Inhibiting Lateral Habenula Improves L-DOPA–induced Dyskinesia

Archival Report

Inhibiting Lateral Habenula Improves L-DOPA–induced DyskinesiaMatthieu F. Bastide, Brice de la Crompe, Evelyne Doudnikoff, Pierre-Olivier Fernagut,Christian E. Gross, Nicolas Mallet, Thomas Boraud, and Erwan Bézard

ABSTRACTBACKGROUND: A systematic search of brain nuclei putatively involved in L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID) in Parkinson’s disease shed light, notably, upon the lateral habenula (LHb), which displayedan overexpression of the ∆FosB, ARC, and Zif268 immediate-early genes only in rats experiencing abnormalinvoluntary movements (AIMs). We thus hypothesized that LHb might play a role in LID.METHODS: ∆FosB immunoreactivity, 2-deoxyglucose uptake, and firing activity of LHb were studied in exper-imental models of Parkinson’s disease and LID. ΔFosB-expressing LHb neurons were then targeted using theDaun02-inactivation method. A total of 18 monkeys and 55 rats were used.RESULTS: LHb was found to be metabolically modified in dyskinetic monkeys and its neuronal firing frequencysignificantly increased in ON L-DOPA dyskinetic 6-hydroxydopamine-lesioned rats, suggesting that increased LHbneuronal activity in response to L-DOPA is related to AIM manifestation. Therefore, to mechanistically test if LHbneuronal activity might affect AIM severity, following induction of AIMs, 6-hydroxydopamine rats were injected withDaun02 in the LHb previously transfected with ß-galactosidase under control of the FosB promoter. Three days afterDaun02 administration, animals were tested daily with L-DOPA to assess LID and L-DOPA-induced rotations.Inactivation of ∆FosB-expressing neurons significantly reduced AIM severity and also increased rotations. Interest-ingly, the dopaminergic D1 receptor was overexpressed only on the lesioned side of dyskinetic rats in LHb and co-localized with ΔFosB, suggesting a D1 receptor-mediated mechanism supporting the LHb involvement in AIMs.CONCLUSIONS: This study highlights the role of LHb in LID, offering a new target to innovative treatments of LID.

Keywords: Daun02, 2-Deoxyglucose, Electrophysiology, Macaque, Parkinson’s disease, Rat

http://dx.doi.org/10.1016/j.biopsych.2014.08.022

Chronic treatment of Parkinson’s disease (PD) patients with thedopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA)induces the development of involuntary movements, knownas L-DOPA-induced dyskinesia (LID) (1,2). The motor nature ofthese manifestations first led to investigating the abnormalitiesof neuronal function in the motor circuits [for review, see (3–5)].Subsequent investigations using metabolic mapping revealedthat nonmotor domains of the basal ganglia and beyond alsoplay a role in these manifestations (6).

Recently, a systematic search of brain nuclei putativelyinvolved in LID characterized ΔFosB, ARC, FRA2, and Zif268immediate-early gene expression patterns, a class of genesrapidly transcribed in response to an external stimulus such asstimulation of the dopamine D1 receptor (D1R) (7–10). Thisapproach shed light notably upon structures located outsidethe basal ganglia. Among those, the lateral habenula (LHb)attracted our attention, as LHb displayed an overexpression ofΔFosB, ARC, and Zif268 (9). Interestingly, Mitchell et al. (11,12)showed in their 2-deoxyglucose (2-DG) seminal studies thatbesides the now classic 2-DG uptake pattern in the basal ganglia(13,14), LHb stood out among several structures as a stronglyaffected nonbasal ganglia nucleus, showing a dramatic increase

in 2-DG accumulation in parkinsonism. We, therefore, postulatedthat LHb might play a role in LID manifestation.

In this study, we analyzed the LHb 2-DG accumulation indyskinetic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated macaques compared with normal, parkinsonian, andL-DOPA-treated parkinsonian ones. We also studied LHbsingle-unit electrophysiological activity in ON L-DOPA dyskinetic6-hydroxydopamine (6-OHDA)-lesioned rats compared with OFFL-DOPA 6-OHDA-lesioned rats, vehicle-treated 6-OHDA rats,and sham-operated rats. Finally, to test the hypothesis that thealtered firing activity of LHb neurons participates in LID gener-ation, we used FosB as a molecular marker of LID to selectivelyexpress ß-galactosidase in FosB/ΔFosB-expressing neuronsand assessed the role of these ΔFosB-expressing neurons inthe rat model of LID in PD (9,15,16) by inhibiting their electricalactivity using Daun02-inactivation (17–21).

