Subthalamic Nucleus Lesions: Widespread Effects on Changes in Gene Expression Induced by Nigrostriatal Dopamine Depletion in Rats

Home , Nigrostriatal pathway, Subthalamic nucleus

The Journal of Neuroscience, October 1995, 75(10): 6562-6575

Jill M. Delfs, Vivian M. Ciaramitaro, Tom J. Parry,a and Marie-Francoise Chesselet Department of Pharmacology and David Mahoney Institute of Neurological Sciences, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Lesions of the subthalamic nucleus block behavioral ef- The subthalamicnucleus has recently gained recognition as a fects of nigrostriatal dopamine depletion in rats and pri- major excitatory drive in the basalganglia (Kitai and Kita, 1987; mates, but the contribution of this region to the molecular FCgeret al., 1991; Parent and Hazrati, 1995). This region con- effects of dopaminergic lesions is unknown. The effects of sists of a small collection of glutamatergic neurons located subthalamic nucleus lesions alone or in combination with above the cerebral peduncle (Smith and Parent, 1988; Rinvik a 6-hydroxydopamine-induced lesion of the substantia ni- and Ottersen, 1993). The subthalamic nucleus projects to the gra were examined in adult rats. Unilateral subthalamic nu- pallidum and substantianigra pars reticulata, two main target cleus lesions caused ipsiversive rotation after peripheral areas of striatal output neurons. Therefore, it is in a strategic administration of apomorphine and a small decrease in glu- position to govern the outflow of motor information from the tamic acid decarboxylase (GAD) mRNA in the ipsilateral basalganglia (Deniau et al., 1978; Nauta and Cole, 1978; Kita globus pallidus (external pallidum). Confirming previous and Kitai, 1987; Parent and Smith, 1987; Takada et al., 1988; results, nigrostriatal dopaminergic lesions caused contra- Smith et al., 1990; Berendseand Groenewegen,1991). Accord- versive rotation after apomorphine injection, and increased ingly, lesionsor high-frequency stimulation of the subthalamic enkephalin mRNA in the striatum, GAD mRNA in the globus nucleus alleviate motor symptomsinduced by nigrostriatal do- pallidus, and somatostatin mRNA in the entopeduncular pamine depletion (Bergman et al., 1990; Aziz et al., 1991; Ben- nucleus (internal pallidum) ipsilateral to the lesion. In ad- azzouz et al., 1993; Limousin et al., 1995). dition, the lesion decreased substance P mRNA in the ip- According to current models of basal ganglia circuitry, silateral striatum compared to the contralateral side, and changesin the activity of both direct and indirect inputs from GAD mRNA in the contralateral entopeduncular nucleus. the striatumto the internal pallidum and the substantianigra pars These effects were abolished in rats with lesions of the reticulata are critical for hypokinetic movement disorders,such subthalamic nucleus and substantia nigra on the same as akinesiain Parkinson’sdisease (Albin et al., 1989; DeLong, side. Thus, the subthalamic lesion prevented changes in 1990). Indirect inputs from the striatum reach the internal pal- gene expression induced by dopamine depletion, not only lidum and substantia nigra via the external pallidum and the in regions receiving a direct input from the subthalamic subthalamicnucleus. Thus, according to this model, the subtha- nucleus (ipsilateral pallidum), but also in regions which do lamic nucleus acts