motor effects and mapping of cerebral alterations in animal models of parkinson's and...

Motor Effects and Mapping of CerebralAlterations in Animal Models of

Parkinson’s and Huntington’s Diseases

M.O. HEBB AND H.A. ROBERTSON*Laboratory of Molecular Neurobiology, Department of Pharmacology, Dalhousie University,

Halifax, Nova Scotia B3H 4H7, Canada

ABSTRACTChanges in stimulant-induced behavioral effects and subcortical c-Fos expression were

compared between rodent models of Parkinson’s disease (PD) and Huntington’s disease (HD).Rats received either a unilateral 6-hydroxydopamine (6-OHDA)-induced lesion of the nigro-striatal dopamine pathway (PD model) or a unilateral infusion of antisense oligodeoxynucleo-tides targeting c-fos into the striatum (HD model). Dopamine-lesioned animals receivedintraperitoneal injections of either d-amphetamine (6-OHDAamp group) or apomorphine(6-OHDAapo group), whereas all animals that received antisense infusions received d-amphetamine (ASF group). All groups exhibited robust circling behavior upon stimulantchallenge. Changes in subcortical activation, as assessed by the induction of Fos-likeimmunoreactivity (Fos-LI), were examined in several brain regions. The 6-OHDAamp andASF groups exhibited robust, ipsiversive circling behavior, with similar changes in Fos-LIin the striatum, entopeduncular nucleus, superior colliculus, and ventromedial thala-mus. The 6-OHDAapo group exhibited contraversive rotation and had reciprocal pat-terns of Fos-LI in these regions. Despite exhibiting the same direction of rotation, the6-OHDAamp and ASF groups had markedly different patterns of Fos-LI in the globus pallidusand the pontine reticular formation. These results suggest that the globus pallidus mayundergo distinct alterations in PD and HD and that the pontine reticular formation isparticularly susceptible to changes in mesencephalic dopamine sources. J. Comp. Neurol.410:99–114, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: basal ganglia; c-Fos; rotation; movement disorders

Parkinson’s disease (PD) and Huntington’s disease (HD)are progressive, neurodegenerative conditions of the basalganglia that produce motor impairment. The pathology ofPD involves the loss of nigrostriatal dopamine neurons inthe substantia nigra pars compacta (SNC) and the develop-ment of rigidity and bradykinesia (Lee, 1987; Agid, 1991).HD produces marked atrophy of the striatum and neocor-tex and is characterized by choreiform movements andcognitive decline, with bradykinesia and rigidity oftenoccurring in late-stage HD (Storey and Beal, 1993;Harper,1996). Different animal models have been devel-oped to study the distinct pathophysiologies of the twodiseases. Whereas 6-hydroxydopamine (6-OHDA) lesion-ing of the nigrostriatal dopamine system has becomewidely accepted as a model of PD in rats (Ungerstedt,1968; Schwarting and Huston, 1996), animal models of HDhave focused on the striatum as a general site of pathology,using lesioning techniques or other methods to suppressregional activity.

The etiology of the motor abnormalities produced in HDand PD remains unclear, but it is apparent that thedistinct symptomatology produced by these conditionsresults from disease-specific alterations in motor circuits.However, the bradykinesia and rigidity produced in bothPD and late-stage HD suggest that the pathological changesthat are induced by these diseases eventually affect com-mon regions or circuits in the brain. Although there havebeen numerous reports of changes in various cerebralnuclei in both PD and HD, currently, there are no descrip-tions of a systematic comparison between the pathophysi-

Grant sponsor: Huntington Society of Canada; Grant sponsor: ParkinsonFoundation of Canada; Grant sponsor: MRC.

*Correspondence to: H.A. Robertson, Laboratory of Molecular Neurobiol-ogy, Department of Pharmacology, Sir Charles Tupper Medical Building,Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.E-mail: [email protected]

Received 29 July 1998; Revised 25 January 1999; Accepted 17 February1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 410:99–114 (1999)

r 1999 WILEY-LISS, INC.

ologies of these conditions based on functional mapping ofactivity in the central nervous system.

Several groups have shown that intrastriatal infusionsof antisense oligodeoxynucleotides that target the immedi-ate-early genes, c-fos or ngfi-a, can effectively suppressprotein expression and produce significant motor abnor-malities and rotational behavior following stimulant chal-lenge (Chiasson et al., 1992, 1994; Dragunow et al.,1993,1994; Heilig et al., 1993; Sommer et al., 1993; Hooperet al., 1994; Hebb and Robertson, 1997a,b). We previouslyshowed that suppression of immediate-early gene expres-sion in the striatum produced an increase in immediate-early gene expression in the ipsilateral globus pallidus(Hebb and Robertson, 1997b). Sommer et al. (1996) alsodemonstrated that striatal infusions of antisense oligode-oxynucleotides targeting c-fos mRNA significantly reducedthe levels of neurotransmitter release in the substantianigra. Thus, suppression of immediate-early gene expres-sion in the striatum inhibits the activity of the striatalprojection neurons and directly affects both gene expres-sion and neurotransmitter release in effector nuclei, suchas the globus pallidus and the substantia nigra. In thecurrent study, we used infusions of antisense oligodeoxy-nucleotides targeting c-fos to create an animal model of thestriatal dysfunction in HD.

In an attempt to correlate motor impairment withalterations in cerebral activity by using animal models ofPD and HD, we compared the changes in neuronal activa-tion that result from 6-OHDA lesioning of the nigrostriataldopamine pathway with the changes produced by anti-sense oligodeoxynucleotide-induced suppression of striatalimmediate-early gene expression. Here, we report thedifferential expression of stimulant-induced Fos-like immu-noreactivity (Fos-LI) in the following motor-related nuclei:striatum, globus pallidus, substantia nigra pars reticulata(SNR), entopeduncular nucleus, ventromedial nucleus ofthe thalamus, superior colliculus, and oral pontine reticu-lar formation (RPO).

