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Experimental Neurology 162, 215–224 (2000)doi:10.1006/exnr.1999.7331, available online at http://www.idealibrary.com on

Functional Activity of Zona Incerta Neurons Is Altered after NigrostriatalDenervation in Hemiparkinsonian Rats1

Celine Perier,* Miquel Vila,* Jean Feger,*,† Yves Agid,* and Etienne C. Hirsch**INSERM U 289, Hopital de la Salpetriere, 47 Boulevard de l’Hopital, 75013 Paris, France; and †Faculte de Pharmacie,

4 avenue de l’observatorie, 75006 Paris, France

Received July 29, 1999; accepted November 22, 1999

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The cellular expression of cytochrome oxidase sub-nit I (COI) mRNA as a metabolic marker for neuronalctivity has recently been used to examine the effectsf nigrostriatal denervation on the functioning of theasal ganglia. However, this technique also allowsunctional changes to be detected in other cerebraltructures in parkinsonian syndromes. Since the zonancerta has been implicated in locomotor activity andas been the site of stereotactic surgery in Parkinson’sisease, the aim of our study was to determine whetherhanges in neuronal activity are observed in thistructure during parkinsonism. Using in situ hybridiza-ion, we analyzed the expression of COI mRNA in ratsith 6-hydroxydopamine unilateral lesion of the sub-

tantia nigra and sham-operated animals. A quantita-ive analysis showed that COI mRNA expression wasncreased in the zona incerta ipsilateral to the lesion4 h and 3 days after lesion, but by day 14 had returnedlmost to the level observed in controls. The hyperac-ivity of zona incerta neurons was confirmed by single-nit electrophysiological recordings. In contrast to theOI mRNA expression, the increase in electric neuro-al activity was still observed 1 month after the lesion.his increase in zona incerta neuronal activity afterigrostriatal denervation might be related to the patho-hysiology of parkinsonism, at least in the early stages,

n agreement with previous reports suggesting annvolvement of the zona incerta in motor functions.2000 Academic Press

Key Words: zona incerta; 6-OHDA-lesioned rats; cyto-hrome oxidase; in situ hybridization; electrophysiol-gy; Parkinson’s disease; basal ganglia.

INTRODUCTION

Parkinson’s disease is a neurodegenerative diseaseharacterized mainly by motor signs such as akinesia,igidity, and tremor. These motor symptoms result from

1 This study was supported by INSERM (France) and the National

tarkinson’s Disease Foundation (Miami, Florida).

215

he severe reduction in striatal dopamine concentra-ions (11). In turn, dopamine depletion provokes aascade of functional changes in the basal gangliaircuitry. In the late 1980s, a model was developed thatroposed a framework for the organization of the basalanglia and provided a plausible explanation for thelinical syndrome of Parkinson’s disease (1, 2). Never-heless, it now appears that this model cannot explainhe whole pathophysiology of the disease, and severalines of evidence indicate that certain connections anderebral structures were omitted in this model (16, 25,6). It was thus necessary to reanalyze the effects ofigrostriatal denervation on the functional changes inhe neuronal circuitry downstream from the lesion. Inhis respect, the zona incerta (ZI), a diencephalictructure, which has recently been described as a relayetween the brain stem and the higher centers (15), isnvolved in locomotor function (21). Increased locomo-or activity has been reported after electrical stimula-ion and injection of pharmacological agents in the zonancerta (20, 30–32). Furthermore, anatomical studiesn rats suggest an involvement of the ZI in motor andculomotor functions, due to its connections with theedunculopontine nucleus, the substantia nigra parseticulata, and the superior colliculus (15, 19, 24, 28,9). In addition, stereotactic surgery aimed at destroy-ng the zona incerta area in parkinsonian patients haseen shown to relieve the motor symptoms (22), whichuggests that this structure may have a role in theathophysiology of the disease. The aim of our studyas to determine whether the neuronal activity of theI is changed in an animal model of Parkinson’sisease. To address this problem, we measured cyto-hrome oxidase expression in order to study the changesn overall metabolic activity of zona incerta neurons inarkinsonism. This method is based on the analysis ofhe cellular expression of the mRNA coding for cyto-hrome oxidase subunit I (COI) (35), which allows annalysis of global changes in functional activity ofeurons, even when markers of neurotransmission areot available (for review, see Wong-Riley (37)). In order

o detect any changes in ZI activity, we first analyzed

0014-4886/00 $35.00Copyright r 2000 by Academic Press

All rights of reproduction in any form reserved.