primarily as a relay between the external not (striatum and contralateral pallidum). This suggests pallidum and the internal pallidurn/substantianigra in the indi- that polysynaptic pathways regulated by the subthalamic rect pathway. nucleus contribute to the effects of dopaminergic lesions The subthalamicnucleus, however, also sendsreciprocal pro- in many regions of the basal ganglia. This pivotal role of jections to the external pallidum (Deniauet al., 1978; Nauta and the subthalamic nucleus may account for the beneficial ef- Cole, 1978; Van der Kooy and Hattori, 1980; Kita and Kitai, fects of subthalamic nucleus lesions on motor symptoms 1987; Takada et al., 1988; Smith et al., 1990). Supporting a resulting from dopamine depletion. functional role for this pathway, pharmacological stimulation [Key words: subthalamic nucleus, Bhydroxydopamine, and lesionsof the subthalamicnucleus alter neuronal firing in globus pallidus, entopeduncular nucleus, basal ganglia, in the external pallidum (Ryan and Sanders, 1993; Soltis et al., situ hybridization, glutamic acid decarboxylase] 1994). In rats and primates, nigrostriatal lesionsincrease burst firing activity and the levels of mRNA for glutamic acid decarboxylase (M, 67,000; GAD67), the enzyme of GABA synthesis, in the external pallidum (Filion, 1979; Miller and DeLong, 1987; Received Apr. 12, 1995; revised May 23, 1995; accepted May 24, 1995. This work was supported by US. Public Health Service Grant MH 44894 Pan and Walters, 1988; Filion and Tremblay, 1991; Kincaid et (M.EC) and Training Grants MH 17168 (J.M.D.) and MH 14654 (T.J.P.). We al., 1992; Soghomonian and Chesselet, 1992; Herrero et al., gratefully appreciate the generosity of Drs. Richard Goodman (Vollum Insti- 1993; Soghomonianet al., 1994). We have previously proposed tute), James Krause (Washington University), Steven Sabol (NIMH), and Allan Tobin (UCLA) for providing the cDNAs used in these studies. (Soghomonian and Chesselet, 1992; see also Kincaid et al., Correspondence should be addressed to Marie-Franqoise Chesselet, M.D., 1992) that theseeffects result from the increasedactivity of sub- Ph.D., University of Pennsylvania School of Medicine, Department of Phar- macology, 3620 Hamilton Walk, Philadelphia, PA 19104. thalamic neuronsobserved after nigrostriatal lesions(Miller and aPresent address: Zynaxis, Inc., Malvern, PA 19355. DeLong, 1987; FCger et al., 1991; Robledo and FCger, 1991; Copyright 0 1995 Society for Neuroscience 0270-6474/95/156562-14$05.00/O Hollerman and Grace, 1992; Bergman et al., 1994). The Journal of Neuroscience, October 1995, 15(10) 6563

Figure 1. Bright-field photomicrographs of cresyl violet-stained sections illustrating a unilateral kainic acid lesion of the subthalamic nucleus. Rats received a unilateral infusion of kainic acid (0.5 nmol in 0.1 pl) and were sacrificed 21 d later. Low-power photomicrograph illustrating (A) the intact subthalamic nucleus (STN) contralateral to the lesion and (B) the kainic acid-lesioned subthalamic nucleus (arrow). C, High-power photomicrograph of intact subthalamic nucleus neurons (arrow) depicted in A. D, High-power photomicrograph of the lesioned subthalamic nucleus depicted in B. Note the neuronal loss and extensive gliosis (glial cell, open arrow) induced by local infusion of kainic acid into the subthalamic nucleus. Scale bars: A and B, 500 pm; C and D, 50 pm.