MATERIALS AND METHODS

Experimental design

All experimental procedures were approved by the Ani-mal Care Committee at Dalhousie University. The subjectswere 29 adult Sprague-Dawley rats (28 male, 1 female)weighing 275–400 grams. Two groups of animals receivedunilateral lesions of the nigrostriatal pathway using6-OHDA (PD model) and were challenged subsequentlywith d-amphetamine (6-OHDAamp group; n 5 7) or apo-morphine (6-OHDAapo group; n 5 11). A third group ofanimals received a unilateral infusion of antisense oligode-oxynucleotides targeting c-fos into the striatum (HD model)followed by administration of d-amphetamine (ASF group;n 5 11).

Surgical procedures

All surgeries were performed under halothane-inducedanesthesia with the animals mounted in a Kopf stereotaxicapparatus (Kopf, Tujunga, CA). Both 6-OHDA and oligode-oxynucleotide infusions were performed by pressure injec-tion using a CMA 100 (Carnegie-Medicin, Stockholm)microinjection pump with 25-gauge steel cannula. Allstereotaxic coordinates are given according to Paxinos andWatson (1997). Animals that received 6-OHDA lesionswere treated with desipramine (25 mg/kg, i.p.) 30 minutes

prior to infusion of 6-OHDA (4 µl of 12 mM solution at 0.5µl/minute) into the right medial forebrain bundle (coordi-nates from Bregma: anteroposterior [AP], 23.6 mm; lat-eral [LAT], 2.0 mm; dorsoventral [DV], 28.8 mm). Afterinfusion, the cannula was left in place for an additional 2minutes to allow for diffusion away from the injection site.After surgery, the skin was sutured, and animals were leftto recover for 21 days before behavioral testing.

Animals in the ASF group received bilateral placementof cannulae into the striata (coordinates from Bregma: AP,1.0 mm; LAT, 63.0 mm; DV, 26.0 mm). The right striatumwas infused with anti-c-fos oligodeoxynucleotide (2 µl of 1mM solution at 0.25 µl/minute), whereas the left striatumreceived an equal volume of vehicle. After infusion, thecannulae were left in place for an additional 2 minutes.The skin was then sutured, and the animals were left torecover for 1 hour before behavioral testing. This postopera-tive latency had been established previously as the periodof optimal suppressive effects of ASF (Hebb and Robertson,1997a).

Infusion of 6-OHDA or oligodeoxynucleotides was per-formed unilaterally to produce motor asymmetry and toleave an intact contralateral system with which to com-pare interhemispheric differences in neuronal activation.The expression of c-fos was used to map the functional(metabolic) activation of neural circuitry (Sagar et al.,1988; Dragunow and Faull, 1989) by the psychostimulantdrugs, d-amphetamine and apomorphine. In 6-OHDA-lesioned animals, administration of indirect (d-amphet-amine) or direct (apomorphine) dopamine agonists stimu-lated opposing motor circuitry and induced rotationalbehavior in different directions, thus permitting compari-son between the activation of specific nuclei and thedirection of rotation. Administration of d-amphetamineinduced ipsiversive rotational behavior in both 6-OHDAampand ASF groups and allowed comparison of alterations inmotor-related nuclei that were produced by either a loss ofmesencephalic dopamine afferents or direct suppression ofstriatal efferent activity.

Behavioral and immunohistochemicalanalyses

After the appropriate recovery periods, animals weregiven intraperitoneal injections of either d-amphetamine(5 mg/kg; 6-OHDAamp and ASF groups) or apomorphine(0.5 mg/kg; 6-OHDAapo group) and were placed in arotometer, where the number and direction of the rotationsmade by the animal were recorded in 10-minute intervalsfor a period of 2 hours. After behavioral testing, animalswere deeply anesthetized with sodium pentobarbital (.100mg/kg) and transcardially perfused, initially with saline,followed by 4% paraformaldehyde in a 0.1 M phosphate-buffer solution, pH 7.4. Brains were removed subsequentlyand postfixed at 4°C until further analysis. Brains wereblocked, cut on a Vibratome in 50-µm coronal sections, andprocessed for c-fos immunoreactivity, as described previ-ously (Robertson et al., 1989). Antibodies to c-fos wereobtained from Genosys (Houston, TX) and were used at adilution of 1:5,000.

Oligodeoxynucleotides

Oligodeoxynucleotides were purchased from Genosysand were single end capped (sulfur modification on thephosphate group between the first and last base pairs).Briefly, standard phosphoramidite chemistry for oligode-

100 M.O. HEBB AND H.A. ROBERTSON

oxynucleotide synthesis was used, and samples of eachsynthesis were examined on polyacrylamide gels to verifyoligonucleotide quality. All oligodeoxynucleotides were ex-tracted and precipitated by the manufacturer (to removeorganics and salts) prior to lyophilization. Oligodeoxy-nucleotides were reconstituted in ultrafiltered (Millipore,Bedford, MA) distilled water at a concentration of 1nmol/µl.

The antisense oligodeoxynucleotide to c-fos, called ASF,was a 15-base oligomer that has been used previously in itspartial and complete phosphorothioate forms (Chiasson etal., 1992, 1994; Dragunow et al., 1993; Heilig et al., 1993;Sommer et al., 1993, 1996; Hooper et al., 1994; Hebb andRobertson, 1997a,b, 1999a,b). Its sequence, 58-GSAA-CAT-CAT-GGT-CGST-38, corresponded to bases 129–143 on themRNA transcript (GenBank accession no. XO6769) andspanned the initiation codon (the subscript S denoteslocations of the sulfur modifications). We previously dem-onstrated the specificity of end-capped ASF in the striatumthrough the comparison of its suppressive actions with theeffects of vehicle infusions and two distinct random oligode-oxynucleotides (Hebb and Robertson, 1997a,b, 1999a). Weand others also have demonstrated the specificity of thesame oligodeoxynucleotide sequence in both partially andfully phosphorothioate forms by using mismatch and sensecontrols (Chiasson et al., 1992; Heilig et al., 1993; Hooperet al., 1994; Sommer et al., 1996). Unilateral infusions ofcontrol oligodeoxynucleotides (random, sense, mismatch)or vehicle into the striatum produced negligible motorasymmetry and minimal suppression of stimulant-in-duced c-fos expression. Thus, the behavioral and neuro-physiological effects described below in the ASF grouphave been attributed to the specific inhibition of c-fosexpression and are not produced by nonspecific oligodeoxy-nucleotide or vehicle infusions.

