Neuroscience 153 (2008) 1213–1224

DIFFERENTIAL BEHAVIORAL EFFECTS OF PARTIAL BILATERAL LESIONS OF OR SUBSTANTIA NIGRA IN RATS

E. Y. PIOLI,a W. MEISSNER,a,b R. SOHR,c C. E. GROSS,a occur across three distinct disorders: parkinsonism, de- E. BEZARDa AND B. H. BIOULACa* pression and negative symptoms of schizophreniaDe ( aUniversite Victor Segalen, Bordeaux 2, Centre National de la Recher-Ajuriaguerra, 1975; Bermanzohn and Siris,). 1992The che Scienctifique, Bordeaux Institute of Neuroscience, UMR 5227, occurrence146 of akinetic symptoms in diverse psychiatric rue Léo Saignat, 33076 Bordeaux Cedex, France disorders supports the notion that akinesia involves not bDepartment of Neurology, CHU de Bordeaux, Hôpital du Haut-only motor behavior but also cognitive and motivational Lévêque Bordeaux, France processes. All these functions are known to be modulated c Institute of Pharmacology and Toxicology, Charité Campus Mitte,by (DA). Indeed, DA in the con- Humboldt Universitaet, Berlin, Germany tributes to the regulation of motor and limbic processes and DA at the cortical level is correlated with cognitive Abstract—Akinesia (or absence of movement) is a prominentfunctions Nieoullon,( 2002; Nieoullon and Coquerel,). 2003 feature of Parkinson’s disease. Akinetic symptoms, however,In this view, clinical evidence shows that motor symptoms are also observed in depression and , whichin neurodegenerative disorders such as PD are accompa- support the hypothesis that akinesia involves more than only nied by cognitive deficitsBrown ( and Marsden, 1990; motor behavior. A common feature of these disorders is the disruption of dopamine homeostasis in the CNS. HereBrown we and Jahanshahi, ).1996 aimed at relating the respective involvement of the nigrostri-Anatomically, the substantia nigra pars compacta atal and mesocortical to akinesia.(SNc) mainly projects to the and the We investigated in the rat the relative effects of selectiveputamen. By contrast, the ventral tegmental area (VTA) bilateral partial lesions of substantia nigra pars compactamainly innervates the frontal cortex, the ventral (SNc) or ventral tegmental area (VTA) which did not andaffect the hippocampal formationFuxe, ( 1965; Lindvall and locomotion, on fine motor, motivational and cognitive behav- iors. Motor impairments were measured by the evaluationBjorklund, of 1974; Fallon et al., 1978; Fallon and Moore, fine motor control in the stepping test and in the paw1978a,b; reach- Haber and Fudge, ). 1997While SNc/VTA projec- ing test. Cognitive functions were assessed by various tionspar- to the forebrain are segregated, a degree of overlap adigms: spontaneous alternation in the Y maze and objectexists in other cortical terminal fields and some subcortical exploration task. Motivational behavior was evaluated by areas.the In particular, neurons projecting to the frontal cor- 100-pellets test. The results suggested that specific behav-tex, the cingulate cortex, the septum rostro-medial caudate ioral impairments are obtained following selective lesions of either SNc or VTA. SNc-lesioned rats exhibited deficits inand fine the are located in the medial SNc motor functions as previously described in animal modelsas ofwell as the lateralFallon VTA and( Moore, 1978b). Parkinson’s disease, whereas VTA-lesioned rats demon- Bilateral DA-containing neuron degeneration of the strated traits of perseveration without significant motorSNc is the main characteristic of human PD. Although impairments. © 2008 IBRO. Published by Elsevier Ltd.damage All to the SNc primarily causes PD, the VTA is also rights reserved. modestly affected, even in early stages Damierof PD ( Key words: akinesia, dopamine, 6-OHDA. et al., 1999a,b), rendering it difficult to attribute a specific deficit to a given nucleus. To solve this ambiguity, we induced selective partial Akinesia is considered to be a principal feature of bilateralParkin- 6-hydroxydopamine (6-OHDA) lesions of either son’s disease (PD) along with rigidity and tremor. theIt lateralis SNc or the medial VTA Pioliin theet al.,rat ( defined as poverty of initiation and execution of willed2004 and) . The rats were subjected to various motor, motivational associated movements in the absence of Lee,paralysis (and cognitive behavioral experiments. Motor impairments 1989). Akinesia represents a clinical syndrome which werecan estimated by the measure of fine motor control in the *Corresponding author. Tel: ϩ33-557-571-551; fax: ϩ33-556-901-421. stepping test and in the paw reaching test. The motiva- E-mail address: [email protected] (B. H. Bioulac). tional behavior was evaluated by the 100 pellets test. Two Abbreviations: ANOVA, analysis of variance; DA, dopamine; DOPAC, paradigms were employed to assess cognitive impair- 3,4-dihydroxyphenylacetic acid; EDTA, ethylenediaminetetraacetic acid; HPLC, high-pressure liquid chromatography; HVA, homovanillic ments: spontaneous alternation in the Y maze and an acid; NE, northeast; NW, northwest; PD, Parkinson’s disease; SE, object