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Behavioural Brain Research 183 (2007) 67–77

Research report

Effect of ventrolateral thalamic nucleus lesions in theunilateral 6-hydroxydopamine rat model

Carina Oehrn, Haydn Allbutt, Jasmine Henderson ∗Department of Pharmacology, Bosch Institute and School of Medical Sciences, University of Sydney, Sydney, NSW 2006, Australia

Received 16 February 2007; received in revised form 18 May 2007; accepted 23 May 2007Available online 29 May 2007

bstract

Whilst dysfunction of basal ganglia-thalamic circuitry is implicated in the genesis of parkinsonian symptomatology, few studies have examinedhe effects of lesioning the motor thalamus in the context of parkinsonism. Forty rats were therefore subdivided into four lesion groups each of 10ats with lesions or sham surgery targeting (1) the medial forebrain bundle and/or (2) motor thalamus, resulting in: Sham/Sham, 6-OHDA/Sham,ham/NMDA and 6-OHDA/NMDA groups. Behavioural testing was performed prior to any surgery and after each surgery including analysis ofosture, drug-induced rotation, sensorimotor and autonomic deficits. As expected 6-OHDA lesions induced abnormalities in posture, locomotion,ensorimotor and pilomotor function, ipsilateral and contralateral rotational asymmetries after amphetamine and apomorphine, respectively. These

ehavioural changes reflect parkinsonism in this model. Additional thalamic lesions virtually abolished apomorphine-induced rotational asymmetrynd improved sensorimotor response latency to tactile stimulation on the contralateral side. These data support the contribution of dysfunctionalotor thalamic circuitry in rotational asymmetry and abnormal sensorimotor function in parkinsonian rats.2007 Elsevier B.V. All rights reserved.

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eywords: Motor thalamus; Thalamotomy; Sensorimotor behaviour; Rotationa

. Introduction

Prior to modern pharmacotherapy with L-dopa, stereotacticurgical interventions, such as pallidotomy and thalamotomyere commonly performed for ameliorating symptoms of PD.hile targeting the internal globus pallidus (GP) to improve

kinesia and rigidity was already suggested as early as the940s, the ventrolateral (VL) nucleus of the thalamus has beenargeted to control tremor and rigidity in PD patients sincehe 1950s [13,17,19]. During the 1970s, L-dopa became the

ajor treatment for PD [14]. While akinesia and bradykinesiaould be significantly improved by application of L-dopa, otherymptoms like postural deficits or tremor did not respond as sat-

sfactorily. Over time L-dopa can lose its effectiveness and sideffects like dyskinesia and the on/off phenomenon commonlyccur in patients [38].

∗ Corresponding author at: Room 211d, Bosch Building, Department ofharmacology, Bosch Institute and School of Medical Sciences, University ofydney, Sydney, NSW 2006, Australia. Tel.: +612 9036 9408;ax: +612 9351 3868.

E-mail address: jasmine@med.usyd.edu.au (J. Henderson).

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166-4328/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2007.05.031

metry; Parkinsonism

In the last decade more attention has been drawn to sur-ical treatment again to decrease side effects of L-dopa andmprove symptoms that cannot be adequately controlled phar-

acologically, like postural instability or tremor. Today, newurgical treatments, based on a better understanding of basalanglia (BG) circuits have been developed, such as targetinghe overactive subthalamic nucleus (STN) by deep brain stim-lation, which results in improvement of akinesia, rigidity andlso tremor of PD patients [4,37]. There are several surgicalreatments for PD, however thalamotomy, is still the most com-

only used method to treat tremor, not only in PD, but also inssential tremor [41,43].

The primate motor thalamus consists of two major subdivi-ions, the ventralis anterior (VA) and ventralis lateralis (VL),he latter of which is composed of an anterior (VLa) and a pos-erior subunit (VLp) [26,32]. Anatomical tracing studies in ratsnd primates and histochemical studies of human thalamus indi-ate that VA receives its major input from the substantia nigra

ars reticulata (SNr) and VLa from the globus pallidus internusGPi) [32–35,47,52]. Furthermore, anatomical tracing studies inacaques demonstrate that both of these motor thalamic nuclei

roject to the supplementary motor area (SMA), premotor area

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nd anterior cingulate [32,47,52]. In addition, the deep cerebel-ar nuclei project via VLp to the motor cortex and this circuitrylays an important role in tremor [32,47,52].

VL is a target of the sensorimotor basal ganglia circuity (BG),hich is affected in PD. The existing model of the pathophys-

ology of PD suggests an overactivity of the GABAergic BGutput nuclei (GPi and SNr) due to dopamine depletion, whichesults in an overinhibition of VL and in turn to an underactivehalamocortical pathway [15]. This is thought to be the mech-nism underlying parkinsonian symptoms such as akinesia andigidity.