METHODS AND MATERIALS

Study Approval

Experiments on rats were performed in accordance with theEuropean Union directive of September 22, 2010 (2010/63/EU)

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on the protection of animals used for scientific purposes. TheInstitutional Animal Care and Use Committee of Bordeauxapproved the present experiments under the license number5012099-A.

Experiments on primate tissues were conducted on apreviously characterized brain bank (6,13,22,23) collected in1999. Experiments were performed in accordance with theEuropean Communities Council Directive of November 24,1986 (86/609/EEC) for care of laboratory animals. No furtherprimate was killed for the present experiments.

2-Deoxyglucose Procedure

Eighteen female Macaca fascicularis monkeys (Shared AnimalHealth, Beijing, China) were housed in individual primate cagesunder controlled conditions of humidity (50 6 5%), temperature(241C), and light (12-hour light/dark cycles); food and waterwere available ad libitum, and animal care was supervised byveterinarians (6). The population corresponds to those used inthe following studies: Guigoni et al. (6) and Bezard et al.(13,22,24). Five animals were kept as untreated control animals(6). The remaining 13 parkinsonian animals received daily MPTP(.2 mg/kg, intravenous) (Sigma, St Louis, Missouri) according toour previously published protocol (22,25,26). Following stabili-zation of the MPTP-induced syndrome, eight animals receivedtwice daily 20 mg/kg of L-DOPA by mouth for 6 to 8 months(Modopar; Roche, Welwyn Garden City, United Kingdom) (L-DOPA/carbidopa ratio, 4:1). Four monkeys displayed dyskine-sia, while four monkeys did not (6). The parkinsonian conditionwas assessed on a parkinsonian monkey rating scale usingvideotape recordings (24,27). The severity of dyskinesia wasrated using the monkey dyskinesia disability scale (28,29).

On the day they were killed, monkeys were given an intra-venous injection of 1 mCi/kg [3H] 2-DG (specific activity, 50Ci/mmol, 185 GBq/mmol) (Interchim, Grenoble, France) in sterilesaline as described previously (6,12,13). After 45 minutes, allanimals were killed by sodium pentobarbital overdose (150 mg/kg,intravenous). L-DOPA-treated animals received L-DOPA 15minutes before 2-DG. Brains were quickly removed, immediatelyfrozen in isopentane (2451C), and stored at 2801C. Tissue wassectioned at 20 mm in a cryostat at 2171C and thaw-mountedonto gelatin-coated slides. Once freeze-dried (2601C; 40.1023

atmospheres) for 2 hours, both serial sections and autoradio-graphic methyl methacrylate standards (Amersham Biosciences,Uppsala, Sweden) were exposed to 3H-Hyperfilm (AmershamBiosciences) for 2 months at 2301C, developed in D-19 devel-oper (Eastman Kodak, Rochester, New York), and fixed in KodakUnifix (Eastman Kodak). Densitometric analysis of autoradio-graphs was performed using an image analysis system (Visio-scan version 4.12; Biocom, Les Ulis, France) as describedpreviously (6,13). An examiner blind with regard to the exper-imental condition analyzed two sections of LHb per animal.Optical densities were averaged for each animal and convertedto the amount of radioactivity bound in comparison with thestandards. Mean bound radioactivity and SEM were thencalculated for each group.

Electrophysiological Single-Unit Experiments

Adult Sprague-Dawley male rats (Charles River Laboratories,Lyon, France), weighing 175 g to 200 g at the beginning of the

experiment, were used. They were housed under standardlaboratory conditions in a 12-hour light/12-hour dark cycle withfree access to food and water. On day 0, under generalanesthesia, unilateral injection of 6-OHDA (2.5 mL at 3 mg/mL)was performed in the right medial forebrain bundle [anterior-posterior (AP) = 23.7 mm; medial-lateral (ML) = 11.6 mm;dorsal-ventral (DV) = 28 mm relative to Bregma (30)] in ratstreated 30 minutes before with citalopram (1 mg/kg intraperito-neal [IP]) and desipramine hydrochloride (20 mg/kg IP) accordingto previously published procedures (9,15,16,31,32). Fifteen ratsdisplaying an impaired stepping test (9,15,31,33,34) assessed ondays 18 to 20 were considered as lesioned and used for thesubsequent experiments. Postmortem analysis confirmed a lossof tyrosine hydroxylase-immunopositive fibers in the striatumgreater than 95% (3,4). Seven rats remained unexposed to L-DOPA. From day 21 onward, eight rats received once daily an IPinjection of a combined dose of benserazide (15 mg/kg) and L-DOPA (6 mg/kg) for 10 days (ON and OFF L-DOPA dyskinetic 6-OHDA-lesioned). On day 31, the baseline abnormal involuntarymovements (AIMs) score was assessed. The four AIMs catego-ries (limb, axial, orolingual, and locomotive) were scored using avalidated rating scale (35,36) for 1 minute every 20 minutes for 2hours (total four observations; maximal score for each observa-tion, 16; maximal total score per session, 64) performed by atrained investigator as previously described (9,15,31,32,37–39).