Quantification of Fos-LI and statisticalanalysis

Sections were selected from each region at approxi-mately the following AP coordinates (from Bregma): stria-tum, 11.5 mm; globus pallidus, 20.8 mm; SNR, 25.8 mm;entopeduncular nucleus, 22.3 mm; ventromedial thala-mus, 22.8 mm; superior colliculus, 26.3 mm; RPO, 27.3mm. Subsequently, each was processed for Fos-LI. Afterimmunohistochemistry, specific areas were scanned anddigitized by using a CCD camera (JVC, Tokyo, Japan). Byusing NIH densitometry software (Bethesda, MD), eachregion of interest was isolated in its entire cross section atthe coordinates indicated above, and the number of Fos-positive nuclei per area was computed. Because tissuefrom several animals was used in unrelated studies, thenumber of animals used to analyze a specific brain regionmay be less than that reported for the behavioral studies.The number of animals used in the analysis of each regionis shown in Figure 9.

Digitally obtained photomicrographs were assembledinto figures for publication by using Adobe Photoshopsoftware (version 4.0; Adobe Systems, Mountain View,CA), with minimal alterations to contrast and background.The regions presented in the figures represent the areasfrom which cell counts were obtained. It should be notedthat changes in the induction of Fos-LI also were observedin the SNC of all animal groups. The population of SNCneurons in which this occurred has been characterizedextensively by using neuroanatomical tracing and immuno-

histochemical techniques. However, a complete discussionof these cells is beyond the scope of this paper, and thesedata will be presented in an another report (Hebb andRobertson, submitted for publication).

Statistical analysis was performed by using SigmaStatsoftware (SPSS Inc., Chicago, IL). All groups of datainitially were tested for normality and equal variance,with subsequent comparisons between ipsiversive andcontraversive values made accordingly by using either aStudent’s t-test or a Mann-Whitney U test. Multiplecomparisons were made between groups by using a one-way analysis of variance (ANOVA) followed by a post-hocNewman-Keuls test. Significance was assumed at P , 0.05.

RESULTS

Behavioral analysis of 6-OHDAand ASF animals

Animals in the 6-OHDAapo group exhibited contraver-sive circling that commenced within 5 minutes of apomor-phine injection, with a peak intensity of 70 turns/10minutes measured at 40 minutes after stimulant adminis-tration (Fig. 1A). The intensity of this behavior rapidlydiminished, and the animals were then sedentary andhypoactive by 90 minutes after drug administration. Thetotal mean rotations for this group was significantlygreater on the contralateral side: 3.2 6 0.9 ipsiversive,281.6 6 38.2 contraversive (P , 0.0001; Fig. 1B). Incontrast to the rapid onset and decline of apomorphine-induced behavior, d-amphetamine elicited ipsiversive cir-cling in 6-OHDAamp animals that commenced approxi-mately 10–20 minutes after drug delivery. The circlingintensity peaked at 60 minutes postadministration, withan intensity of <70 turns/10 minutes (Fig. 1A). Thecircling activity persisted throughout the 120-minute test-ing period, and the animals continued to turn when theywere removed from the rotometer until the anesthetic tookeffect. The total mean number of rotations elicited in the6-OHDAamp group was significantly greater in the ipsiver-sive direction: 585.4 6 100.5 ipsiversive, 2.3 6 1.1 contra-versive (P 5 0.0006; Fig. 1B).

Amphetamine also induced ipsiversive circling in theASF group. The peak circling intensity was less pro-nounced in this group compared with the 6-OHDAampanimals, peaking at approximately 30 minutes postadmin-istration, with a maximum of 51 turns/10 minutes (Fig.1A). Like the 6-OHDAamp group, the stimulant effects ofd-amphetamine persisted throughout the testing period,and the animals usually had some degree of motor asymme-try remaining after 120 minutes. There was a significantlygreater mean number of ipsiversive rotations than contra-versive rotations in the ASF group: 305.3 6 53.0 ipsiver-sive, 21.5 6 7.6 contraversive (P , 0.0001; Fig. 1B).Whereas the ipsiversive rotational behavior elicited byd-amphetamine in the 6-OHDAamp and ASF groups wassimilar in duration, the 6-OHDAamp animals appeared tohave a heightened sensitivity to the stimulant, as reflectedin the significantly greater number of turns elicited in thisgroup. Comparison of absolute numbers of rotations among6-OHDAamp (ipsiversive), 6-OHDAapo (contraversive),and ASF (ipsiversive) groups revealed that 6-OHDAampanimals turned significantly more than 6-OHDAapo (P 50.0045) or ASF (P 5 0.0033) animals. There was nodifference in the absolute number of rotations producedbetween 6-OHDAapo and ASF groups (P 5 0.7856).

CEREBRAL CHANGES IN MODELS OF MOVEMENT DISORDERS 101

Expression of Fos-LI

Striatum. The dose of d-amphetamine used in thesestudies (5 mg/kg) was sufficient to induce robust Fos-LI inthe striatum of naive animals (Hebb and Robertson,1997b). In contrast, the dose of apomorphine (0.5 mg/kg)produced negligible Fos-LI in naive animals but robustexpression in dopamine-depleted animals. In the ipsilat-eral striatum of 6-OHDAamp animals, there were signifi-cantly fewer Fos-positive nuclei than in the contralateralside (P 5 0.0045; Fig. 2A). This expression was distributeddiffusely, without preferential localization to striosomes.In the contralateral striatum, there was robust expressionof Fos-LI in striosomal-like patches (Fig. 2B), as reportedpreviously (Graybiel et al., 1990). In 6-OHDAapo animals,apomorphine induced intense, homogeneous Fos-LI indopamine-depleted (ipsilateral) striata (Fig. 2C) but negli-gible expression in contralateral, intact striata (Fig. 2D;P 5 0.0022; ipsilateral vs. contralateral expression).

Intrastriatal infusion of ASF resulted in a significantsuppression (<65%) of d-amphetamine-induced Fos-LI

(P , 0.0001). This suppression was confined to the infusedstriatum and did not extend into the overlying cortex (notshown) or into the contralateral hemisphere (Fig. 2E,F).The greatest suppressive effects were observed near thecannula tract and extended in a graded fashion to theborders of the striatum. In the area of greatest suppres-sion, there was a near total absence of Fos-LI.