VL however, is more than simply a relay of motor functions.arious clinical studies provided evidence that VL also receivesensory inputs through the cerebellar pathway, projects also tohe somatosensory cortex and might play an important role innitiating, guidance or termination of movement and contributeso making movements in response to sensory input [7,53,57].lso the image of the ventral motor thalamic group as pure relayuclei has been disproved by recent anatomical tracing studiesn macaques which found pronounced projections from VA andL back to the striatum [39,56]. This is also the case in rats

18]. This suggests that the VL also plays a modulating role inG function and provides significant positive feedback to the

ensorimotor striatum [39,56].While recent clinical studies have concentrated on the role

f the VL in the genesis of tremor, the impact of a lesion of VLn sensory, cognitive and autonomic deficits in the context ofD is still largely unexplored. Whilst a prior study examined theffects of VL lesions on motor behaviour in rats, this was donen the absence of nigrostriatal dopaminergic depletion [29]. Theemaining studies of VL lesions in 6-hydroxydopamine-lesioned6-OHDA) rats focused on drug-induced rotational asymmetryut not other aspects of motor dysfunction and parkinsonism36,44,59].

We and others have found, for example, that nigrostri-

tal dopamine depletion in the unilateral 6-OHDA rat modelnduces ipsilateral head position and body axis biases, contralat-ral neglect, motor slowing (in crossing a narrow beam), asell as increased circling behaviour after dopamine agonists

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able 1tudy design and timing

eek Group 1 Group 2N = 10 N = 10

–4 Baseline behavioural testing Baseline behavioural tes

Sham 6-OHDAst surgery 7, 9 Post-op behavioural testing Post-op behavioural test

0 Sham VL Sham VLnd surgery

2, 14, 18 Post-op behavioural testing Post-op behavioural test6 Amphet rotation Amphet rotation

9 Apo rotation Apo rotation2 Sacrifice Sacrifice

inal N 7 5

po = apomorphine; amphet = amphetamine; post-op = post-operative, VL = ventrolat

Research 183 (2007) 67–77

9,20,22,23,51,55,61]. Patients with PD develop bradykinesia,igidity, resting tremor and postural and locomotor abnormal-ties [45], which to some extent are reflected in rats by the

otor slowing, delay in reaction time and abnormal posturesnd locomotion (but not resting tremor). Bilateral 6-OHDAesions in rats are not advisable since they are associated withigher mortality rates and significant morbidity with adipsiand aphagia [55]. Rats with unilateral lesions are able to feednd drink within hours of surgery and there is little mortality55].

For the above reasons, this study assessed the impact of unilat-ral VL lesions alone and in the presence of 6-hydroxydopamine6-OHDA) lesions in a rodent model on a broad range of motornd non-motor behaviours.

. Materials and methods

.1. Animals

Forty young adult female albino Sprague Dawley rats were used. They wereept in an enriched environment with 4–5 animals per cage under controlledonditions of temperature (19–22), humidity (40–70%) and air quality. Standardat chow and water was available ad libitum. The testing was performed duringhe light phase of a 12:12 h light:dark cycle. Experiments were conducted withnstitutional ethics approval from the Animal care and Ethics Committee of theniversity of Sydney, Australia in accordance with the Care and the Use ofnimals in Research guidelines (1996) of the Australian National Health andedical Research Council.

. Experimental and surgical design

The rats weighed 250–300 g at the time of surgery. Each animal received twourgical treatments (separated by a period of 1 month). During the first surgeryhey were given a unilateral 6-OHDA (or sham) lesion of the medial forebrainundle to destroy SNc neurons. In the second surgery unilateral VL thalamicor sham) lesions were made on the same side as during the first surgery. Theseombinations of procedures resulted in four different surgical groups (Table 1).

To evaluate any behavioural changes caused by the different lesions vari-

us tests were performed before surgery, during the first month after 6-OHDAurgery, and for 2 months after thalamic surgery (Table 1). The rats were sac-ificed at the end of testing by deep anaesthesia and transcardial perfusion, therains removed and processed histologically and the magnitude of degenerationn the SNc and VL were examined.

Group 3 Group 4N = 10 N = 10

ting Baseline behavioural testing Baseline behavioural testing

Sham 6-OHDAing Post-op behavioural testing Post-op behavioural testing

NMDA NMDAVL lesion VL lesion

ing Post-op behavioural testing Post-op behavioural testingAmphet rotation Amphet rotation

Apo rotation Apo rotationSacrifice Sacrifice

9 5

eral thalamus; final number included was based on detailed histology.

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.1. Surgery

The rats were deeply anaesthetised with 3% halothane (mixed with oxygen).o prevent any head movement, rats were placed in a stereotactic frame (Stoelt-

ng Stereotactic “lab standard frame”, Illinois, USA) using 45◦ non-puncturearbars with the nosebar position 2.3 mm below the interaural line. Once posi-ioned an incision was made to reveal the bregma. A small hole was drilledo expose the dura. The toxin or vehicle solution was injected by a 23 gauge�l Hamilton microsyringe according to the relevant stereotactic co-ordinates

n the rat brain atlas [46]. After injection of the solution 5 min was allowedor diffusion before the needle was retracted, the wound cleaned and sutured.or prophylactic pain relief, the rats were given 0.05 mg/kg of buprenorphineydrochloride s.c. (Temgesic, Reckitt Benckiser).

.2. SNc lesion

In the first surgery either 4 �g/�l 6-hydroxydopamine hydrobromide (6-HDA; Sigma RBI, St. Louis, MO, USA) dissolved in 0.9% ascorbate-saline orvehicle solution (0.9% ascorbate-saline) was injected unilaterally in the medial

orebrain bundle (mfb) over a period of 4 min at a rate of 1 �l/min, using theo-ordinates: AP = −4.4, L = ±1.1 (both relative to bregma), V = −8.00 (relativeo dura) [23].