Electrophysiological recordings were performed in the rightLHb [AP = 23.5 mm to 24 mm; ML = 1.5 mm to 1 mm; DV =24.2 mm to 25 mm (30)] in anesthetized ON L-DOPAdyskinetic 6-OHDA-lesioned rats with repeated L-DOPA injec-tion each 90 minutes (n 5 8), OFF L-DOPA 6-OHDA-lesioned-rats (i.e., dyskinetic rats that did not receive L-DOPA on therecording day, n 5 8), vehicle-treated 6-OHDA rats (n 5 7), andsham-operated rats (n 5 10). Anesthesia was induced with 3%isofuran and maintained with urethane (1.25 g/kg, IP) andsupplemental doses of ketamine (30 mg/kg, IP) and xylazine(3 mg/kg, IP), as described previously (40–42). Extracellularrecordings of single-unit activity in the LHb were made usingglass electrodes (10–20 MΩ in situ; tip diameter !1.2 mm)containing .5 mol/L sodium chloride (NaCl) solution and neuro-biotin (2% wt/vol) (Vector Laboratories, Burlingame, California).Activity was recorded as previously described (43). Briefly, itwas amplified tenfold with an Axoclamp2B (Molecular Devices,Sunnyvale, California) in the bridge mode versus a referenceelectrode implanted in the neck skin. It was further amplified100fold with differential AC amplifier (model 1700) (A-M Sys-tems, Sequim, Washington) and divided between two channels.One was used for spike recording (300–10000 Hz) and the otherfor local field potential acquisition (.1–10000 Hz). Then, extrac-ellular potentials were digitalized using the Micro1401-3 andanalyzed with Spike2 software (Cambridge Electronic Design,Cambridge, United Kingdom). Following electrophysiologicalrecordings, single neurons were juxtacellularly labeled withneurobiotin as previously described (41,42,44,45).

All electrophysiological recordings were performed in theslow-wave activity brain state. Brain state was qualitativelyassessed for each rat through an electrocorticogram recordedvia a 1-mm-diameter screw juxtaposed to the dura materabove the right motor cortex M1 [AP = 13.5 mm; lateral =13.5 mm (30)] and referenced with another screw inserted inthe skull above the right side of the cerebellum. Raw

Role of Lateral Habenula in Dyskinesia

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electrocorticogram was bandpass filtered (.1–5000 Hz) andamplified 1000fold (model 1700) (A-M Systems) before acquis-ition by Micro1401-3 and Spike2 software. For all electro-physiological recordings, the sampling rate was fixed at20 kHz.

At the end of the recording, rats were perfused trans-cardially with .9% NaCl followed by ice-cold 4% formaldehydein phosphate buffered saline (PBS). Brains were removed,postfixed overnight in the same fixative (41C), then cryopro-tected for 48 hours at 41C in 20% PBS-sucrose. Brains werefrozen in isopentane at 2451C and stored at 2801C untilsectioning followed by neurobiotin staining as describedpreviously (41,42,44,45).

Stereological Data Analysis for Correlation

The number of ∆FosB-immunopositive cells was obtained aspreviously described (9) applying the optical fractionator(9,34,46,47) unbiased stereological method using a LeicaDM6000B microscope (Leica, Nanterre, France) with MercatorPro software (Explora Nova, La Rochelle, France; version7.9.8). Immunolabeled cells were counted by a blinded inves-tigator on every sixth section previously used (9) with stereo-logical parameters adapted to LHb (counting frames: 60 3 60mm, spacing: 100 3 100 mm, number of sections: 3). Theanimal population corresponded to rats used in our formerpublished study: dyskinetic 6-OHDA-lesioned rats (n 5 5) andnondyskinetic 6-OHDA-lesioned rats (n 5 5), in which behaviorwas rated over 120 minutes (9).