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he expression level of COI mRNA by in situ hybridiza-ion in rats with 6-OHDA unilateral lesion of theubstantia nigra and sham-operated animals and thennalyzed electrical activity by unit extracellular electro-hysiological recordings in the same model of Parkin-on’s disease.

METHODS

xperimental Animals

Male Sprague–Dawley rats weighing 280–350 gCERJ, Le Genest St-Isle, France) were housed on a2-h light/dark cycle under constant temperature andumidity conditions and given free access to food andater. All studies were carried out in accordance with

he Declaration of Helsinki and the Guide for the Carend Use of Laboratory Animals adopted and promul-ated by National Institutes of Health. Ten groups ofnimals were analyzed (five groups with 6-OHDA le-ion and five groups with sham lesion). One group with-OHDA lesion and one group with sham lesion wereacrificed at each of the following times: 24 h, 3 days, 7ays, 14 days, and 1 month after lesion. A minimum ofve animals from each group was used both for thelectrophysiological recordings and the in situ hybrid-zation study. Due to possible tissue alteration duringlectrophysiological recording, different animals weresed for electrophysiology and in situ hybridization.

-Hydroxydopamine Lesions

Rats were pretreated with desipramine hydrochlo-ide (25 mg/kg, ip) and pargyline (50 mg/kg, ip) in ordero protect noradrenergic neurons and inhibit mono-mine oxidase, respectively, 30 min prior to infusion of-OHDA (12). They were then anesthetized with 0.2l/100 g (im) of a solution containing 50 mg/ml of

etamine and 80 mg/ml of xylazine. The rats were fixednder stereotaxic conditions with the incisor bar 3.3m below the interaural line (27). A stainless steel

anula, outer diameter 0.3 mm, connected with aatheter to a microsyringe (10 µl airtight, Hamilton)ounted on a motorized driver, was placed in the

ubstantia nigra at the following stereotaxic coordi-ates: anteroposterior (AP) 25.2 mm from the bregma,ediolateral (MD) 1.8 mm. The tip of the canula was

owered 7.3 mm below the dura. A single unilateralicroinjection of 6-OHDA (8 µg in 2 µl of saline

ontaining 0.01% ascorbic acid) was performed for 5in and the canula was left in place for a further 5 min

efore withdrawal. Sham-lesioned animals receivedhe same volume of 0.01% ascorbic acid dissolved inaline. Animals were killed with a lethal dose ofentobarbital at different times after lesion (from 24 ho 14 days for in situ hybridization and from 3 days to 1

onth for electrophysiological recording). t

issue Preparation

Immediately after death, brains were removed fromhe skull and rapidly frozen in isopentane at 240°C.or in situ hybridization, 20-µm sections were cut at20°C in the frontal plane using a cryostat (Reicher,eidelberg, Germany), mounted on gelatin double-

oated slides, and stored at 280°C until processing.

mmunoautoradiography of Dopamine Transporter

The degree of nigrostriatal denervation was assessedy immunoautoradiography using the striatal dopa-ine transporter (DAT) as a marker of dopaminergic

bers, as previously described (23). Briefly, nonspecificinding sites were saturated by incubating fresh frozenrain sections for 30 min in a buffered saline solutionPBS; 50 mM, pH 7.4) containing 1% bovine serumlbumin (BSA). The sections were incubated for 12 h at°C with the primary antibody (anti-DAT polyclonalntibody, raised in rabbits; 1:20,000 dilution). Anti-AT antibodies were raised against the N-terminalortion of the rat DAT protein (amino acids 42–59), asescribed elsewhere (7). After three 10-min washes inBS, the tissue sections were incubated for 2 h in aolution of PBS/BSA containing 35S-labeled anti-rabbitgG (Amersham, Buckinghamshire, United Kingdom;p act 780 Ci/mmol; 1:100 dilution; initial concentra-ion: 20 µg/ml) and finally rinsed three times for 10 minn PBS. For autoradiography, Hyperfilm bmax photo-raphic film (Amersham) was placed in contact withhe slides in X-ray cassettes and exposed at roomemperature for 3 days.