exploration task. Object exploration is associated southeast; S.E.M., standard error of the mean; SNc, substantia nigra with an index of cognitive activity, especially in rodents. pars compacta; SW, southwest; TH, tyrosine hydroxylase; TH-IR, tyrosine hydroxylase immunoreactive; VTA, ventral tegmental area; Both paradigms (in which rats explore without contingent 6-OHDA, 6-hydroxydopamine. reinforcement) allow the study of various behavioral func- 0306-4522/08$32.00ϩ0.00 © 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% ascor- bic 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; Ϯ2.2 mm from midline; Ϫ7.7 mm from dura; and for the Fig. 1. Experimental schedule. ␮ ␮ VTA injection (1 l, 0.125 l/min): 5.2 mm posterior to bregma; field, a frame placed at 3 cm height with 16 photocell beams per Ϯ Ϫ 0.8 mm from midline; 7.6 mm from dura. After each injection, side ensured movement detection. Total motor activity was re- the needle was left in position for 10 min to allow absorption of corded over a period of 7 min on three consecutive days. Animals the injected solution and to minimize spread of the toxin along the from sham and lesioned groups were tested in random order. After needle tract. Sham-operated controls received an identical proce- each test, the open field was cleaned with a moistened sponge. dure with the exception that no toxin was infused but only vehicle Results are expressed as mean activity of the 3 daysϮstandard (0.9% NaCl containing 0.01% ascorbic acid). This procedure was error of the mean (S.E.M.) and analyzed using a one-way analysis selected based on numerous pilot studies which reliably demon- of variance (ANOVA) with group as independent variable. strated selective lesions of the target region. Stepping adjustments. Stepping adjustments were mea- Experimental protocol sured as described by Olsson et al. (1995). Briefly, the experi- menter took the rat with one hand holding both hind limbs and the The animals were allowed to recover for 3 weeks before behav- other hand holding one of the forelimbs. The free paw was placed ioral testing commenced. This period of recovery has been de- in contact with a flat surface. The experimenter then moved scribed to produce stable 6-OHDA lesions (Schwarting and Hus- the animal slowly sideways in forehand and backhand directions. ton, 1996a,b). All rats were subjected to a sequence of behavioral The number of adjusting steps was counted for both paws in the tests as summarized in Fig. 1. After completion of the behavioral backhand and forehand directions. The test was repeated twice a evaluations, the rats were killed with an anesthetic overdose of day for three consecutive days. Animals from sham and lesioned urethane (Sigma) and the were rapidly removed. groups were tested in random order. Results are expressed as the The selectivity of the 6-OHDA lesions was confirmed by ste- mean number of adjusting steps during the six sessionsϮS.E.M. reological counting of mesencephalic neurons characterized by The means were subjected to a two-way ANOVA with group as tyrosine hydroxylase (TH) immunochemistry and by measurement the independent variable and paw as the dependent factor. When of DA and 5-HT content and their metabolites in cortical and appropriate, ANOVAs were followed by post hoc t-tests corrected striatal areas. For this purpose, the cortex and striatum were for multiple comparisons by the method of Bonferroni. dissected, flash-frozen on dry ice and stored at Ϫ80 °C. The caudal part of the containing the mesencephalon was post- Food consumption test. Food consumption was assessed fixed in PFA 4% for 2 days at 4 °C and transferred to 20% sucrose as the time taken to eat 100 pellets as described by Baunez et al. in PBS buffer for cryoprotection. After 1 day, this part of the brain (2002). First, the rats were familiarized with the environment and was frozen by immersion in Ϫ40 °C isopentane and stored at with the food to prevent neophobia. Animals were fed ad libitum Ϫ80 °C. The entire mesencephalon was then cut at Ϫ20 °C in during all the time of the test. The test started with 5 min of 20 ␮m coronal sections. The sections were collected free floating exploration in the bowl. Then, 100 sweet pellets (45 mg, Phymep, for further immunohistochemical processing. Paris, France) were introduced and the time taken by each rat to eat the pellets was measured in seconds. The bowl was then Behavioral testing cleaned with water and left for a few minutes before the next rat was placed in the bowl with another 100 pellets. Animals from Locomotor activity. Spontaneous motor activity was mea- sham and lesioned groups were tested in random order. Results sured using a photocell apparatus (Panlab, Barcelona, Spain) as are expressed as the mean time required for eating the 100 described by Diguet et al. (2004). The apparatus consisted of a pelletsϮS.E.M. The data were submitted to a one-way ANOVA squared open field (44ϫ44ϫ32 cm). On each side of the open with group as the independent variable. When appropriate, E. Y. Pioli et al. / Neuroscience 153 (2008) 1213–1224 1215