To test this hypothesis, we have examined the effects of uni- weight (prepared following the protocol of Jensen-Salbutry Laborato- lateral lesions of the subthalamicnucleus, alone or in combi- ries, Kansas City, MO). Twenty ratsdesignated as controls received an injectionof 0.1 p,lof nation with a 6-hydroxydopamine (6-OHDA)-induced lesion of 0.9% sterile saline into the left subthalamic nucleus. Twenty rats re- the substantia nigra, on the level of expression of GAD67 ceived an injection of 0.5 nmol of the neurotoxin kainic acid (Sigma mRNA in the globus pallidus (external pallidum) in rats. In ad- Chemical Co., St. Louis, MO) dissolved in 0.1 pl of 0.9% sterile saline dition, we investigated the role of the subthalamicnucleus in into the left subthalamic nucleus. All injections were performed over other changesin neurotransmitter-relatedgene expression ob- 48 set and the cannula was left in place for an additional 5 min to allow for diffusion. The stereotaxic coordinates of the injection were served in striatum and pallidum after nigrostriatal lesions. AP: 4.9 mm from interaural zero; ML: 2.5 mm from midline; DV: -7.4 mm from the dura based on the atlas of Paxinos and Watson (1986). Materials and Methods While recovering from anesthesia, rats with kainic acid injections in the Animals and tissue preparation. Forty adult male Sprague-Dawley rats subthalamic nucleus, but not the saline-injected controls, exhibited ste- (Charles River Laboratories, Wilmington, MA; initial weight 280-300 reotyped extension-flexion movements of the contralateral forelimb, gm) were used for the following experiments. Upon arrival, animals orofacial dyskinesias consisting of vacuous chewing and jaw tremors, were handled, weighed and subsequently housed in groups of three until postural asymmetry toward the intact side, and spontaneous contraver- surgical procedures were performed. Rats were maintained on a 12 hr/ sive circling. These behaviors gradually subsided during the 4-6 hr 12 hr light/dark cycle (lights on from 07:OO to 19:00) with food and postoperative period. water available ad libitum. All surgical procedures and behavioral test- Six days after the subthalamic nucleus lesions, 10 kainic acid-le- ing were performed between 08:OO and 14:00 hr, during the light cycle. sioned animals and 10 control animals received an injection of 8 pg of All procedures were done in accordance with the NIH Guide for the the neurotoxin 6-hydroxydopamine(6-OHDA, bromidesalt, Sigma Care and Use of Laboratory Animals and were approved by the local ChemicalCo., St. Louis, MO) dissolvedin 4 ~1 of 0.9% sterilesaline animal care committee. All surgical procedures were performed under containing 1% ascorbic acid over 2 min into the left substantia nigra. anesthesia with equithesin at a dose of 0.3 ml per 100 gm of body The stereotaxiccoordinates of the injectionsite were AP: 3.4 mm from 6564 Delfs et al. l Widespread Effects of Subthalamic Nucleus Lesions in Rats

Figure 2. Bright-field photomicrographs of cresyl violet-stained sections illustrating a unilateral 6-hydroxydopamine (6-OHDA) lesion of the substantia nigra pars compacta. Rats received a unilateral infusion of 6-OHDA (8 p,g in 4 p.1) and were sacrificed 14 d later. Low-power photomicrographs of (A) the intact substantia nigra pars compacta (SNpc) contralateral to the lesion and (B) the 6-OHDA lesioned substantia nigra (arrow). Note that there is some damage to the adjacent ventral tegmental area (VTA), but that the underlying substantia nigra pars reticulata (SNpr) is unaffected by the lesion in B compared to control (A). High-power photomicrographs of (C) the intact substantia nigra pars compacta and (D) the lesioned substantia nigra pars compacta illustrate the complete loss of dopaminergic neurons (arrow in C) and extensive gliosis (open arrow