uantification of DAT Autoradiograms

In the striatum, the area of DAT radioimmunolabel-ng was estimated on autoradiograms by image analy-is (Biocom, Les Ulis, France). For each animal, mea-urements of the optical density of the striatum fromhe nonlesioned hemisphere and the striatum on the‘lesioned’’ side were performed (3), and the ratio be-ween the two measurements was calculated so as toake into account any interindividual variations. Eachesioned group was compared to its sham-lesionedontrol group using an unpaired t test (Sigma Stat 3.2.,andel Scientific, San Rafael, CA).

ynthesis of COI RNA Probe

For the in situ hybridization of COI, a cRNA probeas synthesized from a double-stranded DNA frag-ent, corresponding to nucleotides 5308–6218 of the

at mitochondrial genome (EMBO databank, Ref.IRNXX) within the gene coding for COI, produced byCR and subcloned in the pGEM-T vector (Promega,adison, WI). Sense and antisense cRNA probes were

ranscribed from 1 µg plasmid, as described by Fon-

aine et al. (6).

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217ZONA INCERTA ACTIVITY IN PARKINSONISM

n Situ Hybridization

In situ hybridization with a 35S-labeled cRNA probeas performed as previously described (35). Briefly,nfixed slide-mounted frozen sections were postfixedor 5 min in 3% paraformaldehyde (wt/vol) and thencetylated with 0.25% acetic anhydride in 0.1 mMthanolamine, followed by 0.1 M Tris–glycine treat-ent for 30 min and dehydration in graded ethanol

olutions. Sections were incubated for 3.5 h at 50°C in aumid chamber with 50 µl of hybridization solutionontaining either the antisense or the sense 35S-labeledRNA probe (2.5 3 106 cpm). After hybridization, sec-ions were washed at 50°C in 50% formamide/23 standardaline citrate (SSC), incubated for 30 min at 37°C withNaseA(100 µg/ml in 23 SSC) in order to digest unhybrid-

zed probe, rinsed again at 50°C in 50% formamide/23SC, and then washed overnight at room temperature.fter a final rinse in 23 SSC, the sections were dehydrated

n graded ethanol solutions (70, 80, and 95%) preparedith 300 mM ammonium acetate, delipidated in xylene,

insed in 100% ethanol, and air-dried. Autoradiogramsere generated by exposing the slides to X-ray films

Hyperfilm bmax, Amersham) for 1 day at room tempera-ure. Sections were then dipped in NTB-2 emulsion (Ko-ak, Integra Biosciences, Eaubonne, France), diluted 1:1,ir-dried, and stored at 4°C in lightproof boxes for 1 week.xposed slides were developed in Kodak D-19 for 4 min at5°C and counterstained with hematoxylin 0.1% (w/v) toocalize cell nuclei. For each experiment, two sections perubject were utilized and each experiment was duplicated.

uantification of COI In Situ Hybridization

The level of expression of COI mRNA per neuron wasuantified by computer-assisted image analysis (Histo00, Biocom, Les Ulis, France) as previously described35). The number of silver grains over the neuronal cellody was estimated under polarized light by measuringptical density with respect to a standard curve of aefined number of silver grains. Grain density (numberf silver grains per surface area of the neuron) was thenalculated. Nonspecific labeling was estimated withense probes. In order to delimit the boundaries of theI, CO histochemistry was performed on adjacentections (Fig. 1A) according to Vila et al. (34). Thenalysis was performed for the ZI taken as a whole andor each half of the structure divided in a dorsoventral

FIG. 1. (A) Cytochrome oxidase histochemistry on a frontal ratection passing through the zona incerta. (B) At a higher magnifica-ion the staining appears heterogeneous in the zona incerta. (C)chematic representation of the subdivision of the zona incerta into

our parts: (dl) dorsolateral, (dm) dorsomedian, (vm) ventromedian,vl) ventrolateral. ML, medial lemniscus; ZI, zona incerta; ZId, dorsalona incerta; ZIv, ventral zona incerta; STN, subthalamic nucleus.

cale bar indicates 2 mm in A and 1 mm in B, C, and D.