.3. Thalamic lesion

Four weeks later the VL of the same side of the brain as in the first surgeryas targeted and either 1 �l of 0.12 M N-methyl-d-aspartate (NMDA) or theehicle solution (sterile saline) were injected at a rate of 0.5 �l/min for 2 minsing the corresponding co-ordinates: AP = −2.3, L = ±1.7 (both taken fromregma), V = −5.6 (taken from dura) [46].

.4. Behavioural testing

All behavioural assessments were conducted in the same manner described inetail in previous papers [20,23]. The timing is given in Table 1 unless otherwisepecified below.

.5. Sensory tests

.5.1. Sensorimotor testThe sensorimotor test evaluates the reaction time the rat requires to respond

o a tactile stimulus applied to the vibrissae. After the isolated rat was given timeo habituate in a separate test cage a small wooden probe passed through the barsently stimulated the whiskers whilst minimising any visual and auditory cues.ts’ reaction time to respond was recorded by a stopwatch bilaterally. A responseas defined as a turning of the head and touching the probe with its mouth, snoutr paw [23,51].

.5.2. Disengage testThe disengage test employed the same methods as the sensorimotor test, with

he exception that the rat was tested whilst eating a 2.5 g piece of chocolate.he rats were familiarized with the chocolate for 3 days before testing. Bothensory tests were performed bilaterally every test week [23,51].Test weeksor sensorimotor and disengage tests were prior to any surgery, 2 weeks after-OHDA lesions and 2, 4 and 8 weeks after thalamic surgery.

.5.3. Drug-induced rotationRotational asymmetry caused by the injection of amphetamine and apomor-

hine was tested once per rat towards the end of the study, with 3 weeks inetween the injections. After injection of the relevant drug the rat was imme-iately placed in a white, hemispheric, plastic rotation bowl, filmed and the

umber of ipsilateral and contralateral 360◦ turns per hour were counted byideo playback to evaluate any asymmetry in rotational behaviour. During weekafter thalamic surgery the rats were given 2.5 mg/kg d-amphetamine sulphate

Sigma) i.p. dissolved in sterile 0.9% saline and rotation recorded. Three weeksater they were tested again, this time after injection of 0.2 mg/kg apomorphine

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Research 183 (2007) 67–77 69

ydrochloride (Sigma) dissolved in 0.1% ascorbate-saline s.c. The number ofet rotations (ipsilateral – minus contralateral rotations) were then calculatedor each drug [23,51].

.6. Postural measures

Test weeks for the following behavioural measures (curling and head posi-ion) were prior to any surgery, 2 weeks after 6-OHDA lesions and 2, 4 and

weeks after thalamic surgery. The beam run was conducted approximately 1onth after thalamic surgery.

.6.1. CurlingThe curling test evaluates any asymmetry in body posture. The rat was lifted

ently 2 cm above the bedding for 5 s and any deviation from its’ vertical bodyxis of 10◦ or greater was recorded. If deviation was <10◦ it was recorded aseutral, otherwise as either left or right. The net body axis bias was defined ashe sum of the number of ipsilateral minus contralateral deviations. This test waserformed 5 times in succession per test week [9,23].

.6.2. Head positionThree times in succession every test week the preference of turning the head

n either direction was recorded. Turning was defined by a deviation greater than0 (degrees) from the midline. The head position relative to its body axis wasisted every second for a 60 s interval and the net head positions were calculatedtotal number of seconds the head was turned towards the ipsilateral side minusotal number of seconds the head was held contralaterally during the entire 180 seriod) [20].

.6.3. Beam runEach rat was placed on a wooden beam (3 cm thick, 4 cm wide, 105 cm long)

hat was elevated 80 cm from the ground. The time required to traverse 1 m toeach its home cage which was located on the opposite end of the beam waseasured by a stopwatch. Also the latency to initiate the movement was recorded

nd reported as ‘timed-out’ if it exceeded 2 min. This test was performed 5 timesuring week 4 after thalamic surgery [2,60].

.6.4. Elevated plus mazeThis test was performed to evaluate whether any locomotor impairment

n the beam run might be due to anxiety. The rat was placed in the middlequare (10 cm × 10 cm) of a 50 cm elevated maze that consisted of two open10 cm × 40 cm) and two closed arms (10 cm × 40 cm). Along the arms thereere lines drawn at every 10 cm and the number of times the animal crossedne of the lines or entered one of the arms was recorded. An arm entry wasefined by the rat crossing the first line of an arm with all four feet. The totalmount of time the rat spent in each arm was recorded as well as any behavioursxhibited, such as the number of stretch attends, head dipping, rearing, groomingnd the time period it groomed for. All the tests were filmed for later play backvaluation. The test was performed once approximately 2 weeks after thalamicurgery.

.6.5. Autonomic functionWe have previously reported subtle alterations in pilomotor function after 6-

HDA and/or thalamic lesions in rats [23]. This measure increases in a variety oftates including illness, stress, pain and may be used as an indicator of autonomicysfunction. To detect any changes in autonomic function as well as generalealth, fur colour, tail colour and piloerection was observed and rated fromone = 0, mild = 1, moderate = 2 to severe = 3, alterations on a rating scale [23].he rats were also weighed every week.