Globus pallidus. Administration of d-amphetamineproduced scant but distinct Fos-LI in the globus pallidus ofnaı̈ve animals. We observed approximately 60 Fos-positivenuclei/coronal section. In the 6-OHDAamp group, how-ever, this expression was reduced significantly in theipsilateral globus pallidus (P 5 0.0314; Fig. 3A,B). Asimilar reduction of Fos-LI was observed in the contralat-eral globus pallidus of 6-OHDAapo animals (P 5 0.0079;Fig. 3C,D). In contrast, animals in the ASF group had asignificant increase in the number of Fos-positive nuclei inthe ipsilateral globus pallidus but had typical induction ofFos-LI on the contralateral side (P 5 0.0317; Fig. 3E,F).

Entopeduncular nucleus. The ipsilateral entopedun-cular nucleus of both 6-OHDAamp and ASF groups exhib-ited significantly greater numbers of Fos-positive nucleithan the contralateral side (P 5 0.0079 and P 5 0.0023,respectively; Fig. 4). However, the contralateral entopedun-cular nucleus of 6-OHDAapo animals exhibited signifi-cantly more Fos-positive nuclei than the same region in6-OHDAamp or ASF groups (P 5 0.00035 and P 5 0.00045,respectively). The ipsilateral entopeduncular nucleus of6-OHDAapo animals had an abundance of small, lightlystained nuclei, with fewer large, darkly stained nuclei.When absolute numbers of Fos-positive nuclei were com-pared between hemispheres, these smaller cells signifi-cantly outnumbered the larger nuclei in the contralateralentopeduncular nucleus (P 5 0.0317).

Substantia nigra pars reticulata. In the 6-OHDAampgroup, there was a significant increase in the number ofFos-positive nuclei in the ipsilateral SNR, particularly inthe ventromedial region (P 5 0.0152; Fig. 5A,B). Thenuclear labeling in these animals was intense and delin-eated large, round nuclei. In 6-OHDAapo animals, therewere also significantly greater numbers of cells withFos-LI in the ipsilateral SNR than in the contralateralSNR (P 5 0.0215; Fig. 5C,D). However, as in the ipsilateralentopeduncular nucleus of these animals, the majority ofFos-LI in the ipsilateral SNR was localized to small,lightly stained nuclei. In contrast, the contralateral SNRof 6-OHDAapo animals expressed abundant Fos-LI thatwas localized solely to large-diameter, darkly stainednuclei. This contralateral labeling appeared similar to thatobserved in the ipsilateral SNR of 6-OHDAamp animals(compare Fig.5A with Fig. 5D).

In contrast to the significant differences in Fos-LI in-duced in the SNR between hemispheres of 6-OHDA-lesioned animals, ASF animals had minimal Fos-LI in bothipsilateral and contralateral SNR, with no apparent differ-ences between sides (P 5 0.2424; Fig. 5E,F). The expres-sion of Fos-LI in the SNR of ASF animals was indicative ofthat induced by d-amphetamine (5 mg/kg) in naive ani-mals (not shown).

Ventromedial thalamus. The ventromedial thalamushad marked alterations in Fos-LI in all animal groups.Consistent with an increased inhibitory output from theSNR, the ipsilateral ventromedial thalamus had a signifi-cant reduction in Fos-LI compared with the contralateralside in 6-OHDAamp animals (P 5 0.0146; Fig. 6A,B). A

Fig. 1. Rotational data obtained from animals that received eithera unilateral 6-hydroxydopamine (6-OHDA) lesion or intrastriatalinfusion of antisense oligodeoxynucleotide to c-fos (ASF). Both groupsthat received d-amphetamine (6-OHDAamp and ASF groups) exhib-ited robust ipsiversive circling, whereas the group that receivedapomorphine (6-OHDAapo) demonstrated a marked contraversivebias. A: Temporal patterns of turning behavior. Line graphs illustratethe intensity of ipsiversive (6-OHDAamp and ASF groups) andcontraversive (6-OHDAapo group) circling as assessed in 10-minuteintervals. Ipsiversive turns are indicated by positive values, andcontraversive turns are indicated by negative values. B: Histogramsshowing the total number of rotations. All groups (denoted by thelegend symbols in A) had a significant bias toward either the ipsiver-sive (6-OHDAamp and ASF groups) or the contraversive (6-OHDAapogroup) direction (indicated by asterisks).


Fig. 2. Fos-like immunoreactivity (Fos-LI) in the ipsilateral (A,C,E)and the contralateral (B,D,F) striata of 6-OHDAamp (A,B), 6-OHDAapo(C,D), and ASF (E,F) animals. The ipsilateral striata in 6-OHDAampand ASF animals had a marked reduction in Fos-LI, with typicalstriosomal expression on the contralateral side. In contrast,

6-OHDAapo animals had robust induction of Fos-LI in the ipsilateralstriata, with negligible expression on the contralateral side. Thephotomicrographs shown in E and F are approximately 0.5 mm rostralto the cannulae insertion sites. Scale bar 5 100 µm.

Fig. 3. Fos-LI in the globus pallidus of 6-OHDAamp (A,B),6-OHDAapo (C,D), and ASF (E,F) animals. A, C, and E represent theipsilateral globus pallidus; B, D, and F represent the contralateralglobus pallidus. Note the modest but consistent expression in thecontralateral globus pallidus of 6-OHDAamp (B) and ASF (F) animalsas well as in the ipsilateral globus pallidus of 6-OHDAapo animals (C).

This expression was virtually eliminated in the ipsilateral globuspallidus of 6-OHDAamp animals (A) and in the contralateral globuspallidus of 6-OHDAapo animals (D). In the ASF group, however, therewas a significant increase in the Fos-LI of the ipsilateral globuspallidus (E). Arrowheads indicate the approximate border between theglobus pallidus and the overlying striatum. Scale bar 5 100 µm.