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218 PERIER ET AL.

lane (Fig. 1C) and in a lateromedial plane (Fig. 1D).hese subdivisions were made using arbitrary defini-ions that were reproducible from one section to an-ther. We first compared the silver grain density on theide contralateral to the lesion between sham-lesionednd 6-OHDA-lesioned animals. Since no significantifference was observed for this parameter, the ratio ofilver grain density (grains/µm2) over labeled cellsetween the side ipsilateral to the lesion and theontralateral side was calculated, in order to take intoccount any interindividual variations, possibly arisingrom differences in metabolic activity between animals.ach lesioned group was compared to its sham-lesionedontrol group using an unpaired t test. The null hypoth-sis was rejected at an a risk of 5%.

lectrophysiological Recordings

Extracellular single-unit recordings were made atifferent times after lesion (3 days, 7 days, 14 days, and

month) in rats anesthetized with urethane (1.2g/kg, ip). Body temperature was maintained at 37 6

.5°C using a thermostatically regulated heating pad.he stereotaxic coordinates used to localize the ZI weredapted from the rat brain atlas of Paxinos and Watson27): AP 23.2 to 23.8 posterior to the bregma; MD 1.7 to.6 and 6.3 to 7.8 below the dura. Unit activity wasecorded using glass micropipettes filled with 1% pon-amine sky blue in 0.5 M sodium acetate. The imped-nce was within the range of 10 to 15 MV measured at00 Hz. The signals were amplified (DAM5 WPI),isplayed on a storage oscilloscope, and recorded onagnetic tape. Single-unit activity was processed usingwindow discriminator providing pulses sent to the

nput of an integrator or processed using a computerInterface micro 1401 and spike 2 software, Cambridgelectronic Design, United Kingdom). The localizationf the tip of the microelectrode was marked by anontophoretic deposit of pontamine sky blue at the endf the experiment. The animal was then killed with anverdose of anesthetic. In order to verify the localiza-ion of the electrode, 50-µm-thick histological sectionsere cut using a freezing microtome and stained with

resyl violet (Fig. 2A). Based upon the location of thelue spot within the zona incerta, it was possible toetermine the approximate locations of the other neu-ons recorded.

ata Analysis of Electrophysiological Recordings

Differences in the discharge rate (spikes per second)f ZI neurons between 6-OHDA-lesioned rats and con-rol animals were analyzed by comparing the meaneuronal discharge rate recorded during a 3-min pe-iod, using an unpaired t test. Differences in the firingattern of the ZI neurons between control and lesioned

ats were determined by comparing their discharge e

ensity histograms according to the method developedy Kaneoke and Vitek (13).

RESULTS

AT Immunoradiolabeling

Dopaminergic denervation was quantified in-OHDA-lesioned rats by striatal DAT radioimmunola-eling measurements. A simple visual examinationndicated that by day 3 after 6-OHDA injection, stain-ng intensity ipsilateral to the lesion had alreadytarted to decline compared to that in sham-lesionednimals and that it declined progressively between dayand day 14 after 6-OHDA injection, indicating striatalopamine depletion induced by the neurotoxin (Fig. 3).hese qualitative data were confirmed by a quantita-

ive analysis in lesioned animals, which showed aecrease in striatal DAT radioimmunolabeling ipsilat-

FIG. 2. (A) Frontal section showing the lower recording site inhe zona incerta (ZI) marked with iontophoretically injected pon-amine sky blue, indicated in the figure by an asterisk. Pontamineky blue was injected at the end of the recording track, at the lowerite of recording. The dotted line delimits the ZI. Scale bar indicates.35 mm. STN, subthalamic nucleus. (B) Chart showing the place-ent of the recording electrodes in the ZI.

ral to the lesion compared to the nonlesioned side from

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219ZONA INCERTA ACTIVITY IN PARKINSONISM