ANOVAs were followed by post hoc t-test corrected for multiple experimental environment, except for a conspicuous striped pat- comparisons by the method of Bonferroni. tern (21 cmϫ29.7 cm). A “videotrack” system (EthoVision, Wageningen, The Netherlands) allowed continuous monitoring of Spontaneous alternation in Y maze. Spontaneous alterna- ® the position of the rat and also permitted selection of events tion was tested in a Y maze made of gray opaque Perspex occurring in a defined area of the open field. The open field was (Syma, Le Haillan, France). It consisted of three identical alleys divided in four areas: northeast (NE), northwest (NW), southeast (named A, B, C). Each was 40 cm long, 15 cm wide and had 20 cm (SE) and southwest (SW) zones (see Fig. 2A, 2B, 2C). Three high walls. The three alleys diverged at a 120° angle from a central objects were used. They differed primarily by shape and tex- point. Rats had free access to the three arms of the maze for 10 ture. Object A was a Perspex tube (8 cm in diameter, 25 cm in min, during which the sequence of arm-entrances was recorded height), object B was a wood squared parallelepiped as described by Taghzouti et al. (1986). The Y maze was cleaned (10ϫ10 cm, 23 cm in height) and object C was a Perrier® glass with water between each run. Alternation was determined from bottle (7 cm in diameter, 28 cm in height). successive entries of the three arms on overlapping triplet sets in Animals were submitted individually to five successive 6-min which three different arms were entered. For example, a se- sessions, separated by 3-min inter-session intervals, during which quence 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 consecu- tives 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 meanϮS.E.M. The raw data were submitted to a Kruskal-Wallis test with group as the inde- pendent factor. When appropriate, this test was followed by a post hoc Dunn’s test for multiple comparisons. Skilled paw reaching test. Skilled paw reaching was mea- sured 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 com- menced. 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 grasp- ing 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 remain- ing at the end of the test on the floor of the compartment indicates the clumsiness, i.e. impaired sensori-motor coordination in grasp- ing 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 compari- sons by the method of Bonferroni. Object exploration task. The object exploration task con- sists of three successive phases: i.e. habituation, spatial change and non–spatial-change. We used a modified version of the par- adigm 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 Fig. 2. The object exploration task. Schematic representation of ob- surrounded the open field in order to provide a visually uniform ject configuration during successive sessions. 1216 E. Y. Pioli et al. / Neuroscience 153 (2008) 1213–1224 they were returned to their cages. The rats were placed at the same rated from each other by 50 ␮m(x) and 40 ␮m(y); and in the VTA initial position (i.e. coordinates and body orientation) in the open field dissectors (120 ␮m length, 80 ␮m width) were separated from arena. Animals from sham and lesioned groups were tested in ran- each other by 75 ␮m(x) and 65 ␮m(y). The following formula was ϭ ⌺ Ϫ dom order. The open field was cleaned only at the end of the series used to estimate the number of TH-IR neurons: N V(SNc) ( Q / ⌺ of experiments for each given rat. For a schematic representation V(dis)); where N is the estimation of cell number, V is the volume of the apparatus and object configuration during successive ses- of the SNc or VTA, ⌺QϪ is the number of cells counted in the ⌺ sions see Fig. 2 (2A, 2B, 2C). The five individual sessions con- dissectors, and V(dis) is the total volume of all the dissectors sisted of: (Theoret et al., 1999). Mean estimated number of neurons and Session 1 (served as a measure of locomotor activity, Fig. S.E.M. were then calculated for each group. Data were subjected 2A). The arena was empty. to a two-tailed Student’s t-test. Session 2 (served as object exploratory activity, Fig. 2B). A striped pattern was added to serve as a landmark within the Neurochemical analysis. The extent of striatal and cortical environment. Three objects were placed in the arena at three DA denervation was assessed by measuring levels of DA, 3,4- corners of a fictitious square: object A in the NW zone, object B in dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), the SE zone and object C in the NE zone. The last corner re- 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in the dorsal and mained empty (i.e. the “neutral” area). ventral part of the striatum as well as in the frontal cortex using Sessions 3 and 4 (measured exploratory habituation, Fig. 2B). high-pressure liquid chromatography (HPLC) with electrochemical As in session 2. detection as previously described (Bezard et al., 2001; Pioli et al., Session 5 (assessed the reaction to spatial change, Fig. 2C). 