Fig. 4. Fos-LI in the entopeduncular nucleus of 6-OHDAamp(A,B), 6-OHDAapo (C,D), and ASF (E,F) animals. A, C, and Erepresent the ipsilateral entopeduncular nucleus; B, D, and F repre-sent the contralateral entopeduncular nucleus. Both 6-OHDAamp andASF groups exhibited abundant Fos-LI on the ipsilateral side butnegligible expression on the contralateral side. A distinct increase also

was observed in the contralateral entopeduncular nucleus of6-OHDAapo animals (D). However, on the ipsilateral side, the6-OHDAapo group also had abundant immunolabeling, but it ap-peared as small, lightly stained nuclei (C). For details, see text. Scalebar 5 100 µm.

Fig. 5. Fos-LI in the substantia nigra pars reticulata (SNR) of6-OHDAamp (A,B), 6-OHDAapo (C,D), and ASF (E,F) animals. A, C,and E represent the ipsilateral SNR; B, D, and F represent thecontralateral SNR. Note the marked interhemispheric asymmetry inthe SNR of 6-OHDAamp animals compared with that of the ASFanimals. Distinct expression of Fos-LI was observed in the contralat-

eral SNR of the 6-OHDAapo group (D). However, on the ipsilateralside, there was abundant immunolabeling, but it appeared as small,lightly stained nuclei (C), similar to that observed in the ipsilateralentopeduncular nucleus of these animals (see Fig. 4C). Scale bar 5 100µm.

significant reduction also was observed in the ipsilateralventromedial thalamus of ASF animals (P 5 0.0137; Fig.6E,F). In contrast, the 6-OHDAapo group had a significantdecrease in Fos-LI expression in the contralateral ventro-medial thalamus (P 5 0.0448; Fig. 6C,D).

Superior colliculus. Administration of d-amphet-amine (5 mg/kg) typically induced modest expression ofFos-LI in the intermediate gray lamina of the superior

colliculus (SCIG) in untreated hemispheres (<80–100cells; Fig. 7B,F). ASF animals had a significant increase inFos-LI in this region, which produced a band of immunore-activity in the mediolateral direction across the superiorcolliculus (P 5 0.0152; Fig. 7E). Animals in the6-OHDAamp group appeared to show a trend of increasedFos-LI in the ipsilateral SCIG, but this increase did notreach statistical significance (P 5 0.2249; Fig. 7A). In

Fig. 6. Fos-LI in the ventromedial thalamus of 6-OHDAamp (A,B),6-OHDAapo (C,D), and ASF (E,F) animals. A, C, and E represent theipsilateral ventromedial thalamus; B, D, and F represent the contralat-eral ventromedial thalamus. There was a significant reduction of

Fos-LI in the ipsilateral hemisphere of 6-OHDAamp and ASF groupsand in the contralateral hemisphere of the 6-OHDAapo group. Fororientation purposes, the mammilothalamic tract is indicated byasterisks. Scale bar 5 100 µm.


contrast, 6-OHDAapo animals had significantly greaterexpression of Fos-LI in the contralateral SCIG (P 50.0003; Fig.7D).

Significant alterations in Fos-LI were observed in theintermediate white lamina of the superior colliculus (SCIW)

of all animal groups. The Fos-LI induced by d-amphet-amine was similar in the 6-OHDAamp and ASF animals,with significant reductions in the ipsilateral SCIW in bothgroups (P 5 0.0192 and P 5 0.0340, respectively; Fig. 7).Like the SCIG, animals of the 6-OHDAapo group had a

Fig. 7. Fos-LI in the superior colliculus of 6-OHDAamp (A,B),6-OHDAapo (C,D), and ASF (E,F) animals. A, C, and E represent theipsilateral superior colliculus; B, D, and F represent the contralateralsuperior colliculus. Animals in the 6-OHDAamp and ASF groups had asignificant reduction in Fos-LI in the ipsilateral intermediate whitelamina of the superior colliculus (SCIW), whereas those of the

6-OHDAapo group had reduced expression in the contralateral SCIW(arrowheads). The changes in SCIW Fos-LI were accompanied by asignificant increase in expression in the overlying intermediate graylamina of the superior colliculus (SCIG) on the ipsilateral (ASF) orcontralateral (6-OHDAapo) side (arrows). Scale bar 5 100 µm.


reciprocal expression pattern, with abundant Fos-LI in theipsilateral SCIW but significantly reduced expression onthe contralateral side (P 5 0.004; Fig. 7).

RPO. Alterations in the expression of Fos-LI in theRPO were prominent in the 6-OHDAamp group. Mostanimals in this group had a near total absence of Fos-LI inthe ipsilateral RPO, with abundant expression on thecontralateral side (P 5 0.0022; Fig. 8A). In contrast, the6-OHDAapo group appeared to have greater numbers ofFos-positive nuclei in the ipsilateral RPO. However, thedifference between hemispheres in this group did notreach statistical significance, and, in many animals, therewere no alterations in this region (P 5 0.2588; Fig. 8B).Animals in the ASF group also showed no differencebetween ipsilateral and contralateral expression of Fos-LIin the RPO (P 5 0.6472; Fig. 8C).

DISCUSSION

In the present study, comparison of the pharmacologicalinduction of Fos-LI in PD (6-OHDA) and HD (ASF) modelsrevealed common changes in several subcortical nuclei. Inboth models, significant, stimulant-induced locomotorasymmetry was manifested as rotational behavior. Mostregions analyzed exhibited reciprocal expression of Fos-LIbetween 6-OHDAamp and 6-OHDAapo groups and, thus,could not be excluded from contributing to the motorasymmetry in dopamine-depleted animals. However, thedisparity between locomotor bias and alterations of Fos-LIin the globus pallidus and RPO of 6-OHDAamp and ASFanimals clearly demonstrated the distinct physiologicaleffects of altering nigral and striatal transmission.