.6.6. HistologyTwelve weeks after the first surgery, the rats were deeply anaesthetized with

alothane and perfused transcardially with phosphate buffered saline (PBS), fol-owed by 4% buffered paraformaldehyde (Sigma). The brain tissue was quicklyemoved and immediately placed in 4% buffered paraformaldehyde and storedor 24 h or until sunken. After fixation, the brains were transferred and in 30%ucrose for cryoprotection. Brains were frozen and sectioned at 40 �m on a

7 Brain

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eica (Germany) freezing sledge microtome and collected free floating. Theections were divided into 5 series (i.e. intersection distance of 200 �m). One ofhe series was collected and stained with cresyl violet to examine the locationnd magnitude of the lesions in the VL and spread to surrounding regions. On aecond series, tyrosine hydroxylase (TH) immunohistochemistry was performedo enable quantification of the amount of cell loss in the SNc.

For TH immunohistochemistry, a biotinylated monoclonal antibody againstyrosine hydroxylase raised in mice (1:2000, T2928, Sigma–Aldrich, USA) wassed and TH finally visualized by using a second antibody against Mouse IgG-iotin raised in sheep (1:500) in combination with streptavidin peroxidase andiaminobenzidine reactions [60].

To quantify the cell loss of the SNc, all of the surviving dopaminergic cells ofach serial section (approximately 9 sections per rat) spanning the entire SN wereounted on a light microscope (B3 professional series, Australian laboratoryervice) using a 10 × 10 grid of 2.49 mm2 at 200 times magnification usingfractionator technique as previously reported [23]. The number of cells was

dded and multiplied by 5 to calculate the total number of cells in the SN,s one out of 5 series was counted. The entire nigra was quantified in every ratilaterally. The cell loss was expressed as a percentage relative to the unlesionedide [23]. After examination of the location and volume of the lesions 10 rats thatad incomplete 6-OHDA lesions or demonstrated mistargeted thalamic lesionsn the cresyl violet stained sections were excluded from analysis (see Table 1or final group numbers), plus one rat that died within the testing period. Alsohree rats from the 6-OHDA/Sham group were excluded to match the degreend range of cell loss in both 6-OHDA lesioned groups.

.6.7. Statistical analysisThe data was statistically evaluated using STATVIEW (Version 5). Cell loss

nd rotation were analysed using a one-way ANOVA, all other behavioural

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ig. 1. Photomicrographs of substantia nigra from sections from representative rats fhose which received 6-OHDA lesions ((B) 6-OHDA/Sham and (D) 6-OHDA/NM

B and D, respectively) when compared to animals which received sham surgery toar = 200 �m.

Research 183 (2007) 67–77

arameters were evaluated by the two way-repeated measures analysis of vari-nce (ANOVA). Post hoc analysis was conducted with Fisher’s progressive leastignificant differences test to assess any difference in behaviour in betweenhe groups [23]. Data is expressed as mean ± standard error (S.E.M.).Theehavioural variables after each surgery were compared separately to estab-ish which phase of the study the groups became different. The data at baseline,nd at week 2 and 4 post 6-OHDA surgery were blocked and analysed. This wasepeated for week 2, 4, and 8 post thalamic surgery. Results were consideredignificant when p < 0.05.

. Results

.1. Histology

There was substantial dopaminergic cell loss in those groupshat received 6-OHDA lesions (Fig. 1). Whilst the magnitudef 6-OHDA-induced dopaminergic cell loss was greater in the-OHDA/Sham group (84.7%) than in the 6-OHDA/NMDAroup (63.2%, Fig. 2), this did not achieve statistical signifi-ance. However, both groups which received nigral lesions wereignificantly different from the double Sham and Sham/NMDAroup, which both demonstrated negligible dopaminergic cell

oss (Figs. 1 and 2). The VL thalamus was completely ablatedn the targeted side in the majority of animals included whicheceived thalamic lesions (in 10 of 14). Of the remaining ani-als three had partial VL lesions affecting mainly the ventral

rom each group as visualised via tyrosine hydroxylase immunohistochemistry.DA) exhibited significant loss of dopaminergic neurons on the lesioned sidethe medial forebrain bundle ((A) Sham/Sham and (C) Sham/NMDA). Scale

C. Oehrn et al. / Behavioural Brain

Fig. 2. Histogram illustrating mean cell loss (%) in the SNc of Sham/Sham,6-OHDA/Sham, Sham/NMDA and 6-OHDA/NMDA groups. The asterixesindicate significant differences between groups with dopaminergic lesions com-pared to groups with sham lesions to the medial forebrain bundle (ANOVA:FOp

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= 22.530, p < 0.0001, post hoc: 6-OHDA/Sham vs. Sham/Sham: p < 0.0001, 6-HDA/Sham vs. Sham/NMDA: p < 0.0001, 6-OHDA/NMDA vs. Sham/Sham:= 0.0001, 6-OHDA/NMDA vs. Sham/NMDA: p = 0.0002).

L and one had a lesion of the dorsal VL (Fig. 3; Table 2). Allnimals with VL lesions had variable lesion spread to involvearts of the laterodorsal nucleus (LD). In some instances theedial dorsal (MD), centrolateral (CL) and/or paracentral (PC)

halamic nuclei, reticular nucleus and posterior thalamus werelso involved to a lesser extent (Table 2).