2004). Frontal cortex, anterior and posterior striatum were dis- Object C was moved to the “neutral” area. sected and homogenized in 1 ml of 0.1 M perchloric acid. Protein All sessions were videotaped to be analyzed off-line after- concentrations were measured according to Lowry et al. (1951) ward. The distance run during session one reflected locomotor with human serum albumin as standard. A volume of 20 ␮l was activity. The raw data were submitted to ANOVAs for statistical injected with a manual injection valve (RH 7725, Rheodyne, Co- analysis. tati, USA) and separated by HPLC on a reverse phase column Object exploration activity was assessed during session two (ProntoSil 120 C18 SH, 5 ␮m, 150ϫ3 mm, VDS Optilab, Ger- by comparing the mean exploratory activity in the three zones NW, many). The mobile phase consisted of 100 mM sodium dihydro- NE, SE containing the A, B, C objects, respectively [(NWϩNEϩ gen phosphate, 0.8 mM EDTA (disodium salt), 1.3 mM 1-octane- SE)/3] (object zone (OZ) in Fig. 6B) with the exploratory activity in sulfonic acid (sodium salt) and the pH was adjusted to 3.9 with the empty zone (EZ in Fig. 6B). These values were subjected to a phosphoric acid. The buffer solution was mixed with 40 ml 2-pro- two-way ANOVA with group and zone as the main variables. This panol and distilled water was added to a final volume of 1000 ml. analysis was followed, when appropriate, by post hoc t-tests cor- DA, DOPAC, HVA, 5-HT and HIAA were quantified electrochem- rected for multiple comparisons by the method of Bonferroni. ically by a glassy carbon electrode at a potential of 0.8 V (Ag/AgCl Exploratory habituation was assessed by comparing the mean electrode). The chromatograms were recorded with a chromato- exploratory activity toward the objects [(NWϩNEϩSE)/3] from graphic data system (CSW 1.7, Data Apex Ltd., Czech Republic) session 2 to session 4 (Fig. 6C). After repeated expositions of and quantified by determination of peak areas in relation to the the same environment, object exploration should decrease from known concentration of respective standards. Mean and S.E.M. the first session with objects (session 2) to the session before the values were calculated for both striatal and cortical levels. Neu- spatial change (session 4). Data were subjected to a two-way rochemical data were submitted to a two-tailed Student’s t-test. ANOVA with group and session as the main variables. When appropriate, the analysis was followed by post hoc t-tests cor- rected for multiple comparisons by the method of Bonferroni. The RESULTS reaction to the spatial change was assessed by comparing the Extent of lesion mean exploratory activity toward objects during sessions 3 and 4 with that during session 5 for the displaced object C in the SW Given the aim of the present study, it was imperative that zone (Fig. 6D) and the non-displaced objects A and B (Fig. 6E) our lesions be large enough to cover the target area, but separately. Data were subjected to a two-way ANOVA with group strictly restricted to either the SNc or the VTA. We have and session status as the main variables. When appropriate, post hoc t-tests corrected for multiple comparisons by the method of previously shown with the same model that (i) bilateral Bonferroni were conducted. lesions were highly comparable on both hemispheres and that (ii) partial lesions of the SNc and VTA were restricted Assessment of lesion extent to the targeted nucleus (Pioli et al., 2004). The extent of Stereological counting of tyrosine hydroxylase immunoreac- 6-OHDA-induced lesion in SNc and VTA was quantified by tive (TH-IR) neurons. Free-floating mesencephalic serial sec- unbiased stereological counting of the total number of tions (20 ␮m) were processed for TH immunohistochemistry and TH-IR cell bodies in SNc and in VTA and by the neuro- subsequently counterstained with Cresyl Violet (Nissl staining) as chemical assessment of the content of DA, 5-HT and theirs previously described (Pioli et al., 2004). Unbiased stereological metabolites in the dorsal striatum, ventral striatum and techniques (Gundersen et al., 1988; West, 1999) were used to frontal cortex (see Table 1). estimate the number of mesencephalic DA-containing neurons. In every eighth section, the boundaries of the SNc and VTA were Bilateral 6-OHDA injections in the SNc induced a sig- determined by examining the size and shape of the different TH-IR nificant decrease (i) in the number of TH-IR neurons in the neuronal groups (German and Manaye, 1993; Oorschot, 1996; SNc (tϭ6.28, PϽ0.05), but not in the VTA (tϭ0.35, Paxinos and Watson, 1997). The volume was calculated by using PϾ0.05) and (ii) in DA (tϭ6.34, PϽ0.05), DOPAC (tϭ5.03, ϭ⌺ ⌺ the formula: V Std; where S is the sum of the surface areas, PϽ0.05) and HVA content (tϭ3.00, PϽ0.05) along with an t is the average section thickness and d is the number of slices increase in 5-HT (tϭ4.70, PϽ0.05) and HIAA content in between two consecutives analyzed sections measured (Theoret ϭ Ͻ et al., 1999). A total of 11 sections were counted. The optical dorsal striatum (t 3.72, P 0.05; Table 1). dissectors were distributed using a systematic sampling scheme. Bilateral 6-OHDA injections in the VTA induced a sig- In the SNc, dissectors (80 ␮m length, 60 ␮m width) were sepa- nificant decrease in the number of TH-IR neurons in the E. Y. Pioli et al. / Neuroscience 153 (2008) 1213–1224 1217