Daun02/ß-Galactosidase Inactivation Method

Twelve dyskinetic L-DOPA-treated 6-OHDA-lesioned ratswere obtained as described above except that at the sametime as 6-OHDA injection, all the animals were injected with2 mL of a lentiviral vector expressing LacZ (coding forß-galactosidase) under control of a FosB promoter with a finaltiter of 1.18 3 109 infectious particles per milliliter as pre-viously used (21) in LHb (AP 5 23.48 mm; ML 5 1.65 mm;DV 5 24.4 mm). Guide cannulae were implanted as previouslydescribed (15,21) (AP = 23.48 mm; ML = 1.65 mm; DV =24.2 mm) and cemented to the skull for subsequent Daun02injections (21). Thirty-one days post 6-OHDA and lentiviralinjections, baseline AIM score was assessed as describedabove. On day 32, animals received a 6 mg/kg L-DOPAinjection 1 hour before a 1 mL Daun02 injection (4 mg/mL in5% dimethyl sulfoxide, 5% Tween-80 in PBS at .5 mL/min) inLHb under light isoflurane anesthesia before being placedin their home cage for 3 days as described (18,19,21). Fromthe third day after Daun02 injection, all rats received a daily6mg/kg L-DOPA injection and AIMs were scored over 120minutes (21). To ensure reversibility of Daun02-induced inacti-vation, a control solution (5% dimethyl sulfoxide, 5% Tween-80 in PBS at .5 mL/min) was injected in the same animals6 days after Daun02 injection and AIMs were evaluated.

Both before and at the end of the Daun02 experiment,1 hour after the last L-DOPA injection, i.e., at the peak ofbehavioral effect, rats were deeply anesthetized with chloralhydrate (400 mg/kg, IP) (VWR, Fontenay sous Bois, France)and perfused transcardially with .9% NaCl followed by ice-cold4% formaldehyde in PBS. Brains were removed, postfixed

overnight in the same fixative (41C), then cryoprotected for 48hours at 41C in 20% PBS-sucrose. Brains were frozen inisopentane at 2451C and stored at 2801C until sectioning.

Histological Data Analysis

Cryostat-cut coronal rat brain sections (50 mm thick) werecollected and processed for tyrosine hydroxylase (MAB318)(Millipore, Darmastadt, Germany), ∆FosB (sc-48) (Santa CruzBiotechnology, Santa Cruz, California), D1R (D2944) (Sigma,St. Louis, Missouri) as previously described (9,21,46), and ß-galactosidase (AB1211-5MG) (Millipore) immunohistochemis-try (21).

Data Analysis

2-DG and electrophysiological neuronal frequency data wereanalyzed using one-way analysis of variance to estimateoverall significance, followed by post hoc t tests correctedfor multiple comparisons by Bonferroni method (48). Electro-physiological analyses were conducted on neurons thatpresent at least 500 spikes during epochs of cortical slow-wave activity selected as previously described (45,49). Firingrate was calculated using Neuroexplorer (Nex Technologies,Madison, Alabama), while overall neuron firing patterns (27,50)were analyzed using a density histogram method (51) aspreviously described (27,52). The distribution of electrophy-siological neuronal pattern was analyzed using chi-squaredtest (27). Behavioral data were analyzed with Wilcoxon signed-rank t test (53). All data are presented as mean 6 SEM with athreshold for statistical significance at p , .05. Correlationsbetween LID severity and ∆FosB immunopositive counts wereperformed using Spearman correlation (9).

RESULTS

LID Involves Metabolic, Electrophysiological, andTranscriptional Alterations in LHb

2-DG uptake was measured in LHb to assess the metabolicactivity induced by LID manifestation in monkeys (6,13). In-terestingly, 2-DG accumulation in LHb (F3,14 = 35.71, p , .001)significantly decreased in dyskinetic monkeys compared withnondyskinetic (p , .05) but also compared with MPTP-lesioned(p, .001) and control monkeys (p, .05) (Figure 1). No significantmodification was found between control and nondyskineticmonkeys, while 2-DG uptake was markedly enhanced inMPTP-lesioned monkeys compared with dyskinetic, nondyski-netic, and control monkeys (p , .001 vs. all) (Figure 1) inaccordance with the original report (11). Those data suggest thatthe parkinsonism-induced enhancement in the activity of LHbinputs is dramatically decreased in the dyskinetic animals. Suchdecrease in activity inputs is further visible when comparingdyskinetic and nondyskinetic animals. While 2-DG experimentsindicate that LHb inputs are dysregulated in LID, we did not knowwhat could be the LHb output activity in LID.

