lesion on sucrose consumption (Shimura et al., 2002; MartinezHernandez et al., 2006). Another explanation for the higher performances may be that VTA-lesioned rats failed to inhibit the response, suggesting perseveration in the task. Such perseveration could be the result of the competition between the target areas of VTA and SNc for control of behavior. While VTA projects mainly to portions of the striatum controlling goal-directed actions, SNc projects to areas controlling habits, then both of these results could be a sign of enhanced automation or habit formation (perseverance or stimulus response learning in the Y maze task and enhanced automation of the reaching in the skill task). We initially hypothesized that VTA lesions might result in some form of cognitive akinesia, instead we found that the lesions led to the occurrence of perseveration behavior. Brown and Marsden (1990) have shown that Parkinson patients have problems changing response strategy which gives rise to some thought stereotypies. This paradox especially occurs in some cases of PD associated with an obsessive– compulsive disorder (Mallet et al., 2002). Perseveration behavior can also be observed in schizophrenic patients (Crider, 1997). This kind of perseveration may be due to an attention deficit (Goldman et al., 1991) and might reflect a dysfunction of the frontal (Weinberger et al., 1988; Morice, 1990; Shallice et al., 1991) or the prefrontal cortices (Liddle and Morris, 1991), which both are innervated by the mesocortical pathway. This lack of flexibility can interfere with action planning and consequently can produce inappropriate behavior. The fact remains that those behavioral changes are expressed even though the 6-OHDA-induced lesion of the VTA is only 25% without any change in PFC DA levels. While DA levels were not affected (despite a trend), there was a significant loss of DOPAC suggestive of a breakdown of DA homeostasis in the PFC. The mesocortical pathway is composed of both dopaminergic and nondopaminergic, GABAergic, projections (Carr and Sesack, 2000a,b). GABAergic inhibition in the frontal cortex is critical for controlling the timing of neuronal activities that process ongoing information and plan appropriate actions at a future time (Constantinidis et al., 2002). A majority of the projections of the VTA to the frontal cortex is nondopaminergic. In fact, only 30% of the mesocortical path-

way is dopaminergic while 85% of the mesolimbic pathway to ventral striatum contains DA (Swanson, 1982). It is likely that the GABA containing axons correspond to the 6-OHDA resistant, fast-conducting fibers observed by Thierry et al. (1980). Since this GABA-containing mesocortical pathway provide the substrate for both inhibitory and disinhibitory influences on PFC neuronal activity under DA regulation, a subtle tough significant breakdown of DA homeostasis as in our VTA-lesioned animals might explain that PFC activity changes in a way leading to the perseverative behavior (Weinberger et al., 1986; Carter et al., 1998). The study of the differential behavioral effects of partial bilateral lesions of VTA or SNc and of their mechanistic basis requires further attention in order to elucidate the respective role of the structures and of the neurotransmitter interplays. Acknowledgments—We wish to thank L. Cardoit and S. Dovero for the technical assistance. E.Y.P. was supported by a fellowship from the Ministère de la Recherche et de la Technologie (MRT) and W.M. was a Marie Curie Fellow of the European Community (HPMF-2001-01300). The University Victor Segalen, the CNRS and the IFR (INSERM No. 8; CNRS No. 13) funded this study.