in D). Scale bars: A and B, 500 pm; C and D, 50 pm. interaural zero; ML: 2.0 mm from midline; DV: +2.2 mm from the the substantia nigra and subthalamic nucleus were stained either with interaural zero, based on the atlas of Paxinos and Watson (1986). Thirty hematoxylin and eosin or with cresyl violet and observed with bright minutes before the stereotaxic injection into the substantia nigra, all rats field microscopy to assess the location and extent of the lesions. received an injection of desipramine (25 mg/kg, s.c., Sigma Chemical ‘H-BTCP receptor autoradiography for dopamine uptake sites. In Co., St. Louis, MO) to protect noradrenergic neurons (Wooten and Col- order to assess the extent of the lesion of dopamine terminals in the lins 1981). The remaining 20 rats received an injection of 4 ul of 0.9% striatum, the loss of dopamine uptake sites was determined by autora- sterile saline, 0.1% ascorbic acid into the substantia nigra after pretreat- diography with the selective ligand 3H-N-[ I-92-benzo(b)thiophenyl)- ment with desipramine. To select rats with appropriate lesions, all ani- cyclohexyll-piperidine (BTCP) (Vignon et al., 1988). Using a protocol mals received a subcutaneous injection of apomorphine (0.5 mg/kg, s.c., modified from Filloux et al. (1989), slides containing sections of the Sigma Chemical Co., St. Louis, MO) 8 d after the 6-OHDA lesions of striatum were brought to room temperature from -70°C under a stream the substantia nigra to test for turning behavior as an index of the extent of cool air and were preincubated in ice cold 50 mM Tris HCl buffer of the lesion of dopaminergic neurons (Hudson et al., 1993). containing 200 mrvr NaCl, pH 7.0 at 4°C for 20 min to remove endog- All four groups of rats were sacrificed by decapitation one week after enous dopamine. This was followed by incubation in the same buffer behavioral testing. Thus, the groups are as follows: control rats which as above containing 2 nM 3H-BTCP (NEN/Du Pont, Boston, MA, spe- received sham injections into the subthalamic nucleus and substantia cific activity 49 Ci/mmol) at 4°C for 90 min. Nonspecific binding was nigra, rats sacrificed 21 d after a unilateral kainic acid lesion of the determined by the addition of 10 p,M GBR 12909 (Research Biochem- subthalamic nucleus, rats sacrificed 14 d after a unilateral 6-OHDA icals Inc., Natick, MA), a selective dopamine uptake inhibitor (Janow- lesion of the substantia nigra, and rats with combined lesions sacrificed sky et al., 1986). Sections were washed four times for 8 min each in 21 d after a subthalamic nucleus lesion and 14 d after a substantia nigra ice-cold buffer at 4°C. After a quick dip in ice cold distilled water, lesion. After decapitation, the brains were rapidly removed, frozen on slides were dried rapidly with a stream of cool air, desiccated overnight, powdered dry ice and stored at -70°C until processing. Brains were and exposed to Ultrofilm-3H (Leica Inc., Malvem, PA) for 30 d. cut into 10 pm sections with a Reichert-Jung cryostat, thaw mounted In situ hybridization histochemistry. The cDNA for preproenkephalin onto gelatin-coated slides, and stored at -70°C until use. Sections from was generously provided by Dr. S. L. Sabol (NIMH). A 970 base pair The Journal of Neuroscience, October 1995, 75(10) 6565

Figure 3. Reverse images of film autoradiograms illustrating the effects of subthalamic nucleus and substantia nigra lesions on 3H-BTCP-labeled dopamine uptake sites in the striatum. Representative striatal sections from control animal (A); rat with a unilateral 6-hydroxydopamine lesion of the substantia nigra (B); rat with a unilateral kainic acid lesion of the subthalamic nucleus (C); and rat with a combination of the two lesions (D). Note that the extent of 3H-BTCP labeling is similar in B and D. Scale bars: A-D, 2 mm.