To define if LHb output activity is also dysregulated in LID,we thus analyzed the LHb neuronal discharge frequency andpattern in the 6-OHDA-lesioned rat model of PD and LID(Figure 2A). While dyskinetic 6-OHDA animals recorded whenOFF L-DOPA did show a LHb firing frequency comparablewith that of drug naïve 6-OHDA and sham-operated rats


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(Figure 2B), the ON L-DOPA dyskinetic 6-OHDA-lesioned ratsdisplayed a dramatic increase in firing frequency significantlydistinguishing them from all other groups (F3,88 5 10.30, p, .05);Figure 2B). Regarding LHb neuronal pattern, both ONL-DOPA dyskinetic 6-OHDA-lesioned rats and OFF L-DOPA6-OHDA-lesioned rats displayed a significant difference com-pared with sham-operated rats with a greater proportion ofbursting neurons at the expense of those firing randomly (p, .05)(Figure 2C). These data further suggest that dyskinetic manifes-tations are associated with pathologic changes both in the firingrate and patterns of LHb neurons.

Finally, we established that the ∆FosB-transcriptionalresponse in LHb induced by chronic L-DOPA in 6-OHDA-

lesioned rats (9) linearly correlated with the severity of abnor-mal involuntary movements (R2: 0.91, p , .001) (Figure 2D).

Altogether, the data suggest that LID-related changesin LHb metabolic, transcriptional, and electrophysiologicalactivities distinguish the dyskinetic animals from the non-dyskinetic ones, making the LHb a putative key relay in thegenesis of dyskinesia manifestation.

Inhibition of Habenular ∆FosB-Expressing NeuronsAlleviates LID

To directly assess the casual role of LHb on AIM severity in therodent analog of dyskinesia, we inactivated the electricalactivity of ∆FosB-expressing LHb neurons using the selectiveDaun02/β-galactosidase inactivation method. This methodconsists of the local administration of the prodrug Daun02,which is converted to daunorubicin by ß-galactosidase, readilyexpressed in mammalian cells previously transduced withthe Escherichia coli LacZ gene under the control of a cell-specific promoter (17–19). A FosB-LacZ lentivirus, expressingß-galactosidase only in FosB/∆FosB-expressing neurons (21),was injected in vivo in LHb of 6-OHDA-lesioned rats chroni-cally treated with L-DOPA (5,9,15,16). After the establishmentof stable AIMs, a single intra-LHb administration of Daun02significantly decreased AIMs compared with baseline score(p , .05; Figure 3A). AIMs reduction lasted 3 days comparedwith baseline score (21%, 24%, and 15%, respectively; p ,.05 for all; Figure 3A) in keeping with previous demonstrationof the Daun02-mediated behavioral span (19). After a return tobaseline AIMs score, a control solution (vehicle without Daun-02) was injected in LHb of the same rats. No significantdifference in AIMs score was found between vehicle-treatedrats and baseline scores. Moreover, Daun02 increased rota-tional behavior, an index of the antiparkinsonian and hyper-kinetic effect of L-DOPA (5,36) also associated to LID,compared with both baseline and control-treated rats (69%;p , .05 for all; Figure 3B).

Figure 1. L-3,4-dihydroxyphenylalanine(L-DOPA)–induced dys-kinesia impacts meta-bolic response in lateralhabenula in the maca-que model of L-DOPA–induced dyskinesia.Densitometric analysisof 2-deoxyglucose (2-DG) accumulation incontrol (n 5 5), parkin-sonian (1-methyl-4-phenyl-1,2,3,6-tetrahy-dropyridine [MPTP];n 5 5), L-DOPA non-dyskinetic (MPTP-NON-DYSK; n 5 4),and L-DOPA dyskineticmacaque monkeys (MPTP-DYSK; n 5 5). Dataare expressed in termsof tissue equivalent

ratios of the amount of radioactivity in the considered structure to that in thewhite matter of the same section (*p , .05 from control, $p , .05 from MPTP,#p , .05 from MPTP-NON-DYSK).