FIG. 3. Characterization of the unilateral lesion of the substantia nigra according to the animal’s survival time, assessed byutoradiography of the dopamine transporter (DAT) and COI mRNA expression detected by in situ hybridization on the lesioned andonlesioned side. In 6-OHDA-lesioned rats, striatal (Str) DAT radioimmunolabeling ipsilateral to the lesion decreased progressively from 3ays to 14 days after 6-OHDA injection in the substantia nigra pars compacta. In the zona incerta (ZI) ipsilateral to the 6-OHDA lesion, COIRNA expression was increased 24 h and 3 days after the lesion compared to the nonlesioned side. Scale bars indicate 2.5 mm in the left

olumn and 0.5 mm in the right column.

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2 h (260%) to 14 days (285%) after 6-OHDA injectionTable 1). In sham-lesioned animals, no side-to-sideifferences in DAT radioimmunolabeling were found athe striatal level.

O Histochemistry on Rat Sections

We observed a heterogeneous pattern of labeling,eproducible from one brain to another (Figs. 1A andB). This allowed the ventral portion of the structure toe clearly identified from its intense staining for CO,ompared to the dorsal portion.

OI mRNA Expression in 6-OHDA-Lesioned Rats

The specificity of the labeling was attested by theollowing criteria. (i) A reproducible pattern of hybrid-zation was obtained in the ZI with the antisense COIRNA probe on film autoradiograms and, at the cellularevel, on emulsioned sections. (ii) Control sectionsybridized with the sense COI cRNA did not show anylustered silver grains or any labeling above back-round levels on macroscopic or microscopic examina-ion. (iii) Examination of hematoxylin-counterstainedections showed that COI mRNA was concentratedainly in the neuronal cell bodies, with a signal well

bove the background level. Much weaker labeling wasetected outside the neuronal cell bodies due to theresence of mitochondria in the neuropil.In sham-lesioned animals, no side-to-side differences

n COI mRNA expression were found in the ZI. Noifferences were found between the side contralateralo the lesion in the lesioned animals and the correspond-ng side in sham-lesioned animals (Table 2). In the ZIpsilateral to the 6-OHDA lesion, a significant increasen COI mRNA expression was observed, compared toham-lesioned animals, from 24 h after 6-OHDA intoxi-ation (126%) reaching a peak at 3 days (146.5%), thenecreasing to a level close to that observed in controls111.2% at 7 days and 14.5% at 14 days) and no longerignificantly different (Fig. 3, Table 3).An analysis of the different sectors of the ZI showed

hat COI mRNA expression was significantly increasedn the dorsal and lateral parts of the ZI compared to

TABLE 1

Optical Density Ratio of Dopamine Transporter (DAT)mmunoautoradiographic Labeling in the Striatum, at Differ-nt Survival Times after Unilateral 6-OHDA Lesion of theubstantia Nigra

Control 24 h 3 days 7 days 14 days

AT 1.07 6 0.04 0.98 6 0.03 0.43 6 0.04* 0.22 6 0.02* 0.17 6 0.03*

Note. The data correspond to the ratio of the values on the lesionedide to those on the nonlesioned side.

* P , 0.05 (unpaired t test). l

ther subdivisions for rats sacrificed 24 h and 3 daysfter the lesion (Fig. 4). These data suggest that not allI neurons are hyperactive after nigrostriatal denerva-ion.

lectrophysiological Recordings

The mean discharge rate, expressed as means 6EM, of ZI neurons was 2.16 6 0.27 spikes/s (Table 4)nd ranged from 0.17 to 6.79 spikes/s (n 5 37 neurons).Compared to the control rats, a marked increase in

he mean discharge rate of ZI neurons was detectedrom 3 days after 6-OHDA nigral lesion (1104.6%,

5 27 neurons) and remained significantly higher 7ays (1198.6%, n 5 43 neurons), 14 days (1280.1%,5 36 neurons), and 1 month (1306%, n 5 24 neu-

ons) after the lesion. We found that the mean dis-harge rate was significantly different between animalsilled 3 and 14 days (P , 0.005) and between thoseilled 3 days and 1 month (P , 0.01) after the lesion.