Table 1. Effect of subthalamic nucleus and substantia nigra transcript from the gene encoding preproenkephalin A was isolated from lesions on striatal dopamine uptake sites labeled with 3H-BTCP a rat striatal library and incorporated into the plasmids pSP64 and pSP65 downstream from SP6 promoters oriented in opposite directions (Yoshikawa et al., 1984). This provided the templates for the synthesis Lesion Ipsilateral Contralateral of RNA probes which were either “antisense” (complementary to cel- CONT 0.2344 t 0.009 0.2346 ? 0.011 lular mRNA) or “sense” (identical to cellular mRNA). The cDNA for P-preprotachykinin (substance P) was kindly provided by Dr. J. E. Krau- (100.0 t 3.6%) (100.0 + 4.7%) se (Washington University, St. Louis MO). This cDNA consists of a SN 0.0164 + O.OOS* 0.2138 2 0.005 560 base sequence isolated from a Sprague-Dawley rat striatal library (7.0 + 3.3%) (98.8 5 2.1%) and was incorporated into a pGEM1 vector with SP6 and T7 promoter STN 0.2235 + 0.013 0.2474 t 0.013 regions (Krause et al., 1987). The GAD67 cDNA, which consists of a full-length 2.3 kilobase sequence isolated from a cat occipital cortex (95.3 t 5.5%) (105.2 -r- 5.6%) library and incorporated into the pSP65 (antisense) and pSP64 (sense) SN/STN 0.0201 2 0.005* 0.2350 2 0.007 vectors (Kaufman et al., 1986; Erlander et al., 1991), was kindly pro- (8.6 + 1.9%) (100.2 + 3.0%) vided by Dr. A.J. Tobin (UCLA, Los Angeles, CA). It showed a 97% sequence identity with the cloned rat cDNA encoding GAD67 (Erlander Mean optical density measured from film autoradiograms of rats with sham lesion (CONT), a unilateral lesion of the substantia nigra (SN), a unilateral et al., 1991). The somatostatin cDNA consists of a 400 base sequence lesion of the subthalamic nucleus @TN), or a combination of the two lesions isolated from a rat medullary thyroid carcinoma cDNA library and in- (SN/STN). Data are expressed as absolute values ? SEM of the ipsilateral and corporated into the pSP64 transcription vector. This cDNA, which was contralateral striatum from five rats per group. Numbers in parentheses are a generous gift of Dr. R. H. Goodman (Vollum Institute, Portland, OR), percentage of control k SEM. encodes the tetradecapeptide somatostatin and somatostatin 28 in ad- * p < 0.05 when absolute values were compared to the corresponding side of dition to a 51 amino acid NH, terminal extension (Goodman et al., control using ANOVA with Dunnett’s post hoc test. 1982). The protocol for probe synthesis and in situ hybridization has 6566 Delfs et al. *Widespread Effects of Subthalamic Nucleus Lesions in Rats

hematoxylin and eosin and coverslipped with Eukitt mounting media 0 ipsiversive turns (Calibrated Instruments, Hawthorne, NY). The anatomical specificity of autoradiographic labeling with each of * q contraversive turns v) the probes has been previously characterized and was verified in each -- t individual experiment (Chesselet et al., 1987; Soghomonian and Ches- .i 5 selet, 1991; Salin and Chesselet, 1992). No specific labeling was ob- 5 i 300 served in control sections processed with sense RNA probes. 5”’ Quantification. The level of labeling over individual neurons in the +I globus pallidus and entopeduncular nucleus was measured on emulsion- 6, coated slides with a MORPHON image analysis system (Smolen and $zL d 200 Beaston-Wimmer, 1990). This system consists of a MT1 65 video camera, a Leitz microscope, a Numonics GraphicMaster image digitizer and an IBM 212 computer. The anatomical structure of interest was outlined 5 ‘E under low-power and the area covered by silver grains over individual labeled neurons was quantified. Neurons were visualized under bright =0 field illumination with a 40X objective and projected onto a video mon- c” 100 itor with a resulting magnification of 1200X. Each neuron of interest $ .E was outlined using the digitizing tablet and the threshold gray value 8’ was adjusted to distinguish grains from background staining. The por- tion of the enclosed area which was above threshold illumination was determined and expressed as the number of pixels occupied by silver 0 grains. Linear regression analysis has shown a direct correlation (+0.96 CONT SN STN SN/STN for 40X magnification) between the number of pixels