Figure 2. L-3,4-dihydroxyphenylalanine (L-DOPA)–induced dyskinesia (LID) impacts electrophysiological and transcriptional responses in lateral habenula(LHb) in the rat model of abnormal involuntary movements. (A) Representative example of LHb neurobiotin-injected neuron in the rat after electrophysiologicalrecording, scale bar 300 mm (with an inset magnification, scale bar 20 mm). (B) LHb neuronal firing frequency (spike/sec) analysis between sham-operated rats(n 5 30 neurons), vehicle-treated 6-hydroxydopamine (6-OHDA) rats (n 5 21 neurons), ON L-DOPA dyskinetic 6-OHDA-lesioned rats (6-OHDA LID ON, n 5 20neurons), and OFF L-DOPA 6-OHDA-lesioned-rats (6-OHDA LID OFF, n 5 16 neurons) (*p , .05 from sham, $p , .05 from 6-OHDA, #p , .05 from 6-OHDALID OFF) with representative rasters distinguishing LID ON and LID OFF activity over 1 second of recording. (C) LHb neuronal firing pattern (% of neuronsrecorded) analysis between sham-operated rats (n 5 30 neurons), vehicle-treated 6-OHDA rats (n 5 21 neurons), ON L-DOPA dyskinetic 6-OHDA-lesionedrats (6-OHDA LID ON, n 5 20 neurons), and OFF L-DOPA 6-OHDA-lesioned-rats (6-OHDA LID OFF, n 5 16 neurons) (*p , .05 from sham-operated rats).(D) Correlation between abnormal involuntary movement severity and number of LHb ∆FosB immunopositive neurons (R2: 0.91, p , .001) in dyskinetic (red)and nondyskinetic (blue) 6-OHDA-lesioned rats. MHb, medial habenula.





Increased Habenular D1R Expression Co-localizeswith ∆FosB Expression

∆FosB expression is, inter alia, known to be driven by D1Rstimulation (7,9,54–56). We, therefore, investigated the possi-bility of such a mechanism in the LHb by determiningthe LHb content of D1Rs. Immunofluorescence assay revealeda dramatically increased expression of D1R only on thelesioned side of dyskinetic 6-OHDA-lesioned rats comparedwith untreated 6-OHDA-lesioned and sham-operated ones(Figure 3C), suggesting that chronic L-DOPA drives D1Rexpression in LHb neurons that normally express very lowlevels (Figure 3C). In addition, double immunofluorescence ofD1R/∆FosB revealed a co-localization of ∆FosB and D1Rin LHb of the lesioned side of dyskinetic rats (Figure 3D),suggesting that, as in other areas receiving dopaminergicinput, ∆FosB rise is induced by D1R stimulation. Of note is thefact that the number of ∆FosB immunopositive neurons in LHbwere stable in those animals terminated at the end of theDaun02 experiment compared with dyskinetic animals termi-nated before the Daun02 experiment (p . .5), suggesting thattransient (but relatively prolonged) silencing of LHb neuronsdoes not impact ∆FosB expression.

ß-galactosidase immunofluorescence confirmed thatexpression of the FosB/LacZ lentivirus was restricted to LHb(Figure 3E), confirming the specificity of LHb in the observedbehavioral manifestations.

DISCUSSION

LID has been associated with both presynaptic and postsy-naptic mechanisms at the striatal level in the basal ganglia (3–5).In this study, we report that LHb is functionally and behavior-ally involved in LID pathophysiology in accordance withgrowing evidence supporting the involvement of structuresoutside the basal ganglia in LID (6,9,57,58). First, we demon-strated a LID-related pathologic activity of LHb at differentfunctional levels including metabolic, transcriptional, andelectrophysiological readouts, indicating that increased LHbactivity in response to L-DOPA treatment is associatedwith LID expression. Then, selective inactivation of ∆FosB-expressing habenular neurons both alleviated LID severity andenhanced L-DOPA antiparkinsonian action, suggesting aninvolvement of LHb both in LID severity and in the antiparkin-sonian effect of L-DOPA therapy. Taken altogether, our resultshighlight a key role of LHb in the genesis of dyskinesiamanifestation outside of the basal ganglia.

The amount of 2-DG uptake correlates directly with themagnitude of the mean synaptic activity and is, therefore,considered to be a measure of the global afferent activity of astructure (59–61). However, this technique does not allowdistinction between a modification in excitatory and in inhib-itory afferent activity (62). Furthermore, 2-DG uptake reflectsthe activity of all cellular elements in the region of interest, i.e.,perikarya, dendrites, axonal fibers, and glia. Despite theselimitations, a classic example of dissociated 2-DG uptake andelectrical activity is given by the subthalamic nucleus (STN) inPD that becomes hyperactive and bursty while displaying adecreased 2-DG accumulation (6,11–13,63). In addition, dis-inhibition of STN neurons by local injection of bicuculline, a

gamma-aminobutyric acid antagonist, increases the firing rateof STN neurons, as well as the firing rate and 2-DG uptake inthe globus pallidus, the entopeduncular nucleus, and thesubstantia nigra pars reticulata in the rat (62). The oppositecan be observed when locally injecting muscimol, a gamma-aminobutyric acid agonist, suggesting that inhibition of STNneurons decreases the mean afferent activity and firing rate inits target nuclei (62).