REFERENCES Barneoud P, Descombris E, Aubin N, Abrous DN (2000) Evaluation of simple and complex sensorimotor behaviours in rats with a partial lesion of the dopaminergic nigrostriatal system. Eur J Neurosci 12:322–336. Barneoud P, Parmentier S, Mazadier M, Miquet JM, Boireau A, Dubedat P, Blanchard JC (1995) Effects of complete and partial lesions of the dopaminergic mesotelencephalic system on skilled forelimb use in the rat. Neuroscience 67:837– 848. Baunez C, Amalric M, Robbins TW (2002) Enhanced food-related motivation after bilateral lesions of the subthalamic nucleus. J Neurosci 22:562–568. Bermanzohn PC, Siris SG (1992) Akinesia: a syndrome common to parkinsonism, retarded depression, and negative symptoms of schizophrenia. Compr Psychiatry 33:221–232. Bezard E, Dovero S, Prunier C, Ravenscroft P, Chalon S, Guilloteau D, Bioulac B, Brotchie JM, Gross CE (2001) Relationship between the appearance of symptoms and the level of nigrostriatal degeneration in a progressive MPTP-lesioned macaque model of Parkinson’s disease. J Neurosci 21:6853– 6861. Brown RG, Jahanshahi M (1996) Cognitive-motor dysfunction in Parkinson’s disease. Eur Neurol 36 (Suppl 1):24 –31. Brown RG, Marsden CD (1990) Cognitive function in Parkinson’s disease: from description to theory. Trends Neurosci 13:21–29. Buhot MC, Naili S (1995) Changes in exploratory activity following stimulation of hippocampal 5-HT1A and 5-HT1B receptors in the rat. Hippocampus 5:198 –208. Carr DB, Sesack SR (2000a) Dopamine terminals synapse on callosal projection neurons in the rat prefrontal cortex. J Comp Neurol 425:275–283. Carr DB, Sesack SR (2000b) GABA-containing neurons in the rat ventral tegmental area project to the prefrontal cortex. Synapse 38:114 –123. Carter CS, Perlstein W, Ganguli R, Brar J, Mintun M, Cohen JD (1998) Functional hypofrontality and working memory dysfunction in schizophrenia. Am J Psychiatry 155:1285–1287. Constantinidis C, Williams GV, Goldman-Rakic PS (2002) A role for inhibition in shaping the temporal flow of information in prefrontal cortex. Nat Neurosci 5:175–180.

E. Y. Pioli et al. / Neuroscience 153 (2008) 1213–1224 Crider A (1997) Perseveration in schizophrenia. Schizophr Bull 23:63–74. Damier P, Hirsch EC, Agid Y, Graybiel AM (1999a) The substantia nigra of the human brain. I. Nigrosomes and the nigral matrix, a compartmental organization based on calbindin D(28K) immunohistochemistry. Brain 122 (Pt 8):1421–1436. Damier P, Hirsch EC, Agid Y, Graybiel AM (1999b) The substantia nigra of the human brain. II. Patterns of loss of dopamine-containing neurons in Parkinson’s disease. Brain 122 (Pt 8):1437–1448. De Ajuriaguerra J (1975) The concept of akinesia. Psychol Med 5:129 –137. Dember WN, Fowler H (1958) Spontaneous alternation behavior. Psychol Bull 55:412– 428. Diguet E, Fernagut PO, Wei X, Du Y, Rouland R, Gross C, Bezard E, Tison F (2004) Deleterious effects of minocycline in animal models of Parkinson’s disease and Huntington’s disease. Eur J Neurosci 19:3266 –3276. Einat H, Szechtman H (1995) Perseveration without hyperlocomotion in a spontaneous alternation task in rats sensitized to the dopamine agonist quinpirole. Physiol Behav 57:55–59. Fallon JH, Koziell DA, Moore RY (1978) Catecholamine innervation of the basal forebrain. II. Amygdala, suprarhinal cortex and entorhinal cortex. J Comp Neurol 180:509 –532. Fallon JH, Moore RY (1978a) Catecholamine innervation of the basal forebrain. III. Olfactory bulb, anterior olfactory nuclei, olfactory tubercle and piriform cortex. J Comp Neurol 180:533–544. Fallon JH, Moore RY (1978b) Catecholamine innervation of the basal forebrain. IV. Topography of the dopamine projection to the basal forebrain and neostriatum. J Comp Neurol 180:545–580. Faure A, Haberland U, Conde F, El Massioui N (2005) Lesion to the nigrostriatal dopamine system disrupts stimulus-response habit formation. J Neurosci 25:2771–2780. Fuxe K (1965) Evidence for the existence of monoamine neurons in the central nervous system. IV. Distribution of monoamine nerve terminals in the central nervous system. Acta Physiol Scand 64 (Suppl) 247:237. German DC, Manaye KF (1993) Midbrain dopaminergic neurons (nuclei A8, A9, and A10): three-dimensional reconstruction in the rat. J Comp Neurol 331:297–309. Goldman RS, Axelrod BN, Tandon R, Berent S (1991) Analysis of executive functioning in schizophrenics using the Wisconsin Card Sorting Test. J Nerv Ment Dis 179:507–508. Gundersen HJ, Bendtsen TF, Korbo L, Marcussen N, Moller A, Nielsen K, Nyengaard JR, Pakkenberg B, Sorensen FB, Vesterby A, et al. (1998) Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 96: 379 –394. Haber SN, Fudge JL (1997) The primate substantia nigra and VTA: integrative circuitry and function. Crit Rev Neurobiol 11:323–342. Haber SN, Fudge JL, McFarland NR (2000) Striatonigrostriatal pathways in primates form an ascending spiral from the shell to the dorsolateral striatum. J Neurosci 20:2369 –2382. Hay JF, Moscovitch M, Levine B (2002) Dissociating habit and recollection: evidence from Parkinson’s disease, amnesia and focal lesion patients. Neuropsychologia 40:1324 –1334. Holter SM, Tzschentke TM, Schmidt WJ (1996) Effects of amphetamine, morphine and dizocilpine (MK-801) on spontaneous alternation in the 8-arm radial maze. Behav Brain Res 81:53–59. Hughes RN (2004) The value of spontaneous alternation behavior (SAB) as a test of retention in pharmacological investigations of memory. Neurosci Biobehav Rev 28:497–505. Jog MS, Kubota Y, Connolly CI, Hillegaart V, Graybiel AM (1999) Building neural representations of habits. Science 286:1745–1749. Kirik D, Rosenblad C, Bjorklund A (1998) Characterization of behavioral and neurodegenerative changes following partial lesions of the nigrostriatal dopamine system induced by intrastriatal 6hydroxydopamine in the rat. Exp Neurol 152:259 –277.