measured with the MORPHON system and visual grain counting (Salin et al., 1990; Location of lesion Chesselet and Weiss-Wunder, 1994). For the present experiments, spe- Figure 4. Rotational behavior in lesioned animals following peripheral cific labeling was arbitrarily defined as the presence of 10 or more administration of apomorphine (0.5 mg/kg, s.c.). Rats received either a grains per pallidal neuron. sham lesion (CONT), a unilateral 6-hydroxydopamine lesion of the sub- A random sample of 50 neurons per animal was measured in each stantia nigra (SN), a unilateral kainic acid lesion of the subthalamic anatomical structure on the sides ipsilateral and contralateral to the le- nucleus @TN), or a combination of the two lesions (SN/STN). The bars sion. This was accomplished by moving nonoverlapping frames over represent the mean number + SEM of ipsiversive (towards the lesion, the region and measuring neurons with at least 10 grains over the cell open bars) or contraversive (away from the lesion, hatched bars) ro- body until 50 labeled cells were analyzed. We have previously verified tations in a 30 min test period. Asterisks indicate statistically significant that- this sample size provides a ver; reliable estimate of the average differences (p < 0.05) between absolute values (n = S/group) compared level of labeling in uallidal neurons (J. J Soghomonian and M. E Ches- with ANOVA and Dunnett’s post hoc comparison. selet, unpublishid observations; see Soghom&ian and Chesselet, 1992). In the present experiments, autoradiographic background in areas de- void of labeled cells was extremely low and thus the values were not subtracted. An average level of labeling per neuron was determined for been described previously (Chesselet et al., 1987). Briefly, the synthesis both the ipsilateral and contralateral side of each region for each rat, mixture consisted of 10 PM UTe 2.5 IJ,M ?-UTP (1000 Ci/mmol, and this value was used for statistical analysis. In addition, frequency NENlDu Pont, Boston, MA), ATP, CTP, and GTP in excess, SP6 or T7 distributions of the level of labeling were constructed for each pallidal RNA polymerase, dithiothreitol (DTT), ribonuclease inhibitor (RNA- segment. The density of labeled neurons in the globus pallidus and sin), and 2 pg of linearized DNA containing the insert. Due to its large entopeduncular nucleus was determined from camera lucida drawings. size, the GAD67 RNA probe was partially hydrolyzed into 100-200 For striatal sections from in situ hybridization and receptor autora- base pair fragments for increased tissue penetration (Chesselet et al., diographic studies, regional analysis was performed using the Macin- 1987). Following the synthesis, the probes were extracted in phenol/ tosh-based NIH IMAGE analysis system (version 1.44). Kodak optical chloroform/isoamyl alcohol and precipitated overnight in ethanol at density (0.D) standards were used to construct a curve from which O.D. -70°C. values could be determined from gray levels on the film. Optical density For in situ hybridization histochemistry, experiments were performed measurements were taken separately for the ipsilateral and contralateral on frontal sections (10 pm) through the striatum (A-P: 1.7 mm), globus striatum. The results from two sections per rat were averaged and the pallidus (A-P: -0.92 mm), and entopeduncular nucleus (A-P: -2.8 mean O.D. for the ipsilateral and contralateral striatum was calculated. mm) based on the atlas of Paxinos and Watson (1986). Sections were Data analysis. Only slides processed concurrently in individual ex- brought to room temperature from -70°C under a stream of cool air, periments were compared statistically and all statistical analyses were postfixed in 3% paraformaldehyde, acetylated, and dehydrated. Sections done on absolute values with the STATVIEW 512+ INTERACTIVE STATIS- TICS AND GRAPHICS PACKAGE (version 1.0, Abacus Concepts). For au- were incubated with 3-5 ng probe (400,000 dpm/ng) in humid chambers toradiography and in situ hybridization data, comparisons were made at 50°C for 3 l/2 hours. Posthybridization treatments included three between the ipsilateral or contralateral side of lesioned rats and the washes in 50% formamide, 2 X SSC (0.3 M NaC1/0.03 M sodium