Alterations in c-fos expression

Striatum. Dopamine that is released into the striatumexerts its effects on striatonigral (direct pathway) andstriatopallidal (indirect pathway) neurons predominantlyby activation of D1 and D2 receptors, respectively (Robert-son and Robertson, 1987; Gerfen et al., 1990; Graybiel,1990, 1991; LeMoine et al., 1990, 1991). Because D1Rs arecoupled positively and D2Rs are coupled negatively to bothadenylate cyclase and inositol phospholipid signaling path-ways (Kebabian and Calne, 1979; Stoof and Kebabian,1981; Mahan et al., 1990), stimulation of these receptorsincreases transmission through direct striatal projectionsbut decreases transmission through indirect striatal projec-tions. Because the ipsilateral striata of 6-OHDA animalswere devoid of tyrosine hydroxylase-positive terminals,the Fos-positive cells in this region most likely projected tothe globus pallidus and were stimulated by a reduction inD2R-mediated inhibition that was produced by the loss ofdopamine. However, because there were only sparse num-bers Fos-positive nuclei in unstimulated striata (notshown), other influences (i.e., corticostriatal afferents)likely contributed to the stimulation of these neurons in6-OHDAamp animals. For example, the decrease in D2R-mediated inhibition of striatopallidal neurons in dopamine-depleted striata may have rendered these cells hypersensi-tive to glutamatergic stimulation from the cortex. Thus,the reduction in D2R activity and the increase in glutama-tergic transmission (produced by stimulant administra-tion) could synergistically activate striatopallidal neurons,whereas the reduction in D1R-mediated stimulation ofstriatonigral neurons would antagonize the cortical influ-

Fig. 8. Fos-LI in the oral pontine reticular formation (RPO) of6-OHDAamp (A), 6-OHDAapo (B), and ASF (C) animals. The experi-mental side is on the left in A–C. Note the marked suppression ofFos-LI in the ipsilateral RPO of 6-OHDAamp animals (A). Thisreduction in expression was not observed in either the 6-OHDAapogroup or the ASF group. Scale bar 5 100 µm.


Fig. 9. Histograms of Fos-positive cells counts in several subcorti-cal areas. The region analyzed is given at the top of each graph. Thenumbers at the bottom of each histogram denote the animal group: 1,6-OHDAamp; 2, 6-OHDAapo; 3, ASF. Numbers in parentheses indi-cate the number of animals used in the analysis of that region. Solidbars indicate ipsilateral cell counts, and open bars indicate contralat-eral cell counts. Note the differences in the ordinate scale in each

histogram. Asterisks indicate significant differences between ipsilat-eral and contralateral hemispheres (P , 0.05). EPN, entopeduncularnucleus; GP, globus pallidus; RPO, oral pontine reticular formation;SCIG, intermediate gray layer of superior colliculus; SCIW, intermedi-ate white layer of superior colliculus; SNR, substantia nigra parsreticulata; STR, striatum; VMT, ventromedial thalamus.

ence. This hypothesis is supported by previous studies thatdemonstrated dopamine-glutamate interactions in thestriatum (Lannes and Micheletti, 1997; Morelli, 1997;Ozer et al., 1997) and also the D2R antagonist-mediatedincrease in extracellular glutamate concentrations in theentopeduncular nucleus of 6-OHDA-lesioned animals(Biggs et al., 1997).

Globus pallidus. Both 6-OHDAamp and ASF groupshad modest but distinct expression of Fos-LI in the contra-lateral globus pallidus, typical of that induced by d-amphetamine. Although the mechanism by which dopa-mine agonists induce pallidal Fos is unclear, one possibilityis that apomorphine (in dopamine-depleted striata) andd-amphetamine (in intact striata) reduce striatopallidaltransmission through increased D2R activation. This no-tion is supported by a study by Floran et al. (1997) thatshowed that methamphetamine or the D2 agonist, quin-pirole, inhibited the release of g-aminobutyric acid (GABA)in the globus pallidus of the rat. Also, Black et al. (1997)used positron emission tomography in the baboon todemonstrate that cerebral blood flow to the globus palliduswas reduced significantly after intravenous administra-tion of a D2-selective agonist. Finally, in PD patients,Hutchinson et al. (1997) demonstrated that administra-tion of apomorphine increased the firing rate of neurons inthe external segment of the globus pallidus.

In the ipsilateral globus pallidus of 6-OHDAamp ani-mals, Fos-LI was reduced by approximately 60% but was

increased by greater than tenfold in ASF animals. Becausestriatopallidal neurons release GABA, which suppressesglobus pallidus activity, it is difficult to attribute thedecrease in Fos-LI observed in 6-OHDAamp animals to areduction in striatal activity. Alternatively, a reduction instriatal dopamine and a subsequent decrease in D2R-mediated inhibition of striatopallidal neurons would in-crease the inhibitory tone to the globus pallidus. Becauseintrastriatal infusion of ASF directly suppressedd-amphetamine-induced Fos-LI, presumably without af-fecting dopaminergic innervation, this treatment was notexpected to produce a D2-mediated increase in striatopalli-dal activity. The significant Fos induction in the ipsilateralglobus pallidus after intrastriatal ASF infusion indicatedthat ASF-induced inhibition of striatopallidal activity wassufficient to disinhibit pallidal neurons and increase theFos-LI in this region. It is interesting that increasedglobus pallidus activity in ASF animals has been corre-lated previously with the intensity of circling behavior inrats, such that elimination of this pallidal influence tripledthe rotational intensity (Hebb and Robertson, 1999a).Thus, by influencing basal ganglia output nuclei (i.e.,SNR), the increase and decrease in globus pallidus activityof ASF and 6-OHDAamp groups, respectively, could ac-count for the current variation in behavioral responses tostimulant challenge (Fig.10).