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Knowlton BJ, Mangels JA, Squire LR (1996) A neostriatal habit learning system in humans. Science 273:1399 –1402. Koenig O, Thomas-Anterion C, Laurent B (1999) Procedural learning in Parkinson’s disease: intact and impaired cognitive components. Neuropsychologia 37:1103–1109. Kokkinidis L (1987) Amphetamine-elicited perseverative and rotational behavior: evaluation of directional preference. Pharmacol Biochem Behav 26:527–532. Lalonde R (2002) The neurobiological basis of spontaneous alternation. Neurosci Biobehav Rev 26:91–104. Le Moal M, Simon H (1991) Mesocorticolimbic dopaminergic network: functional and regulatory roles. Physiol Rev 71:155–234. Lee CS, Sauer H, Bjorklund A (1996) Dopaminergic neuronal degeneration and motor impairments following axon terminal lesion by instrastriatal 6-hydroxydopamine in the rat. Neuroscience 72: 641– 653. Lee RG (1989) Pathophysiology of rigidity and akinesia in Parkinson’s disease. Eur Neurol 29:13–18. Liddle PF, Morris DL (1991) Schizophrenic syndromes and frontal lobe performance. Br J Psychiatry 158:340 –345. Lindvall O, Bjorklund A (1974) The organization of the ascending catecholamine neuron systems in the rat brain as revealed by the glyoxylic acid fluorescence method. Acta Physiol Scand Suppl 412:1– 48. Loh EA, Smith AM, Roberts DC (1993) Evaluation of response perseveration of rats in the radial arm maze following reinforcing and nonreinforcing drugs. Pharmacol Biochem Behav 44:735–740. Loughlin SE, Fallon JH (1984) Substantia nigra and ventral tegmental area projections to cortex: topography and collateralization. Neuroscience 11:425– 435. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193: 773–780. Mallet L, Mesnage V, Houeto JL, Pelissolo A, Yelnik J, Behar C, Gargiulo M, Welter ML, Bonnet AM, Pillon B, Cornu P, Dormont D, Pidoux B, Allilaire JF, Agid Y (2002) Compulsions, Parkinson’s disease, and stimulation. Lancet 360:1302–1304. Marinus J, Visser M, Verwey NA, Verhey FR, Middelkoop HA, Stiggelbout AM, van Hilten JJ (2003) Assessment of cognition in Parkinson’s disease. Neurology 61:1222–1228. Martinez-Hernandez J, Lanuza E, Martinez-Garcia F (2006) Selective dopaminergic lesions of the ventral tegmental area impair preference for sucrose but not for male sexual pheromones in female mice. Eur J Neurosci 24:885– 893. Mollion H, Ventre-Dominey J, Dominey PF, Broussolle E (2003) Dissociable effects of dopaminergic therapy on spatial versus nonspatial working memory in Parkinson’s disease. Neuropsychologia 41:1442–1451. Montoya CP, Campbell-Hope LJ, Pemberton KD, Dunnett SB (1991) The “staircase test”: a measure of independent forelimb reaching and grasping abilities in rats. J Neurosci Methods 36:219 –228. Morice R (1990) Cognitive inflexibility and pre-frontal dysfunction in schizophrenia and mania. Br J Psychiatry 157:50 –54. Nieoullon A (2002) Dopamine and the regulation of cognition and attention. Prog Neurobiol 67:53– 83. Nieoullon A, Coquerel A (2003) Dopamine: a key regulator to adapt action, emotion, motivation and cognition. Curr Opin Neurol 16:S3–S9. Ogura T, Ogata M, Akita H, Jitsuki S, Akiba L, Noda K, Hoka S, Saji M (2005) Impaired acquisition of skilled behavior in rotarod task by moderate depletion of striatal dopamine in a pre-symptomatic stage model of Parkinson’s disease. Neurosci Res 51:299 –308. Olsson M, Nikkhah G, Bentlage C, Bjorklund A (1995) Forelimb akinesia in the rat Parkinson model: differential effects of dopamine agonists and nigral transplants as assessed by a new stepping test. J Neurosci 15:3863–3875. Oorschot DE (1996) Total number of neurons in the neostriatal, pallidal, subthalamic, and substantia nigral nuclei of the rat basal