In our study, the metabolic (2-DG) and electrophysiologicalcharacteristics of the LHb are suggestive of a role of LHb inthe pathophysiology of LID. LHb is mainly innervated by theoutput structures of the basal ganglia, while minor afferentsarise from the ventral tegmental area, lateral hypothalamus,and lateral preoptic area (64–69). LHb indeed receives inhib-itory afferents from the ventral pallidum (65,66) but alsoreceives excitatory afferents from the border cells of theinternal part of the globus pallidus (GPi) (64,65). The elevatedLHb 2-DG uptake in parkinsonian monkeys (compared withcontrol and L-DOPA-treated ones) therefore suggests anincrease in afferent activity converging toward the LHb, inkeeping with initial studies (11,12). The GPi overactivity in PD(70–75) was thought to be responsible from such an increase.Contrary to most GPi neurons, LHb-innervating excitatory GPiborder cells (64,65) show a decreased firing rate in parkinson-ism (76), possibly explaining the lack of difference in LHbneuronal discharge frequency between control and 6-OHDA-lesioned rats. In LID, however, GPi border cells show asignificant increase in firing rate associated with a patternmodification compared with parkinsonism and control states(76). Consequently, it can be speculated that increasedhabenular neuron firing rate in LID is driven by border cellinput or alternatively to the general decrease in inhibitory basalganglia output, GPi in primates and substantia nigra parsreticulata in rodents, characteristic of LID in dyskinetic PDpatients (72–75), in dyskinetic MPTP monkeys (27,71), and indyskinetic 6-OHDA rats (38).

LHb is primarily considered to be a relay connecting thelimbic system and the basal ganglia with monoaminergiccenters (77). LHb projects mainly to monoaminergic brainregions including dopaminergic areas (i.e., ventral tegmentalarea and substantia nigra pars compacta) serotoninergic areas(i.e., dorsal and medial raphe) and also to the cholinergiclaterodorsal tegmentum (77–80). Recent evidence suggeststhat LHb plays a critical role in dopaminergic-related proc-esses including drug abuse and reward (77,80–83). Interest-ingly, cocaine administration increases LHb neuronal firingfollowing D1R and D2 receptor stimulation (82), while specificLHb inactivation through deep brain stimulation decreasescocaine-seeking behavior (84). Dopaminergic receptor activa-tion through systemic apomorphine injection strongly enhan-ces spontaneous activity of distinct habenular neuron subsets(85). Those data are reminiscent of the present results withhyperdopaminergic-induced increased activity and inhibitionof LHb neurons resulting in improvement of the hyper-dopaminergic-induced behavior.

Interestingly, increased expression of D1R in LHb of dys-kinetic 6-OHDA-lesioned rats ipsilateral to the lesion suggestsa D1R-related mechanism in engaging LHb in LID pathophysi-ology. LID derives from sensitized D1 receptors due to chronicL-DOPA stimulation (15,86). Therefore, while the key role of





striatal D1R in LID has been well described, proposing a rolefor extrastriatal (e.g., intra-LHb) D1R may seem provocative. Itdoes, however, share enough similarity with striatal involve-ment to be a realistic hypothesis (3–5). Indeed, as in thestriatum, L-DOPA induces an overexpression of ∆FosB in LHb,which 1) correlates with LID severity, 2) co-localizes with D1R,and 3) drives, at least in part, LID expression. Fos-likeimmediate early genes are directly related to the D1R pathwayboth in the striatum (7,54–56) and LHb (87), as their expression

is directly enhanced by specific D1R agonist. Role of D1Rremains, however, to be experimentally demonstrated.

Therefore, we suggest that the alleviation of AIM severitythrough the inactivation of habenular ∆FosB-expressing neu-rons occurs mainly in D1-positive neurons, putatively involvinghabenular D1R/∆FosB in LID pathophysiology. How LHbneurons impact upon the expression of LID remains unre-solved. LID pathophysiology involves postsynaptic mecha-nisms but also presynaptic dysfunction. Notably, in advancedPD, it is believed that exogenous L-DOPA is mostly taken upby serotonergic terminals, dopamine becoming the falseneurotransmitter of those serotonin neurons (88,89). LHbprojects heavily upon serotonin neurons of the raphe (78). Inboth PD and toxin-induced parkinsonism, while the dopami-nergic areas are markedly lesioned, the serotonergic ones arerelatively preserved (90,91). Thus, increased LHb electrophy-siological activity in AIMs might participate in the aberrantdopamine release from serotonin terminals (90,92–95) andhence impact LID.