.2. Sensory tests

.2.1. Simple sensorimotor testAt the baseline there was no difference between groups in

ensorimotor reaction time. After 6-OHDA surgery the slight

ncrease in latency observed on the contralateral side in animalshat received a dopaminergic lesion, was not statistically sig-ificant. Thalamic surgery did not lead to any further changesn any group. On the ipsilateral side no significant changes in

able 2xtent of thalamic lesions

ata Group VL lesion LD CL/PC Other

4 Complete + +/− MD4 Ventral + −/− VP, RT4 Ventral + −/− MD4 Complete + +/+ MD4 Complete + +/− MD, VM, C3 Complete + −/− AT3 Complete + +/+ VA/AV, VM3 Ventral + +/+ MD3 Complete + −/− VM, VP, RT, Po, ZI, Pf3 Complete + −/− VP, RT3 Complete + +/+ MD, VM3 Complete + −/− MD3 Complete + +/+ MD3 Dorsal + −/− C, Pf

+) also involved; (−) not involved. Other thalamic nuclei involved:D = mediodorsal, VP = ventral posterior, RT = reticular, AT = anterior,

A = ventral anterior, VM = ventral medial, Pf = parafascicular, Po = posterior.ther regions: C = some motor cortical degeneration noted, ZI = zona incerta.a Total: 14.

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Research 183 (2007) 67–77 71

ensorimotor latency were observed throughout the study (Dataherefore not shown).

.2.2. Disengage testSimilarly, there was no sensorimotor neglect observed at

aseline in this task. After the animals received the first surgery,oth 6-OHDA lesioned groups were significantly impaired butot different from each other (Fig. 4A). Two and 4 weeks afterhalamic surgery only animals with pre-existing nigral lesions6-OHDA/Sham) still exhibited a pronounced deficit, which wasy now not only significantly different to the Sham/Sham andham/NMDA group, but which also differed significantly fromnimals that received a thalamic lesion in addition to the 6-HDA lesion (Fig. 4B). The latency of these 6-OHDA/NMDA

esioned rats was improved considerably and now did not differrom Sham/Sham and rats that only received thalamic lesionsFig. 4B). Lesioning VL did not have any deleterious effect inhe Sham/NMDA group, as these animals still did not differ fromaseline values and Sham/Sham lesioned rats (Fig. 4A and B).y week 8 after thalamic surgery, rats of the group that only

eceived nigral lesions showed evidence of behavioural recov-ry and dropped to a mean deficit comparable to animals ofhe 6-OHDA/NMDA lesioned group, which exhibited a slightncrease in sensory neglect again by this time (Fig. 4B). Thereere no significant effects of either surgery on the ipsilateral

ide (data not shown).

.3. Drug-induced rotation

.3.1. AmphetamineRats in the 6-OHDA/Sham group showed rotational asymme-

ry after amphetamine injection (average of 76 rotations/30 min)owards the lesioned side. Those that received both lesion typesid not show any significant rotational behaviour, similar toham/NMDA or Sham/Sham groups (Fig. 5). These differencesid not however, achieve statistical significance.

.3.2. ApomorphineApomorphine injection caused marked contralateral turning

f 6-OHDA/Sham rats. In contrast, rats in the Sham/Sham,ham/NMDA and 6-OHDA/NMDA groups did not develop anyotational behaviour. Post hoc comparison revealed that thoseroups were significantly different to the 6-OHDA/Sham group,ut did not differ from each other (Fig. 6).

.4. Postural measures

.4.1. Curling and head positionAs expected, 6-OHDA lesioning induced some ipsilateral

iases in curling and head position. The biases were variable,owever, and did not achieve statistical significance. There waso further effect of thalamic surgery on either variable (data nothown).

.4.2. Beam runWhilst 6-OHDA lesioned rats from both groups appeared

lightly slower to start crossing the beam, these groups

72 C. Oehrn et al. / Behavioural Brain Research 183 (2007) 67–77

Fig. 3. (A–I) Extent of lesioning of VL is shown diagrammatically in two rats with the smallest (light grey) and largest VL lesions (dark grey), respectively at ninea ]. Ven( terior( c), ha

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nteroposterior levels (−1.3 mm to −4.2 mm), adapted from the rat brain atlas [46Pf) and paraventricular (PV) nuclei; ventral posterior nucleus (VP), ventral anPO), caudate-putamen (CPu), lateral globus pallidus (LGP), internal capsule (i

ere not statistically significantly different to those of the

ham/NMDA or Sham/Sham groups for this parameter (data nothown).

When the total time required to cross the beam was analysed,oth the 6-OHDA/Sham and the 6-OHDA/NMDA groups took

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trolateral thalamus (VT), laterodorsal (LD), medial dorsal (MD), parafascicularnucleus (VA), anterior nucleus (AT), reticular nucleus (Rt), posterior thalamusbenula (Hb). Black shading indicates ventricles.

tatistically significantly longer to complete the task than either

ham/Sham or Sham/NMDA groups. Rats that only received the

halamic lesion (Sham/NMDA) did not exhibit any difference inotal time to cross the beam in comparison to Sham/Sham ratsFig. 7).