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 SNc Sham SNc VTA Sham VTA

TH-IR neurons SNC 8516Ϯ1037 2938Ϯ458* 8525Ϯ1099 8045Ϯ635 VTA 8282Ϯ933 7971Ϯ718 8264Ϯ550 6165Ϯ737* HPLC (nmol/g of protein) Frontal cortex DA 11.6Ϯ1.1 5.4Ϯ0.9* 13.1Ϯ1.5 10.8Ϯ2.1 DOPAC 5.5Ϯ0.7 1.8Ϯ1.9* 7.7Ϯ0.7 3.7Ϯ0.6* HVA 8.1Ϯ4.3 2.4Ϯ0.2 3.4Ϯ0.4 3.2Ϯ0.4 5-HT 2.4Ϯ0.2 9.8Ϯ1.3* 1.7Ϯ0.1 2.6Ϯ0.8 HIAA 2.3Ϯ0.1 10.3Ϯ1.3* 2.8Ϯ0.2 3.2Ϯ0.56 Ventral striatum DA 157.7Ϯ21.9 93.9Ϯ14.6* 108.2Ϯ22.4 141.4Ϯ17.5 DOPAC 53.5Ϯ10.3 31.3Ϯ4.9 58Ϯ7.8 46.1Ϯ3 HVA 20.4Ϯ2.9 15.2Ϯ2.1 18.6Ϯ3 19.2Ϯ2.2 5-HT 4.8Ϯ0.5 16.9Ϯ2.6* 4.2Ϯ0.9 4.9Ϯ0.7 HIAA 9.9Ϯ1.6 21.7Ϯ2.3* 10Ϯ0.6 10.8Ϯ1.2 Dorsal striatum DA 340Ϯ11.4 143.2Ϯ40.7* 313.3Ϯ18.3 379.9Ϯ31.9 DOPAC 77.2Ϯ2.6 33.3Ϯ6.5* 89Ϯ7.7 82.8Ϯ6.7 HVA 29.7Ϯ1.4 16.9Ϯ3.2* 29.5Ϯ1.9 33.2Ϯ3.3 5-HT 3.1Ϯ0.2 11.1Ϯ1.3* 3.4Ϯ0.4 4.6Ϯ1 HIAA 10.1Ϯ0.4 20.8Ϯ2.2* 13.4Ϯ1 14.1Ϯ1.7 Actimetry (Mean beam break) 2379Ϯ371 2357Ϯ200 2606Ϯ418 2386Ϯ407

Data are expressed as meanϮS.E.M. * PϽ0.05, a significant difference with the respective sham-operated animals.

VTA (tϭ2.75, PϽ0.05), but not in SNc (tϭ0.67, PϾ0.05; stepping test for the forehand (see Fig. 3A) and for the Table 1). The VTA lesions had no significant effect on DA backhand direction (see Fig. 3B) in animals with partial (frontal cortex: tϭ0.86, PϾ0.05; ventral striatum: tϭ1.82, bilateral SNc lesion. By contrast, partial bilateral VTA le- PϾ0.05; dorsal striatum: tϭ0.24, PϾ0.05), HVA (frontal sion had no effect on the number of stepping adjustments cortex: tϭ0.44, PϾ0.05; ventral striatum: tϭ0.16, PϾ0.05; in either condition (see Fig. 3A and 3B). dorsal striatum: tϭ0.93, PϾ0.05), 5-HT (frontal cortex: tϭ1.06, PϾ0.05; ventral striatum: tϭ0.85, PϾ0.05; dorsal Food consumption test. An overall estimation indi- striatum: tϭ1.13, PϾ0.05) and HIAA levels (frontal cortex: cated differences between experimental groups on the F ϭ PϽ tϭ0.64, PϾ0.05; ventral striatum: tϭ0.58, PϾ0.05; dorsal time needed to eat the 100 pellets ( (3,28) 4.4, 0.05). striatum: tϭ0.36, PϾ0.05; Table 1). However, VTA lesion Post hoc comparisons revealed that rats with partial bilat- reduced DOPAC content in the frontal cortex (tϭ4.28, eral SNc lesion were significantly slower to eat 100 food PϽ0.05), suggesting that DA homeostasis was disrupted pellets than shams (see Fig. 4A). In contrast, rats with in these animals. partial bilateral VTA lesions consumed food reward pellets as fast as shams (see Fig. 4A). Behavioral assessment Spontaneous alternation in Y maze. As expected, Locomotor activity. To ensure that our partial rats alternated at levels significantly above chance indicat- 6-OHDA lesions were not associated with changes in ing their readiness to explore novel environmental stimuli locomotion, horizontal activity was measured in an open (see for review Dember and Fowler, 1958; Lalonde, 2002; field. As expected, there was no significant difference in Hughes, 2004). An overall effect was found on the spon- ϭ Ͼ ϭ Ͻ horizontal activity between groups (F(3,28) 0.19, P 0.05, taneous alternation in the Y maze (F(3,28) 5.4, P 0.05). A Table 1), validating the partial lesion approach, i.e. partial post hoc comparison revealed that partial bilateral VTA lesion of either the SNc or the VTA did not affect lesioned rats made significantly less spontaneous alterna- locomotion. tions that their sham controls (see Fig. 4B). In contrast, partial bilateral SNc-lesioned rats alternated in the Y maze Stepping adjustments. The stepping test assessed at a comparable level as their sham controls (see Fig. 4B). fine motor control. An overall estimation showed differ- ences between treatment groups on stepping adjustment Skilled paw reaching test. The skilled paw-reaching ϭ Ͻ in all conditions (forehand: F(3,57) 35.8; P 0.05; back- test in the rat is a complex sensorimotor task analogous to ϭ Ͻ hand: F(3,57) 63.5; P 0.05). Further post hoc testing re- skilled reaching in humans (Whishaw and Pellis, 1990). vealed a significant decrease in the performance in the The criteria of success, attempts and sensorimotor coor- 1218 E. Y. Pioli et al. / Neuroscience 153 (2008) 1213–1224