1224

E. Y. Pioli et al. / Neuroscience 153 (2008) 1213–1224

ganglia: a stereological study using the Cavalieri and optical disector methods. J Comp Neurol 366:580 –599. Owen AM, Iddon JL, Hodges JR, Summers BA, Robbins TW (1997) Spatial and non-spatial working memory at different stages of Parkinson’s disease. Neuropsychologia 35:519 –532. Paille V, Henry V, Lescaudron L, Brachet P, Damier P (2007) Rat model of Parkinson’s disease with bilateral motor abnormalities, reversible with levodopa, and dyskinesias. Mov Disord 22:533– 539. Paxinos G, Watson C (1997) The rat brain in stereotaxic coordinates. London: Academic Press. Pioli E, Sohr R, Meissner W, Barthe N, Gross CE, Bezard E, Bioulac BH (2004) Partial bilateral mesencephalic lesions affect D1 but not D2 binding in both the striatum and cortex. Neurochem Int 45:995–1004. Roedter A, Winkler C, Samii M, Walter GF, Brandis A, Nikkhah G (2001) Comparison of unilateral and bilateral intrastriatal 6-hydroxydopamine-induced axon terminal lesions: evidence for interhemispheric functional coupling of the two nigrostriatal pathways. J Comp Neurol 432:217–229. Roncacci S, Troisi E, Carlesimo GA, Nocentini U, Caltagirone C (1996) Implicit memory in parkinsonian patients: evidence for deficient skill learning. Eur Neurol 36:154 –159. Sabol KE, Neill DB, Wages SA, Church WH, Justice JB (1985) Dopamine depletion in a striatal subregion disrupts performance of a skilled motor task in the rat. Brain Res 335:33– 43. Saint-Cyr JA, Taylor AE, Lang AE (1988) Procedural learning and neostriatal dysfunction in man. Brain 111 (Pt 4):941–959. Sakai K, Gash DM (1994) Effect of bilateral 6-OHDA lesions of the substantia nigra on locomotor activity in the rat. Brain Res 633:144 –150. Salmon DP, Butters N (1995) Neurobiology of skill and habit learning. Curr Opin Neurobiol 5:184 –190. Schneider JS, Kovelowski CJ 2nd (1990) Chronic exposure to low doses of MPTP. I. Cognitive deficits in motor asymptomatic monkeys. Brain Res 519:122–128. Schwarting RK, Huston JP (1996a) The unilateral 6-hydroxydopamine lesion model in behavioral brain research. Analysis of functional deficits, recovery and treatments. Prog Neurobiol 50:275–331. Schwarting RK, Huston JP (1996b) Unilateral 6-hydroxydopamine lesions of meso-striatal dopamine neurons and their physiological sequelae. Prog Neurobiol 49:215–266. Shallice T, Burgess PW, Frith CD (1991) Can the neuropsychological case-study approach be applied to schizophrenia? Psychol Med 21:661– 673.