C. Oehrn et al. / Behavioural Brain Research 183 (2007) 67–77 73

Fig. 4. (A) Contralateral disengage test before and 4 weeks after 6-OHDAsurgery (6-OHDA wk 4). The graph illustrates the mean latency in seconds toreact to a tactile stimulus while eating chocolate. The 6-OHDA/NMDA groupdiffers significantly from Sham/Sham group (ANOVA: F = 12.106, p = 0.0021,post hoc: (*) 6-OHDA/NMDA vs. Sham/Sham: p = 0.0282; other comparisonsnot significant). (B) Contralateral disengage test after subsequent thalamicsurgery. The graph illustrates the mean latency (sec) on the contralateral side 2(mot thal wk 2), 4 (mot thal wk 4) and 8 weeks (mot thal wk 8) after thalamic (VL)surgery. The asterix indicates the significant difference between 6-OHDA/ShamaOp

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Fig. 5. Amphetamine rotation. Graph illustrating the mean net number of turns(ipsilateral minus contralateral turns) within a 30 min interval after amphetamineinjection. Whilst animals of group 2 (6-OHDA/Sham) were the only ones to showrotational asymmetry, ANOVA failed to attain significance (ANOVA: F = 2.941,p = 0.0555).

Fig. 6. Apomorphine rotation. Graph illustrating the mean net number of turnsthe four groups exhibited within 30 min. The 6-OHDA/Sham group demon-s(g

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nd the other three groups (ANOVA: F = 3.317, p = 0.0387, post hoc: 6-HDA/Sham vs. Sham/Sham: p < 0.0001, 6-OHDA/Sham vs. Sham/NMDA:= 0.0002, 6-OHDA/Sham vs. 6-OHDA/NMDA: p = 0.0141).

.4.3. Elevated plus mazeThere were no statistically significant differences between

roups for total line crosses, arm entries, time spent in thepen/closed arms (data not shown). All groups exhibited simi-ar behaviours in the closed arm regarding head dipping, rearingnd grooming. However, rats with NMDA lesions performedore stretch attends in the closed arm (which achieved statis-

ical significance for NMDA/Sham group versus Sham/Shamroup, Fig. 8).

.4.4. Autonomic function and general healthThere were no significant differences between groups in

ither the degree of piloerection, tail or fur colour for the duration

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trated a significant degree of contralateral rotation relative to the other groupsANOVA: F = 9.094, p = 0.0004, post hoc analysis: 6-OHDA/Sham vs. all otherroups all p < 0.0005).

f the study. The rats remained in good health for the durationf the study and there was no difference in weight gain betweenroups.

. Discussion

Rats given a unilateral lesion of the SNc developed strongrug-induced rotational behaviour and an increased reactionatency in the disengage test on the contralateral side of theesion. They also were slower to cross the balance beam. Theame deficits have been reported previously, supporting theeproducibility of the dopaminergic lesions and behavioural data20,48,51,61,62]. In animals with pre-existing dopaminergicesions, further lesioning of the VL abolished apomorphine-nduced rotational responses and reduced the latency of theontralateral response in the disengage test, but did not alter

ocomotor deficits in the balance beam test. Nor were posturalr non-motor parameters affected.

Based on the established model of PD which suggests anveractivity of the BG output nuclei which in turn leads to

74 C. Oehrn et al. / Behavioural Brain

Fig. 7. Total time to cross the beam in seconds. The groups receiving 6-OHDA lesions took significantly longer than either Sham/Sham or Sham/NMDAlesioned groups (asterixes). 6-OHDA-Sham operated animals did not differ inlatency to cross the beam from 6-OHDA/NMDA lesioned animals; neither didthe two groups of animals without 6-OHDA lesions differ from each other(ANOVA: F = 4.308, p = 0.0156, post hoc: 6-OHDA/Sham vs. Sham/Sham:pS

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= 0.0100, 6-OHDA/Sham vs. Sham/NMDA: p = 0.0315, 6-OHDA/NMDA vs.ham/Sham: p = 0.0134, 6-OHDA/NMDA vs. Sham/NMDA: p = 0.0406).

n excessive inhibitory input to VL, one would expect andditional VL lesion to worsen the deficits caused by DA deple-ion (bradykinesia, rigidity). In contrast, thalamotomy has beenound clinically to be an effective form of treatment in PD,ot only to improve tremor, but also for rigidity to a lesserxtent [41,43]. We did not observe any worsening of deficitsfter lesioning VL in parkinsonian rats. Surprisingly our resultsven show an improvement in some behavioural tests (reducedpomorphine-induced rotational asymmetry and sensorimotoreglect in “disengage” test). Also a VL lesion alone did notesult in significant deficits, relative to sham-operated controls,n most of the behavioural parameters studied. For example, one

ight have expected locomotor deficits as a result of a decreasedhalamocortical activation. However, the rats may have compen-

ated to some extent using the non-injured side since lesionsere unilateral.

ig. 8. Stretch attends in the closed arms of the elevated plus maze duringesting time (5 mins). ANOVA: F = 3.154, p = 0.0452, post hoc: Sham/Sham vs.ham/NMDA: p = 0.0076, Sham/Sham vs. 6-OHDA/NMDA p = 0.0580; otheromparisons were non-significant.

6

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Research 183 (2007) 67–77

.1. Technical issues

The lack of significant results with postural measures (headosition and curling) as well as latency to initiate movementn most parameters of the elevated plus maze, may relate tohe degree of variability of data, lesion size and the relativelymall numbers of animals. We do not believe investigatorias was problematic since the investigator who performedhe behavioural tests was blinded to the surgical status of thenimals.