ci- corresponding side of control rats using ANOVA and the Dunnett’s post trate) at 52°C and a 30 min incubation in 100 kg/ml RNase A (Sigma hoc test with p < 0.05 considered significant (Bruning and Kintz, 1987). Chemical Co., St. Louis, MO) at 37°C. After an overnight rinse in 2 X Comparisons between the ipsilateral and contralateral sides of animals SSC, 0.05% Triton X-100, sections were dehydrated in graded ethanols, were made using a paired t test with p < 0.05 considered significant. defatted in xylene, and desiccated. In the striatum, there is a high den- For the purpose of comparing parallel experiments graphically, data are sity of cells expressing the mRNAs of interest, therefore film autora- expressed as the percentage of the corresponding side of control diography (3H-Ultrofilm, Leica, Malvern, PA) was used for quantifica- (?SEM). tion. In the globus pallidus and entopeduncular nucleus, the neurons expressing GAD67 and somatostatin mRNAs are scattered among my- elinated fibers and autoradiographic signals cannot be optimally detect- Results ed on film (Chesselet et al., 1987). Therefore, sections were coated with Kodak NTB3 emulsion diluted 1: 1 with 300 mu ammonium acetate for Histological verijcation of the lesions single-cell analysis. Test slides or strips of film were developed at reg- Rats usedin these studieswere selectedon the basisof histo- ular intervals to determine optimal exposure time, that is, when specific logical examination of the lesions. The subthalamicnucleus of labeling was robust but not saturating based on O.D. standards and rats with local injections of kainic acid (0.5 nmol in 0.1 ~1) previous emulsion experiments. This corresponds to 3-10 d for the pallidum and 14-30 d for the striatum. Both films and emulsion autora- showednearly completeloss of neuronsand extensive gliosis in diograms were developed in Kodak D-19 developer and fixed in Kodak the entire nucleus (Fig. l&D) comparedto controls (Fig. lA,C), Rapid Fix. Emulsion coated sections were lightly counterstained with except in a few caseswhere there was sparingof neuronsin the The Journal of Neuroscience, October 1995, 75(10) 6567

Figure 5. Photomicrographs illustrating the effects of subthalamic nucleus and substantia nigra lesions on the expression of GAD67 mRNA in the globus pallidus ipsilateral to the lesion. Sections of the globus pallidus were processed for in situ hybridization and emulsion autoradiography. Dark-field photomicrographs of neurons (white arrows) labeled with the GAD67 RNA probe in the globus pallidus of rats with sham lesion (A), a unilateral 6-hydroxydopamine (6-OHDA) lesion of the substantia nigra (B), a unilateral kainic acid lesion of the subthalamic nucleus (C), or a combination of the two lesions (D). Note the increase in the number of silver grains over individual neurons in B indicating an increase in GAD67 gene expression in the globus pallidus ipsilateral to a unilateral 6-OHDA lesion the substantia nigra. A decrease in the number of silver grains is observed in C indicating a decrease in GAD67 mRNA levels in the ipsilateral globus pallidus following a unilateral subthalamic nucleus lesion. Scale bars, 50 km. most medial pole of the nucleus.In somecases, gliosis extended uptake inhibitor 3H-BTCP was performed on striatal sections. into the most ventral part of the zona incerta, although the le- Loss of 3H-BTCP binding sites has been shown to be highly sionsdid not affect the adjacentlateral hypothalamus(Fig. 1B). correlated with decreaseddopamine level in the striatum after All animalswith 6-hydroxydopamine injections retained for the nigrostriatal lesions (Filloux et al., 1988; Vignon et al., 1988). study showeda profound lossof neuronalcell bodies and gliosis The loss of dopamineuptake siteswas greater than 90% in the in the substantianigra pars compacta (Fig. 2B,D) compared to striatum ipsilateral to the lesion in rats with either a unilateral controls (Fig. 2A,C). The adjacent substantianigra pars reticu- lesion of the substantianigra or a combination of subthalamic lata was not affected by the lesion (Fig. 2B). In some animals, nucleus and substantianigra lesions(Fig. 3B,D; Table 1) com- there was a moderate loss of neurons in the lateral aspectsof pared to controls (Fig. 3A). There was no significant difference the ventral tegmental area adjacentto the substantianigra (Fig. in the level or topographical distribution of the remaining 3H- 2B). BTCP sitesafter single or combined lesions(Fig. 3B,D; Table In order to confirm adequatedepletion of dopaminergicnerve 1). Unilateral lesionsof the subthalamicnucleus had no effect terminalsin nigrostriatal and combinedsubthalamic-nigrostriatal on dopamineuptake sites in the striatum on either side of the lesioned animals, autoradiography for the selective dopamine brain (Fig. 3C, Table 1). 