SNR and entopeduncular nucleus. Animals in the6-OHDAamp group had significant elevation of Fos-LI in

Fig. 10. Generalized schematic of the alterations that occurthroughout the extended basal ganglia after stimulation with d-amphetamine in naive (A), 6-OHDA-lesioned (B), and ASF-infused (C)animals. Arrow thickness indicates relative levels of neuronal activity.The symbols at each arrowhead indicate whether that particularcircuit is excitatory (1) or inhibitory (2). Solid circles indicate normalor hyperactive neurons, and open circles indicate neurons in whichactivity was suppressed directly or abolished by the treatment. A: Inthe striatum of naive animals, d-amphetamine has a predominantlystimulatory effect on striatonigral and striatoentopeduncular neurons(direct pathway), with little or no effect on the indirect, striatopallidalpathway. The net result is an inhibition of basal ganglia output fromthe SNR and entopeduncular nucleus. B: After 6-OHDA-mediatedablation of the nigrostriatal dopamine system, activity of striatonigral(and striatoentopeduncular) neurons is suppressed due to a reductionin D1 receptor (D1R)-mediated stimulation. In contrast, the striatopal-lidal neurons become hyperactive, presumably due to the reduction inD2R-mediated inhibition of these cells. These changes in activity

synergistically increase the output of the entopeduncular nucleus andsubstantia nigra pars reticulata (SNR) through reduced striatalinhibition and increased subthalamic nucleus (STN)-mediated stimu-lation. C: Intrastriatal infusion of ASF effectively reduces transmis-sion through both the direct circuit and the indirect circuit. Althoughthere is reduced striatal inhibition of the entopeduncular nucleus andSNR through the direct pathway, there also is a compensatoryincrease in globus pallidus (GP) activation and subsequent reductionin STN-mediated stimulation of these output nuclei. Thus, the SNRand entopeduncular nucleus are stimulated to a greater extent in6-OHDA animals than in ASF animals. This is consistent with thegreater locomotor asymmetry and SNR Fos-LI in 6-OHDAamp ani-mals observed here. Also, it is conceivable how an increase in globuspallidus activity, without a reduction of striatonigral transmission (asin choreic Huntington’s disease), could produce the opposite effect onthe entopeduncular nucleus and SNR, reducing the inhibitory drive tothe thalamus and superior colliculus, thereby increasing thalamocorti-cal and tectal stimulation. SNC, substantia nigra pars compacta.


both the ipsilateral SNR and the entopeduncular nucleus,whereas ASF animals had a marked increase in only theentopeduncular nucleus. The activation pattern in thestriatum and the globus pallidus suggested that ASFanimals had a decrease in striatonigral-mediated inhibi-tion but also a reduction in subthalamic nucleus-mediatedstimulation of the SNR (produced by increased globuspallidus output). In contrast, 6-OHDAamp animals hadinhibition of striatonigral transmission and an increase insubthalamic nucleus-mediated stimulation of the SNR(produced by decreased globus pallidus output; Fig.10).The altered Fos-LI observed in several subcortical nucleiindicated that the smaller SNR response in ASF animalswas not due to a lack of asymmetrical motor activation andthat the difference in SNR stimulation between thesegroups may have produced the disparity in rotationalintensity.

Animals in the 6-OHDAapo group exhibited a greaternumber of Fos-positive nuclei in both the ipsilateral SNRand the entopeduncular nucleus relative to the contralat-eral side. This increase was inconsistent with the changesin other regions of the basal ganglia and brainstem andwith the observed behavioral effects (i.e., contralateralrotation). The Fos-LI on the ipsilateral side was confinedpredominantly to small, lightly stained nuclei, which mayhave been the result of a sensitized response to directdopaminergic stimulation. The appearance of the sametype of staining in both SNR and entopeduncular nucleusof the 6-OHDA-lesioned hemisphere suggested that dopa-mine may play a major role in the regulation of thesenuclei. This may have significant clinical implications, asPD therapy with apomorphine or L-dopa would be ex-pected to produce similar effects.

The pattern of Fos-LI observed in the ipsilateral SNRand entopeduncular nucleus of 6-OHDAapo animals wasnot seen in 6-OHDAamp animals, excluding the possibilitythat it was caused by a gliosis-induced increase in endog-enous peroxidase and increased diaminobenzidine reac-tion product. The typical activation of downstream nuclei(i.e., thalamus, superior colliculus) indicated that theresponse of the ipsilateral SNR and entopeduncular nucleusdid not influence the activity of these other regions. It isnoteworthy, however, that the contralateral SNR andentopeduncular nucleus of 6-OHDAapo animals expressedan increased level of Fos-LI in large, darkly labeled nuclei.The numbers of Fos-positive nuclei were similar to thoseobserved in the ipsilateral regions in 6-OHDAamp ani-mals, suggesting that this induction was mediated byafferent projections and not by direct stimulation of dopa-mine receptors.

Ventromedial thalamus. The changes in Fos-LI inthe ventromedial thalamus were consistent with the basalganglia alterations in all groups and suggested that in-creased inhibitory tone from the entopeduncular nucleusand SNR reduced thalamic stimulation. Because severalASF animals had negligible increases in SNR activity, theentopeduncular nucleus was implicated in conveying thedominant input to the ventromedial thalamus. This isconsistent with the dense innervation of thalamic regions,including the ventromedial thalamus, by entopeduncularnucleus afferents (van der Kooy and Carter, 1981; Jimenez-Castellanos and Reinoso-Suarez, 1985; Moriizumi et al.,1988; Finkelstein et al., 1996).

Superior colliculus. The superior colliculus is in-volved in the initiation of saccadic eye movements, head-

eye coordination, postural control, and species-specificdefensive behaviors (Wurtz and Albano, 1980; Sahibzadaet al., 1986; Dean et al., 1988; Dean and Redgrave, 1992).Two major efferent systems from the superior colliculushave been well characterized: First, there is a contralat-eral projection in which the majority of axons descendmedially from the superior colliculus and cross the midlineat the dorsal tegmental decussation. These fibers join thepredorsal bundle and innervate the medial pons andmedulla, with some fibers projecting directly to the spinalcord. These tectoreticulospinal neurons represent the mainpremotor projection from the superior colliculus to thebrainstem that mediates orientation of the head and eyes.In the second major efferent system, an ipsilateral projec-tion sends fibers that terminate in the dorsolateral ponsand cuneiform nucleus, the activation of which producesincreased avoidance and defensive behaviors (Waldronand Gwyn, 1969; Dean et al., 1986; Ellard and Goodale,1986, 1988; Redgrave et al., 1987). Studies of the spatialdistribution of ipsilaterally and contralaterally projectingcells have shown that they are localized predominantly tothe intermediate and deep layers of the superior colliculus.Specifically, tectoreticulospinal neurons appear to have ahigher concentration in lateral regions of the intermediatelayers, whereas ipsilaterally projecting cells are locatedmore medially (Redgrave et al., 1986; Westby et al., 1990).