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) re- vealed 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 con- firmed by the two-way ANOVA with repeated measures (groupϫzone), which revealed a significant effect of zone ϭ Ͻ ϭ (F(1,57) 94.1, P 0.05), but not of group (F(3,28) 1.51, PϾ0.05). The ANOVA also revealed a significant groupϫ ϭ Ͻ 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 (Pϭ0.0001). 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 repre- sents the time-course of the average mean exploratory activity in areas containing objects for sessions 2–4 ([NWϩNEϩSE]/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 meanϮS.E.M. * Denotes a significant difference with the respective sham-operated animals, PϽ0.05. dination for the different phases of the paw reaching were evaluated. An overall estimation between groups dis- ϭ played significant differences in the success (F(3,57) 9.1, Ͻ ϭ Ͻ P 0.05) and the attempt (F(3,57) 10.0, P 0.05), but not in ϭ Ͼ the sensorimotor coordination (F(3,57) 0.5, P 0.05) of paw reaching. Post hoc testing revealed increased suc- cess (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 ex- hibit 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 Fig. 4. (A) The food consumption test. (B) The spontaneous alterna- evaluation of locomotor activity. Fig. 6A depicts the mean tion. Effect of bilateral partial 6-OHDA-induced lesions in the SNc or in exploratory activity across the arena during sessions 1 and the VTA on the 100 pellets test (A) and on the percentage of sponta- 2. The two-way ANOVA analysis revealed a significant neous alternation (B). Black bar, hatched bar, gray bar and open bar ϭ Ͻ correspond to SNc sham group, SNc lesioned group, VTA sham group effect of session (F(1,57) 12.14, P 0.05) but not of lesion and VTA lesioned group, respectively. Data are expressed as ϭ Ͼ (F(3,28) 1.53, P 0.05), nor a significant interaction be- meanϮS.E.M. * Denotes a significant difference with the respective ϭ Ͼ Ͻ tween the two (F(3,57) 1.8, P 0.05). The session effect sham-operated animals, P 0.05. E. Y. Pioli et al. / Neuroscience 153 (2008) 1213–1224 1219

moved from its initial position (NE, Fig. 2B) to the empty location (SW, Fig. 2C). The other objects were not manip- ulated. In this session, two categories of objects are con- sidered separately for analysis: the displaced object (C) and the nondisplaced objects (A and B). Reaction to spa- tial 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 dem- onstrated that all groups showed an increase in explor- atory 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 (groupϫsession) which revealed ϭ Ͻ an effect of session (F(3,28) 20.9, P 0.05), but no signif- ϭ Ͼ icant effect of group (F(1,57) 1.74, P 0.05), nor a signifi- ϭ cant interaction between those variables (F(6,83) 0.25, PϾ0.05). A two-way ANOVA with repeated measures on the non-displaced familiar objects (groupϫsession) re- ϭ vealed no significant effect of session (F(3,28) 2.64, Ͼ ϭ P 0.05), no significant effect of group (F(1,57) 1.86, PϾ0.05), and no significant interaction between both vari- ϭ Ͼ ables (F(6,83) 0.25, P 0.05). In summary, during the first session of the object ex- ploration 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 The present study shows that distinctive patterns of be- havioral 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 be- havior, while the VTA is preferentially associated with a Fig. 5. The skilled paw reaching test. Effect of bilateral partial perseverative behavior. 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 Methodological considerations testing day. Performances of the rats in the paw reaching test are expressed as meanϮS.E.M. number of eaten pellets (A), number of The aim of this study was to compare the effects of re- steps without pellets (B), and number of missed pellets on the floor (C). stricted lesions of two major dopaminergic mesencephalic Black bar, hatched bar, gray bar and open bar correspond to SNc pathways on different behavioral tasks. To examine the sham group, SNc-lesioned group, VTA sham group and VTA-lesioned group, respectively. * Denotes a significant difference with the respec- extent of cognitive impairment in 6-OHDA-lesioned rats, tive sham-operated animals, PϽ0.05. we employed a bilateral lesion model acting on either the SNc or the VTA, rather than the different dopaminergic showed a habituation response, i.e. decrease of exploration target structures (e.g. striatum, nucleus accumbens, or activity across experimental sessions. A two-way ANOVA ). The SNc and VTA projections are topo- with repeated measures revealed a significant effect of group graphically organized (Haber et al., 2000). However, an ϭ Ͻ (F(3,28) 3.14, P 0.05) and a significant effect of session extensive overlap exists in theirs projections (Fallon and ϭ Ͻ (F(2,83) 9.91, P 0.05), but no significant interaction between Moore, 1978b; Loughlin and Fallon, 1984). Therefore, to ϭ Ͼ both variables (F(6,83) 1.34, P 0.05). identify distinct effects of SNc and VTA projections on Finally, in session 5, the rats are exposed to a new motor and cognitive processes in overlapping target areas, spatial configuration of the objects. A familiar object was the extent of SNc and VTA lesion had to be restricted to the 1220 E. Y. Pioli et al. / Neuroscience 153 (2008) 1213–1224