Several rats were removed from the study due to inadequateesioning/surgical mistargeting. This is not uncommon whenttempting to lesion two small structures in the same rat.njection of NMDA in the VL not only ablated this structureut caused a variable extent of cell loss in other adjacenthalamic nuclei as has been reported in other studies [29,44].n all cases, parts of the laterodorsal, nucleus was involvednd there was variable involvement of the ventromedial motorhalamus (VM), medial dorsal, the centrolateral (CL) and/or thearacentral (PC) thalamic nuclei. A few animals also had lesionpread to involve the reticular and posterior thalamic nuclei.iven the known connectivity of most of these regions and

unctional associations it seems unlikely that the variable extentf their involvement would have accounted for the consistentehavioural changes observed which can be readily explained inhe context of VL connectivity. However, it has been shown thatarkinsonian cats display reduced sensory neuron activationfter facial touch involving not only the VA/VL but also the CLegions [54] so we cannot exclude an additional contribution tomproved disengage response in our 6-OHDA/NMDA lesionedats from CL involvement in a subset of our rats. Other motortudies of CL lesions in rats without 6-OHDA lesions haveound impaired rotarod performance but normal function on atationary beam, holeboard and in swimming to a cued platformn the Morris water maze [28].

. Behavioural analyses

.1. Rotational asymmetry

Injection of apomorphine in unilateral 6-OHDA lesionedats results in pronounced contralateral rotation due to over-ensitivity of DA receptors in the striatum on the lesioned side55,61]. This is thought to reduce the overinhibition of VL viahe BG and therefore to release the thalamocortical pathway. Thexaggerated imbalance of thalamocortical activation betweenemispheres contributes to the rotational asymmetry observed.dministration of amphetamine causes intact dopaminergiceurons to release an increased amount of dopamine, thus theres a larger amount of DA released on the non-lesioned side in

unilateral lesioned parkinsonian rat which leads to distinctpsilateral rotation as reported previously [20,55,62].

.2. Drug-induced rotation

It has been reported that an additional electrolytic ipsilateralesion of ventral thalamus in the unilateral 6-OHDA lesioned

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at resulted in a reduction of apomorphine-induced circling,hereas it did not have any effect on rotation in response

o amphetamine [30,36]. Furthermore, a contralateral thalamicesion decreased rotational responses to amphetamine, but noto apomorphine [30,36]. Similarly, we observed prominent rota-ional responses to amphetamine and apomorphine followingesions of the SNc. Contrary to the previous results [30,36] andditional excitotoxic lesion of the ipsilateral VL in the presenttudy or electrolytic VL lesion in another recent study [44]otally abolished, rather than reduced, any rotational responseo apomorphine in 6-OHDA lesioned rats.

As described above apomorphine releases the overinhibitedL enabling an activation of the cortex. An interruption of thisathway at the level of VL would alter thalamocortical projec-ions and potentially better match cortical activation on bothides, so that net asymmetry and therefore rotational response,isappear. It is unlikely that the reduction of rotational responseesults from differential dopaminergic cell loss between the tworoups that received nigral lesions. Recent studies looking athe effect of graded 6-OHDA lesions on various behavioural

easures found that partially-lesioned animals with a meanell loss of 40% exhibited substantial rotational behaviour48,60]. The strong circling of animals with only nigral lesions

months after 6-OHDA surgery indicates that compensatoryechanisms were unlikely to have substantially influenced this

ask. Also, the rotation rate in the 6-OHDA/NMDA lesionednimals was far less than that observed previously in partially-esioned animals (approximately 50 turns/30 min at 10 weeks)o VL lesioning per se, rather than compensatory mecha-isms associated with smaller lesions, is thought responsibleor the dramatic reduction of rotation in the 6-OHDA/NMDAroup. This supports that an intact motor thalamus in neededo create the rotational response and it forms part of theotational circuitry, in addition to the basal ganglia regionsSTN, GPi and SNr) reported in previous lesioning studies16,20–22,24,25,27].

Whilst there was negligible rotational asymmetry in rats witharkinsonian lesions and subsequent ipsilateral VL lesions inesponse to amphetamine administration, this did not achieveignificance compared to the rats with 6-OHDA lesions alone,robably due to the small sample size. Some authors [44]xplained an observed reduction of both amphetamine- andpomorphine-induced rotational responses by a general effectn gross motor behaviour with lesion of the motor thalamusausing a general hypokinetic state. This explanation does noteem to fit with the fact that our 6-OHDA/NMDA lesioned ani-als did not exhibit any deficits in the other motor tests above

hat seen with 6-OHDA lesions alone. Rats with only thalamicesions did not take longer to initiate or cross the beam in thealance beam test, nor did they exhibit an increased reactionatency in the disengage test.