TABLE 2

COI mRNA Expression Detected by in Situ Hybridizationn the ZI of Unilateral 6-OHDA-Lesioned Rats, on the Sideontralateral to the Lesion

ostlesion delay Sham-lesioned animals 6-OHDA-lesioned animals

1 day 1.61 6 0.24 1.77 6 0.373 days 0.85 6 0.07 1.18 6 0.247 days 1.22 6 0.13 1.02 6 0.16

14 days 1.40 6 0.23 1.53 6 0.36

Note. COI mRNA expression was estimated on emulsioned sec-ions, in the ZI neurons of unilateral 6-OHDA-lesioned animals andham-lesioned animals on the side contralateral to the lesion. Theata correspond to the mean 6 SEM of silver grain density overabeled cells in the zona incerta on the nonlesioned side. No differ-nces were found between the lesioned side and the correspondingide in sham-lesioned animals.

TABLE 3

COI mRNA Expression Detected by in Situ Hybridization inthe ZI of Unilateral 6-OHDA-Lesioned Rats

ostlesion delay Sham-lesioned animals 6-OHDA-lesioned animals

24 h 0.95 6 0.05 1.21 6 0.08*3 days 0.98 6 0.02 1.44 6 0.18*7 days 0.92 6 0.06 1.03 6 0.12

14 days 0.91 6 0.04 0.95 6 0.11

Note. COI mRNA expression was estimated on emulsioned sectionsn the ZI neurons of unilateral 6-OHDA-lesioned animals and sham-esioned animals. The data correspond to the ratio of silver grainensity over labeled cells in the zona incerta on the lesioned side tohat on the nonlesioned side.

* P , 0.05 and indicates a significant difference between the

esioned side and the nonlesioned side.

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221ZONA INCERTA ACTIVITY IN PARKINSONISM

he localization of the microelectrode is illustrated inig. 2B. Most of the recorded neurons were located inhe central part of the zona incerta, but over the wholeorsoventral extent of the structure.The increases in mean discharge rate were not

ssociated with changes in the pattern of spikingctivity. Indeed, an analysis of the density dischargeensity histograms using the method of Kaneoke anditek (13) did not show evidence of any difference in theattern of spiking activity between controls and le-ioned animals. The interspike interval histogramhowed a shift to the right (Fig. 5), reflecting a decreasen the interspike interval, associated with an increasen the mean discharge rate.

DISCUSSION

In this study we found that ZI neurons are hyperac-ive after nigrostriatal denervation in rats. This hyper-ctivity was assessed by two independent methods: initu hybridization of COI mRNA expression and electro-hysiological recordings. The changes in COI mRNA

FIG. 4. COI mRNA expression in different sectors of the ZI in ratsacrificed 3 days after the lesion. COI mRNA expression was signifi-antly increased in the dorsal and lateral parts of the ZI but not in thether parts of the structure. Black bars represent the silver grainensity in 6-OHDA-lesioned rats. White bars represent the silverrain density in sham-lesioned rats. *Significantly different (P , 0.05),aired t test.

TAB

Characteristics of ZI Neuron Activity in Control Rand 1 Mont

Control (n 5 5) 3 days (n 5 5)

iring rate (spike/s) 2.16 6 0.27 4.42 6 0.69*

Note. Characteristics of the firing rate of a population of ZI neuridbrain dopaminergic neurons sacrificed at different times after le

epresent the mean 6 SEM.

* P , 0.05, compared to controls.

xpression were first observed 24 h after nigrostriatalenervation. Whereas the changes in COI mRNA ex-ression were no longer detectable in animals sacri-ced 7 and 14 days after the lesion, changes in electri-al activity persisted 1 month after the lesion. Asuggested by the analysis of the four subdivisions of theI, the hyperactive neurons were preferentially located

n the dorsal and lateral parts of the ZI. These datauggest that, after nigrostriatal denervation, metabolicctivity is differentially regulated in the ventral andhe dorsal parts of the ZI. Differences between theorsal and the ventral parts of the ZI were observed inontrol rats on CO histochemically stained sections, asreviously reported (24). These variations might bexplained by differences of connectivity between thewo parts of the ZI. In this context it is known that mostrain-stem inputs and outputs of the ZI, such as theidbrain reticular nucleus, the pontine reticular