Shimura T, Kamada Y, Yamamoto T (2002) Ventral tegmental lesions reduce overconsumption of normally preferred taste fluid in rats. Behav Brain Res 134:123–130. Swanson LW (1982) The projections of the ventral tegmental area and adjacent regions: a combined fluorescent retrograde tracer and immunofluorescence study in the rat. Brain Res Bull 9:321–353. Taghzouti K, Simon H, Le Moal M (1986) Disturbances in exploratory behavior and functional recovery in the Y and radial mazes following dopamine depletion of the lateral septum. Behav Neural Biol 45:48 –56. Tang C, Pawlak AP, Prokopenko V, West MO (2007) Changes in activity of the striatum during formation of a motor habit. Eur J Neurosci 25:1212–1227. Theoret H, Boire D, Herbin M, Ptito M (1999) Stereological evaluation of substantia nigra cell number in normal and hemispherectomized monkeys. Brain Res 835:354 –359. Thierry AM, Deniau JM, Herve D, Chevalier G (1980) Electrophysiological evidence for non-dopaminergic mesocortical and mesolimbic neurons in the rat. Brain Res 201:210 –214. Ungerstedt U (1971) Adipsia and aphagia after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system. Acta Physiol Scand Suppl 367:95–122. Weinberger DR, Berman KF, Illowsky BP (1988) Physiological dysfunction of dorsolateral prefrontal cortex in schizophrenia. III. A new cohort and evidence for a monoaminergic mechanism. Arch Gen Psychiatry 45:609 – 615. Weinberger DR, Berman KF, Zec RF (1986) Physiologic dysfunction of dorsolateral prefrontal cortex in schizophrenia. I. Regional cerebral blood flow evidence. Arch Gen Psychiatry 43:114 –124. West MJ (1999) Stereological methods for estimating the total number of neurons and synapses: issues of precision and bias. Trends Neurosci 22:51– 61. Whishaw IQ, Pellis SM (1990) The structure of skilled forelimb reaching in the rat: a proximally driven movement with a single distal rotatory component. Behav Brain Res 41:49 –59. White NM (1997) Mnemonic functions of the basal ganglia. Curr Opin Neurobiol 7:164 –169. Yadin E, Friedman E, Bridger WH (1991) Spontaneous alternation behavior: an animal model for obsessive-compulsive disorder? Pharmacol Biochem Behav 40:311–315. Yin HH, Knowlton BJ, Balleine BW (2004) Lesions of dorsolateral striatum preserve outcome expectancy but disrupt habit formation in instrumental learning. Eur J Neurosci 19:181–189. Zigmond MJ, Stricker EM (1972) Deficits in feeding behavior after intraventricular injection of 6-hydroxydopamine in rats. Science 177:1211–1214.

(Accepted 10 January 2008) (Available online 29 February 2008)