.3. Sensimotor tests

The BG do not merely process motor information, but alsoensory, cognitive and associative information [1,11,49,51].uch circuits are important for action selection, choosing a rel-

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Research 183 (2007) 67–77 75

vant input and select an appropriate motor response. Sensorynd associative abnormalities are features of PD, which not onlyontribute to the motor deficits, but also lead to impairments inttention and sensorimotor processing [6,10,49,50,53]. A com-ination of motor, sensory and cognitive deficits contribute toarying extents to the increased latency in the disengage test20,51]. Several experimental and clinical studies provide evi-ence that there is not only motor, but also disturbed sensoryrocessing caused by dopamine depletion and this correlatesith the magnitude of nigral dopaminergic cell loss and stri-

tal dopamine depletion [60]. There are also altered neuronalesponses in the BG to sensory stimulation in the parkinso-ian cat [54]. In an earlier impaired sensory gating mechanismsithin the BG in were proposed to explain increased abnormali-

ies in tests of sensorimotor integration and proprioception in PDatients versus controls [53]. Defective sensory processing on aimanual slow matching task was also attributed to sensimotorisintegration within the BG [40]. Also PET studies in patientsave revealed a decreased activation in response to vibration ofrain regions including the BG and the sensorimotor cortex [8].arkinsonian symptoms have also been attributed to an inability

o select relevant sensory information from irrelevant [10,11]nd is manifested clinically in PD patients when conductingwo tasks simultaneously [5,10,12,31,58]. Regarding our experi-

ent, the inability to integrate sensory information and impairedual task performance might explain why the response to aensory input was significantly slowed in 6-OHDA lesionedats that were engaged in another task involving increased taskomplexity and attentional load (“Disengage” versus simpleensorimotor response).

Anatomical tracing studies in macaques and rats as well aslectrophysiological studies in humans indicate support thatL is not only a relay of motor and sensory information fromasal ganglia and cerebellum to cortex but also projects totriatal sensorimotor regions [7,18,39,56,57]. Dysfunction inuch circuitry could therefore impair sensorimotor function atoth striatal and cortical levels, reflected by slowed responsesnd dual task performance deficits in parkinsonian animalsnd patients. Our data supports the suggestions that VL is alsonvolved in sensorimotor processing.

Rats which received nigral lesions exhibited sensory neglectn the contralateral side of the lesion if engaged in anotherask. After receiving an additional thalamic lesion animalshowed response latencies compatible with controls in the firstonth after lesioning. This effect is very likely to be attributed

o the lesion of VL and not to compensatory mechanisms,s previous studies of graded 6-OHDA lesions reported thatartially-lesioned rats showed a strong contralateral neglect,hat was not compensated for at least 2 months post surgery60]. Furthermore, the reduced latency in the disengage testn our 6-OHDA lesioned rats, was evident within 2 weeks ofesioning VL. Conversely, lesioning VL alone did not causeny sensory neglect in the test performed.

The amelioration of sensory deficits resulting from VLesioning might suggest that the pathology of PD in sensoryrocessing may at least partially result from a change in activityf motor thalamic neurons as described above. Paradoxically,

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he lesion may enable an improvement of sensimotor processingt both cortical and striatal levels and therefore a faster initiationf the movement/response time reflected by reduced disengageeficit.

.3.1. Balance beam testRats of both groups that received lesions of the SNc took

ignificantly longer to cross the beam compared to both theham/Sham and the Sham/NMDA groups. Intriguingly, thereas no difference between the group that only received nigral

esions (6-OHDA/Sham) and the 6-OHDA/NMDA group.e therefore could not demonstrate any further effect of the

halamic lesions on initiation, balance ability and locomotion, inither rats that had received previous 6-OHDA lesions nor anyuch deficits in the rats that only received a lesion of VL. Whilstrecent study of VL/VM lesioned rats (but without 6-OHDA

esions) found impaired acquisition in the rotarod test, whenhe motor was turned off the animals showed no impairment.he latter condition approximates to some extent the beam

un conducted in the present study [29]. Whilst, it is thereforeonceivable that we may have detected subtle changes withreater motor task complexity, other motor tasks such as theoleboard and suspended string tests were also not impaired byhalamic lesions [29], also suggesting that our observations arealid.

.4. Autonomic, postura/changes and elevated plus maze

Whilst, we previously reported that prominent posturaleficits were observed in rats with a mean TH + cell loss ofpproximately 87% and a loss of 97% of striatal DA it isossible to observe more subtle deficits in partially-lesionednimals [60], This also mimics the progression of the diseasen PD patients in which prominent postural instability is gen-rally a late feature [45]. No significant abnormalities wereetected in autonomic measures after VL surgery, suggestinghat other circuits are involved in such deficits. The only signif-cant effect of thalamic lesions in the elevated plus maze wasn increasing stretch-attend behaviour, which is considered aisk-assessment behaviour, suggesting increased anxiogenesis3].

. Conclusion

This study showed that motor thalamic lesions abolishedpomorphine-induced rotational asymmetry and reduced senso-imotor latency in the disengage task in parkinsonian rats. Theata support a role of the VL thalamic circuitry in both sensorynd motor dysfunction in PD and that targeting this region mayave some beneficial effects on Parkinsonian symptoms. Drug-nduced rotational asymmetry has been proposed as a means of

odelling L-dopa induced dyskinesia in PD [27]. Several stud-es report improved L-dopa-induced dyskinesias in PD patientsfter thalamotomy [41,42] Our data may also support a benefi-ial role of thalamotomy to ameliorate this unwanted effect ofrug therapy.

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[

Research 183 (2007) 67–77

cknowledgements

The authors would like to thank Dr Jane Radford and herathology laboratory team for technical assistance. This studyas funded by a grant from the University of Sydney Research

nd Development Scheme.

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