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 (ϮS.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 ([NWϩNEϩSE]/3) with the exploratory activity in the empty zone SW. Data represent mean (ϮS.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 ([NWϩNEϩSE]/3) for sessions 2–4. Data are expressed as mean (ϮS.E.M.) distance run. (D) Reaction to spatial change. Data are expressed as mean (ϮS.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 (ϮS.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 with severe motor deficits. Indeed, extensive SNc lesion the VTA should not impair locomotor activity since it is (Ͼ80%) induce locomotor hypo-activity (Sakai and Gash, impossible to investigate cognitive functions in animals 1994), while extensive VTA lesion induce hyper-activity E. Y. Pioli et al. / Neuroscience 153 (2008) 1213–1224 1221

(Le Moal and Simon, 1991). Moreover, extensive bilateral been an increasing awareness of the associated non- lesions of the SNc and VTA require intensive nursing of the motor symptoms such ad cognitive deficits (Owen et al., rats as they become aphagic and adipsic (Ungerstedt, 1997; Marinus et al., 2003; Mollion et al., 2003). Those 1971; Le Moal and Simon, 1991; Sakai and Gash, 1994). cognitive deficits would even be present before the motor Thus, the majority of studies in rat models of parkinsonism manifestations. For example, loss of DA in the striatum are based on unilateral lesion and only few studies have impairs habit formation (Salmon and Butters, 1995; Knowl- used bilateral lesion without systematically assessing the ton et al., 1996; White, 1997; Hay et al., 2002) and implicit effects on non-motor functions (see for review Schwarting memory (Saint-Cyr et al., 1988; Roncacci et al., 1996; and Huston, 1996a,b). In the present study, rats with par- Koenig et al., 1999) in PD patients. Our results further tial bilateral 6-OHDA lesion of the SNc or VTA did not ground the possibility that those symptoms occur earlier in demonstrate any deficits in locomotion, providing a valu- the disease process than previously thought. able model for studying cognitive deficits (Schneider and A recent study showed that the stepping test appears Kovelowski, 1990). to be a reliable tool to assess the persisting subtle motor dysfunction consequent to dopaminergic deficiency: the Effect of SNc partial bilateral lesions degree of altered performance was well correlated with the Rats with partial bilateral 6-OHDA lesion of the SNc had an severity of the underlying dopaminergic lesion and this impaired performance in the stepping test and in the 100 motor abnormality was improved under L-DOPA treatment pellets test, but displayed normal locomotion and spontane- (Paille et al., 2007). In our study, the performance of the ous alternation, normal performance in skilled paw reaching, partial bilateral SNc-lesioned rats on the stepping test and a level of object exploration similar to that observed in the altered in comparison with that of the sham rats. This result sham group (Table 2). is in line with the behavioral characterization of the Motivation behaviors have been traditionally divided 6-OHDA unilateral lesioned (Lee et al., 1996; Kirik et al., into preparatory and consummatory acts. Preparatory be- 1998; Roedter et al., 2001; Ogura et al., 2005) and more havior consists of the approach, exploration and instru- importantly of the 6-OHDA bilateral lesioned rats as exper- mental responding for a reward, whereas consummatory imental models of PD (Paille et al., 2007). behavior includes eating and drinking. The 100 pellets task Effect of VTA partial bilateral lesions only allows the evaluation of consummatory behavior, which mainly involves the sensorimotor system and DA In the present study, rats with partial bilateral VTA lesion transmission. Rats with partial bilateral SNc lesion had an displayed no motor deficits, but behavioral abnormalities impaired performance in the motivational task, the 100 in spontaneous alternation and in skilled paw reaching pellets test. Our results are in accordance with previous (Table 2). studies showing that DA depletion of the dorsolateral stri- During free exploration of the Y maze, a normal rat atum, but not of the dorsomedial part, disrupts the ability to alternates arm visits to acquire or process novel informa- retrieve small food pellets by using the forepaw (Sabol tion. Spontaneous alternation quantifies an unlearned re- et al., 1985) and that extensive DA depletion of the fore- sponse in rats, which involves different processes such as brain produces aphagia and adipsia (Ungerstedt, 1971; perception, attention, memory and motivation (see for re- Zigmond and Stricker, 1972). Although subtle deficits in view Dember and Fowler, 1958; Lalonde, 2002; Hughes, skilled grasping and eating (an automated skill) could ac- 2004). Here, rats with partial bilateral VTA lesions lost this count for the deficits in this task, the dorsolateral part of innate behavior; their alternation performance remained striatum is known as well