In preclinical models, most antidyskinetic drugs can neg-atively affect the duration and/or magnitude of the therapeuticeffect of L-DOPA, highlighting their lack of strict selectivity forthe underlying mechanisms of LID (96,97). In this study, LIDreduction through selective inactivation of LHb was associ-ated with a remarkable increase in L-DOPA-induced rotationalbehavior. Therefore, even if the increase in rotational behaviorcould be a consequence of a decrease in AIMs and vice versa,LHb should be considered as a key player in mediating theantiparkinsonian effect of L-DOPA through specifically ∆FosBexpressing neurons.

Conclusion

Our results show that LHb is involved both in LID and in theantiparkinsonian effect of L-DOPA. LID is associated withmetabolic, electrophysiological, and transcriptional events inLHb, while the inactivation of habenular neurons alleviates LID.Even though the underlying mechanisms involving LHb in LIDpathophysiology are not yet completely elucidated, our datasuggest that these effects are mediated, at least in part, byD1R/∆Fosb-expressing neurons. Taken altogether, our resultshighlight the role of LHb in LID, offering a new target toinnovative treatments of LID.

ACKNOWLEDGMENTS AND DISCLOSURES

This work was supported by Agence Nationale de laRecherche Grants (EB: ANR-07-MNP-Trafinlid), the Fondationde France (EB), and Grant LABEX BRAIN ANR-10-LABX-43.MFB is the recipient of a Ministry of Higher Education andResearch Grant. The Université Bordeaux Segalen and theCentre National de la Recherche Scientifique provided infra-structural support. The funders had no role in study design,data collection and analysis, decision to publish, or prepara-tion of the manuscript.

We are grateful to Pr. Alan R. Crossman, University ofManchester, United Kingdom, for his input to the manuscript.

Dr. Erwan Bézard has equity stake in Motac Holding Ltdand receives consultancy payments from Motac NeuroscienceLtd. Messrs. Matthieu Bastide and Brice de La Crompe,

Figure 3. Inhibition of ∆FosB-expressing lateral habenula (LHb) neuronsalleviates L-3,4-dihydroxyphenylalanine (L-DOPA)–induced dyskinesia. (A)Cumulated axial, limb, and orofacial (A.L.O.) abnormal involuntary move-ments (AIMs) scores in L-DOPA-treated 6-hydroxydopamine (6-OHDA) rats(n 5 12) before and after Daun02 and after control solution injection(*p , .05 from baseline and control). (B) Cumulated rotation scores inL-DOPA-treated 6-OHDA rats (n 5 12) before and after Daun02 and aftercontrol solution injection (*p , .05 from baseline and control). (C) Repre-sentative LHb mapping of dopamine D1 receptor (D1R) expression in sham-operated (SHAM), 6-OHDA-lesioned (6-OHDA), and Daun02-injected6-OHDA-lesioned dyskinetic rats (Dysk). Scale bar 50 mm. (D) Co-localizationof D1R/∆FosB (*) expression in LHb neurons in the Daun02-injected 6-OHDA-lesioned side of dyskinetic rats. Scale bar: 5 mm. (E) Representative LHbß-galactosidase (ß-Gal) expression in the Daun02-injected side of dyski-netic rats (scale bar: 100 mm) with an inset (scale bar: 10 mm).





Ms. Evelyne Doudnikoff, and Drs. Pierre-Olivier Fernagut,Christian E. Gross, Nicolas Mallet, and Thomas Boraudreported no biomedical financial interests or potential conflictsof interest. Current Grant support includes Agence Nationalede la Recherche (EB, CEG), China Science Fund (EB), MichaelJ. Fox Foundation (EB), FP7 from European Union (EB), FranceParkinson (EB, P-OF), Fondation de France (EB), CariploFoundation (EB), and United Kingdom Medical ResearchCouncil (EB).

ARTICLE INFORMATION

From the Université de Bordeaux (MFB, BdlC, ED, PF, CEG,NM, TB, EB), Institut des Maladies Neurodégénératives;National Centre for Scientific Research (MFB, BdlC, ED, PF,CEG, NM, TB, EB), Institut des Maladies Neurodégénératives,Bordeaux; and Centre Hospitalier Universitaire de Bordeaux(CEG), Bordeaux, France.

Address correspondence to Erwan Bézard, Ph.D., Univer-sité de Bordeaux, Institut des Maladies Neurodégénératives,Campus de Carreire, Bât 3B 1er étage, 146 rue Léo Saignat,Bordeaux 33076, France; E-mail: [email protected].

Received May 23, 2014; revised Aug 27, 2014; acceptedAug 29, 2014.

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Inhibiting Lateral Habenula Improves L-DOPA–induced Dyskinesia