ucleus, the ventral tegmental area, the pedonculopon-ine nucleus, the dorsal raphe, the periaqueductal grayatter, and the substantia nigra pars reticulata, arise

nd terminate in the dorsal parts of the ZI (15). Inontrast, projections from the ZI to the deep layers ofhe superior colliculus arise from the ventral part of thetructure, whereas it is the dorsal part of the ZI thateceives inputs from the deep layers of the superiorolliculus (15, 24). However, our electrophysiologicalata indicate that the hyperactive neurons were alsoocated in the central part of the zona incerta. Thus, its likely that all ZI neurons were hyperactive after theesion, with an even more active subpopulation in theorsolateral part, as indicated by metabolic data. Elec-rophysiological recording in this sector of the ZI wouldelp to clarify this point.

oncomitant Increases in Expression of COI mRNAand Electrophysiological Activity in ZI Neurons afterNigrostriatal Denervation

The use of the mitochondrial enzyme CO as a meta-olic marker for neuronal functional activity is nowell established (for review, see Wong-Riley (37)). Thectivity of this enzyme is regulated by changes in theynaptic firing pattern (8, 9), and the enzyme is respon-

4

s and in Rats Recorded 3 Days, 7 Days, 14 Days,fter Lesion

7 days (n 5 7) 14 days (n 5 6) 1 month (n 5 5)

6.45 6 1.04* 8.21 6 0.74* 8.77 6 1.11*

recorded in control rats and rats with 6-OHDA-induced lesion ofn. n denotes the number of animals studied in each group. All data

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ive to alterations in neuronal activity (34). Moreover, aecent study has shown that both intraneuronal activ-ty and unit electrical activity are increased in theubthalamic nucleus after nigrostriatal denervationnd thus that the measurement of COI mRNA expres-ion represents a good index of functional activity ineurons at the cellular level (36).In the present study, we found a metabolic activation

nd an increase in electrical activity (data not shown)4 h after 6-OHDA intoxication. However, due to theifficulty of keeping the animals alive for electrophysi-logical recordings 24 h after a first anesthesia, theesults of electrophysiological recordings cannot beonsidered significant. An increase in electrical activityas observed from 3 days to 1 month after lesion,hereas metabolic activation, which was markedly

ncreased 3 days after lesion, returned to a level close tohat of controls by day 7 and remained at this level atay 14. Thus, the study shows that the neuronalctivity changes persisted during a time period inhich changes in COI mRNA were no longer observed,

uggesting a dissociation between the two measure-ents. Nevertheless, it has been reported that bursting

ctivity elicits the expression of c-Fos within the post-ynaptic neurons, whereas regular stimulation doesot (4), and that cytochrome oxidase is regulated byhanges in the firing pattern (8, 9). In our study, the ZIeurons did not show any changes in their firingattern after nigrostriatal denervation, which couldxplain why this enzyme is down-regulated while elec-

FIG. 5. Histograms (A, B) showing changes in interspike intervalsischarge rate of unit activity of neurons recorded in the zona incertaB, D). The interspike interval histograms show a shift to the left, refl

rical activity is still high. It is likely that the protein t

ranslated in the first days after the lesion is sufficiento produce the necessary energetic supply for changesn neuronal activity to occur.

The relative changes in neuronal firing activity werereater than the changes in COI mRNA expression.his result is in agreement with two previous studies,

n which changes in metabolic activity, assessed byuantitative 2-deoxyglucose autoradiography (5), or inOI mRNA expression (36), were compared to neuronalctivity, in the subthalamic nucleus. The divergenceetween the relative increase in the metabolic andlectrophysiological activity compared to the controlevel might be due to differences in sensitivity betweenhe two techniques. Alternatively, it could be due to theact that, even if CO levels reflect changes in theynaptic firing pattern, this enzyme also participates inhe production of energy necessary for basal biologicalellular functions not directly linked to the generationf action potentials and neurotransmitter release.