to underpin the development of around chance level. A similar decrease in spontaneous habit response (White, 1997; Jog et al., 1999; Yin et al., alternation has been described in the Y maze and in the 2004; Faure et al., 2005; Tang et al., 2007). Even if PD was eight-arm radial maze for rats with mesocorticolimbic path- seen so far as primarily affecting motor function, there has way lesions (Taghzouti et al., 1986), but also for amphet- amine-treated rats (Y maze (Kokkinidis, 1987); eight-arm Table 2. Summary of the differential behavioral effects induced by partial bilateral lesions of VTA or SNc radial maze (Loh et al., 1993; Holter et al., 1996)) and for rats sensitized to the DA agonist quinpirole (T maze (Einat Behavioral tasks Lesioned groups and Szechtman, 1995)). A deficit in spontaneous alterna- tion is seen as an increased tendency to repeat a choice SNc VTA between two arms, which could be interpreted as impair- Locomotor activity ns ns ment in spatial memory. However, rats with partial bilateral Stepping adjustments 2 ns VTA lesion showed normal performance in the object ex- Food consumption 2 ns ploration task, reaching the sham group exploration level Spontaneous alternation ns 2 of the objects presentation and of the spatial change. This Skilled paw reaching ns 1 result suggested that VTA-lesioned rats are able to re- Object exploration ns ns member a spatial object configuration and to properly ori- ent their behavior to a spatial change. As object explora- “1” Denotes a significant increase of performance, whereas “2” symbolizes a significant decrease of performance compared to sham tion like the spontaneous alternation task is an ethologi- animals; ns signifies that the performances are non-significantly dif- cally based test without involving reward delivery and ferent from the sham animals. taking advantage of animal’s willingness to explore novel 1222 E. Y. Pioli et al. / Neuroscience 153 (2008) 1213–1224 environmental stimuli we can compare both results. Be- way is dopaminergic while 85% of the cause VTA-lesioned rats were not impaired in the object to ventral striatum contains DA (Swanson, 1982). It is likely exploration task, their deficit in the spontaneous alternation that the GABA containing axons correspond to the task cannot be ascribed to some spatial memory impair- 6-OHDA resistant, fast-conducting fibers observed by Thi- ments. Yadin et al. (1991) proposed that the reduction in erry et al. (1980). Since this GABA-containing mesocortical spontaneous alternation in a T-maze could serve as an pathway provide the substrate for both inhibitory and dis- animal model of obsessive-compulsive behavior. They in- inhibitory influences on PFC neuronal activity under DA terpreted the finding that rats keep returning to the same regulation, a subtle tough significant breakdown of DA arm as a form of compulsive “checking.” homeostasis as in our VTA-lesioned animals might explain Furthermore, bilaterally partially VTA-lesioned rats dis- that PFC activity changes in a way leading to the perse- played a higher paw reaching performance compared with verative behavior (Weinberger et al., 1986; Carter et al., sham animals, indicating an abnormal response. This may 1998). be interpreted as the result of increased motivation. How- The study of the differential behavioral effects of partial ever, VTA-lesioned rats did not exhibit any performance bilateral lesions of VTA or SNc and of their mechanistic differences in the 100 pellets test compared with sham basis requires further attention in order to elucidate the animals which is in agreement with no effect of VTA-lesion respective role of the structures and of the neurotransmit- on sucrose consumption (Shimura et al., 2002; Martinez- ter interplays. Hernandez et al., 2006). Another explanation for the higher performances may be that VTA-lesioned rats failed to in- Acknowledgments—We wish to thank L. Cardoit and S. Dovero hibit the response, suggesting perseveration in the task. for the technical assistance. E.Y.P. was supported by a fellowship Such perseveration could be the result of the competition from the Ministère de la Recherche et de la Technologie (MRT) between the target areas of VTA and SNc for control of and W.M. was a Marie Curie Fellow of the European Community (HPMF-2001-01300). The University Victor Segalen, the CNRS behavior. While VTA projects mainly to portions of the and the IFR (INSERM No. 8; CNRS No. 13) funded this study. 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 (perse- REFERENCES verance or stimulus response learning in the Y maze task Barneoud P, Descombris E, Aubin N, Abrous DN (2000) Evaluation of and enhanced automation of the reaching in the skill task). simple and complex sensorimotor behaviours in rats with a partial We initially hypothesized that VTA lesions might result lesion of the dopaminergic nigrostriatal system. 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(Accepted 10 January 2008) (Available online 29 February 2008)