The significant reduction of Fos-LI in the SCIW ipsilat-eral to the hemisphere with increased SNR and entopedun-cular nucleus activity was consistent with elevated, GABA-mediated inhibition from the basal ganglia. This lateralregion corresponds to the termination zones of nigrotectalafferents and contains the majority of tectoreticulospinalneurons, the activation of which produces contralateralhead and eye movements (Berthoz and Grantyn, 1986;Bickford and Hall, 1992). Thus, unilateral suppression ofthese cells would be expected to induce ipsiversive postur-ing due to the influence of the contralateral tectoreticulo-spinal neurons. These expectations were met by all threeanimal groups in the present study.

The mechanism of stimulation and the functional role ofSCIG activation are unclear. Previous studies have demon-strated that the expression of Fos-LI in SCIG cells in-creases with the degree of ipsiversive rotation in theanimal (Hebb and Robertson, 1999b). Westby et al. (1990)used electrophysiological techniques to assign modalitypreferences to cells of the superior colliculus that project tothe ipsilateral cuneiform nucleus. It was determined thatcuneiform nucleus-projecting cells throughout the medial-to-lateral extent of the SCIG respond preferentially tovisual stimuli. However, because the superficial layers ofthe superior colliculus are the primary retinorecipientareas of this structure (Linden and Perry, 1983; Dreher etal., 1985), SCIG stimulation may be mediated indirectly byglutamatergic, corticotectal afferents projecting from thefrontal eye fields or the somatosensory cortex. The para-doxical increase in SCIG activation has not been reportedpreviously in Parkinsonian models, but it may help ex-plain the extensive variations in oculomotor abnormalitiesobserved in both PD and HD (Avanzini et al., 1979;Shibasaki et al., 1979; Kennard and Lueck, 1989).

Pontine reticular formation. Severe oculomotor defi-cits have been reported in both PD and HD. Decreasedsaccade velocity, prolonged saccadic reaction time, andabnormal smooth pursuit are common symptoms in bothdiseases (Avanzini et al., 1979; Shibasaki et al., 1979;


Kennard and Lueck, 1989; Nakamura et al., 1991). Theseabnormalities have been attributed to changes in tectalregulation of reticulospinal neurons that control extraocu-lar muscles.

The RPO is a major relay center in the pons that receivesinput from higher centers as well as from the spinal cord.Anatomical tracing of the afferent projections to the RPOrevealed dense innervation by the medial prefrontal cor-tex, zona incerta of the diencephalon, and ipsilateralsuperior colliculus as well as other regions of both theipsilateral and contralateral reticular formation (Sham-mah-Lagnado et al., 1987). The RPO receives a lesssignificant innervation from the ipsilateral SNR and thecuneiform nucleus. The RPO and its caudal counterpartinnervate many common oculomotor-related regions of thebrainstem, including the abducens nucleus, SCIG, ante-rior pretectal nucleus, ventral lateral geniculate nucleus,and regions of the central gray directly bordering theoculomotor nucleus (Vertes and Martin, 1988). Efferentfibers from the RPO also innervate numerous ipsilateraland contralateral reticular sites and project fibers through-out the cervicosacral extent of the spinal cord (Jones andYang, 1985).

The functional differences between the ipsilaterallyprojecting reticulospinal neurons and the contralaterallyprojecting tectoreticulospinal neurons have been reviewedby Berthoz and Grantyn (1986). In the cat, ipsiversiveorienting behavior was correlated with the activation ofreticulospinal neurons, whereas contraversive orientingbehavior was correlated with tectoreticulospinal neuronactivity. It was hypothesized that these two circuits influ-ence head-eye coordination by the convergence of theirsignals on spinal motoneurons. The final output of reticulo-spinal neurons, which receive input from tectoreticulospi-nal neurons, is dependent on the composite excitatory-inhibitory influences on reticular cells. Those authorscautioned, however, that the correlation of saccade genera-tion with tectoreticulospinal neuron activity is relativelylow compared with that of other nuclei (i.e., periabducensarea), and they suggested that the tectoreticulospinalneuron/reticulospinal neuron influence represents a primi-tive orienting system that has been superseded by moreprecise mechanisms.

Our analysis of changes in Fos-LI in the RPO of6-OHDAamp animals revealed a mean decrease of 66% inthe ipsilateral hemisphere. In this group, every animal(n 5 6) exhibited a marked decrease in ipsilateral Fos-LI,with several animals displaying a near total ipsilateralsuppression in this nucleus. The marked reduction ofFos-LI in the RPO of these animals was likely a combinedeffect of reduced tectoreticular stimulation and increasednigroreticular inhibition. In contrast,ASF and 6-OHDAapoanimals exhibited robust Fos-LI in the RPO of bothhemispheres, with no differences between sides. Thesedisparate results support the comments made by Berthozand Grantyn (1986), who suggested that the orientationbias and rotation in these groups most likely were domi-nated by systems other than the RPO. The influence of theasymmetric RPO activation on motor control in6-OHDAamp animals remains unclear. If these RPO cellsprojected to the ipsilateral spinal cord, then the unilateralsuppression of RPO activity would be expected to producean orientating response in the contralateral direction. Thefunction of the contralateral spinal projection of RPOneurons remains unclear but likely acts to reduce motor

activity in antagonistic musculature. Therefore, the robustipsiversive circling exhibited by these animals suggestedthat 1) these cells did not project to the ipsilateral spinalcord, or 2) the influence of this system was dominated byalternate descending motor circuits.

CONCLUSIONS

The results of this study suggest that the asymmetry instimulant-induced motor behavior produced by unilateral6-OHDA lesioning or ASF infusion in rats is mediated bysimilar changes in the striatum, entopeduncular nucleus,SNR, superior colliculus, and ventromedial thalamus.These findings generally are consistent with clinical de-scriptions of cerebral changes in PD and HD. In addition,the present results indicate that the globus pallidus andthe RPO may provide modulatory signals that influencethe extent of motor deficits. Combinations of metabolicmapping with electrophysiological and tracing techniquesin primate models of PD and HD ultimately may reveal therole of specific cerebral and brainstem nuclei in theproduction of motor symptomatology in these neurodegen-erative diseases.

ACKNOWLEDGMENT

We thank Dr. D.A. Hopkins for his comments on thisarticle.

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