rigin of ZI Hyperactivity

One or more of the following three hypotheses couldxplain the hyperactivity of ZI neurons. First, theopaminergic neurons of the substantia nigra parsompacta may directly influence the activity of ZIeurons. Such a concept would imply a reduced innerva-ion of ZI neurons bearing D2 dopamine receptors.hus, a reduced stimulation of D2 dopamine receptors,egatively coupled to adenylate cyclase, would relay

d electrophysiological recordings (C, D) showing changes in the meancontrol rat (A, C) and a rat recorded 7 days after the 6-OHDA lesion

ng a shortening of the interspike interval.

anof aecti

he activity of ZI neurons. This hypothesis is compat-

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223ZONA INCERTA ACTIVITY IN PARKINSONISM

ble with the description of D2 dopamine receptors onI neurons in the rat brain (38). Second, ZI hyperactiv-

ty might be explained by a decreased activity ofnhibitory afferent fibers to the ZI. The major inhibitoryfferent fibers to the ZI originate from the superiorolliculus, and it is known that the activity of superiorolliculus GABAergic neurons is decreased in parkinso-ian syndromes, probably due to an increase in the

nhibitory influence of the substantia nigra pars reticu-ata (33). Finally, ZI hyperactivity could be due to anncreased excitatory input to ZI neurons. In this contextt has been shown that neurons of the pedunculopon-ine nucleus project to the ZI (15) and that they areyperactive after nigrostriatal denervation, as recentlyemonstrated in an unpublished study performed inur laboratory.

onsequences of ZI Hyperactivity

The consequences of ZI hyperactivity after nigrostria-al denervation are as yet unknown. Nevertheless,rojections from the ZI to some of the basal ganglia andelated structures, including the basal forebrain, thentopedonuclar nucleus, the substantia nigra, and otherrain-stem areas that overlap with some of the termi-al sites of the subthalamic nucleus projections, haveeen described (14). Furthermore, since the ZI is knowno be involved in locomotor function, this hyperactivityould participate in the motor symptoms observed inarkinsonian syndromes. Indeed, before the advent ofevodopa, several structures were destroyed by stereo-actic surgery in order to alleviate the symptoms of theisease. In a study of 173 parkinsonian patients inhom the ZI was surgically lesioned, a dramatic im-rovement was observed in akinesia, rigidity, and evenremor (22). However, the utmost caution needs to bexercised in trying to interpret such results since thelectrolytic lesion may destroy intrinsic neurons of theI but also the passage fibers, such as the fibers arisingrom the internal globus pallidus and the substantiaigra pars reticulata, which are thought to be hyperac-ive in Parkinson’s disease (33). Alternatively, a hyper-ctivity of GABAergic neurons of the ZI, which arenown to project to the neocortex (17), may explain theecreased cortical activity reported in Parkinson’s dis-ase. The latter hypothesis seems unlikely, given thebsence of changes in GABAergic markers after-OHDA lesion in rats (personal unpublished data).hat is now clearly needed is a behavioral analysis of

he effect of a reduction in ZI neuronal activity on theotor signs in animals rendered parkinsonian.In addition, it is now well known that the ZI is

eciprocally connected to the superior colliculus, atructure involved in the control of eye movements, andhe ZI has been shown to participate in the control ofaccades (18). It has been reported that changes in unit

ctivity in various basal ganglia structures are related

o the execution of saccades and that some parkinso-ian patients suffer from different types of eye move-ent abnormality, such as hypometric and slow sac-

ades with somewhat increased latencies (10). Theseata suggest that the alteration in the functioning ofhe ZI neurons reported in our study might be involvedn eye movement abnormalities in parkinsonian syn-romes.In summary, we have shown that, in the rat, ZI

eurons are hyperactive after nigrostriatal denerva-ion and that this hyperactivity persists for at least 1onth after nigrostriatal denervation. Given the known

nvolvement of the ZI in motor and oculomotor func-ions, the increase in ZI activity after nigrostriatalenervation might be related to the symptomatology ofhe disease. Further studies will be needed to deter-ine the precise functional role of the changes in the

ctivity of the ZI and its involvement in the pathophysi-logy of parkinsonian syndromes. Should this structurendeed be involved in the motor symptoms of theisease, it may represent a new therapeutic target forharmacological treatment or deep brain stimulation.

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