6566 Delfs et al. *Widespread Effects of Subthalamic Nucleus Lesions in Rats

this region (Chesseletet al., 1987). Expression of GAD67 Cl ipsilateral mRNA has been correlated with levels or patterns of electro- contralateral q physiological activity in a number of systems(Chesselet et al., 1993) and has been previously shown to increasein the globus pallidus after unilateral lesionsof the nigrostriatal dopaminergic pathway (Kincaid et al., 1992; Soghomonian and Chesselet, 1992). Labeling for GAD67 mRNA in a section of the globus pallidus in a control rat is illustrated in Figure 5A. Unilateral kainic acid lesionsof the subthalamicnucleus resulted in a small (19.2%) decreasein the level of GAD67 gene expressionin the globus pallidus ipsilateral to the lesion (Figs. 5C, 6). Frequency distribution analysisconfirmed this effect by showing a shift to the left of the histogramcompared to the ipsilateral side of controls (Fig. 7A,E). The mediansof the frequency distributions were also significantly decreasedwhen comparedto the corre- spondingside of control rats (Table 2). The decreasein GAD67 mRNA expressionwas not due to changesin the number of labeledneurons in the globus pallidus (Table 2). CONT SN STN SN/STN Confirming previous results(Kincaid et al., 1992; Soghomon- Location of lesion ian and Chesselet,1992) unilateral 6-OHDA lesionsof the substantia nigra induced a marked increase(51.9%) in the level of Figure 6. Effect of subthalamic nucleus and substantia nigra lesions mRNA encodingGAD67 in the globus pallidus ipsilateralto the on the expression of GAD67 mRNA in the globus pallidus. Rats received either a sham lesion (CONZ), a unilateral 6-hydroxydopamine lesion (Fig. 5B, 6), without changesin the number of labeled lesion of the substantia nigra (SV), a unilateral kainic acid lesion of the neurons(Table 2). The frequency distribution of labelingper cell subthalamic nucleus (STN), or a combination of the two lesions (SW in the ipsilateral globus pallidus was shifted to the right, and its SW) as described in the Materials and Methods section. Sections of median increasedwhen compared to the contralateral side and the globus pallidus were processed for in situ hybridization histochemistry and emulsion autoradiography. The bars represent the mean num- to the ipsilateral side of control rats, confirming the increased ber of pixels occupied by silver grains per neuron on the side ipsilateral level of labeling (Fig. 7A,C,D; Table 2). This increasewas abol- (open bars) or contralateral to the lesion (hatched bars) and are ex- ished in rats with a lesion of the subthalamic nucleus on the pressed as percent of control + SEM. Asterisks indicate statistically sameside as the 6-OHDA lesion (Fig. SD, 6). Analysis of fre- significant differences @ < 0.05) between absolute values of the cor- quency distributions of the level of labeling per neuron con- responding sides of control and lesioned animals (n = 5 per group) with ANOVA and Dunnett’s post hoc comparison. firmed the absenceof changesin GAD67 mRNA levels after combinedlesions (Fig. 7A,G; Table 2).

Apomorphine-inducedrotational behavior GAD 67 and somatostatinmRNAs in the entopeduncular Rats with a unilateral lesion of the subthalamicnucleus exhibited nucleus (internal pallidum) robust ipsiversive rotational behavior in responseto peripheral Subthalamiclesions alone did not significantly changeGAD67 administration of the dopamineagonist apomorphine (0.5 mgl mRNA levels in the ipsilateral or contralateralentopeduncular kg, s.c.) administered14 d postlesion(Fig. 4). The direction of nucleus(Fig. 8). Unilateral 6-OHDA lesionsof the substantiani- the rotation was opposite to that observed after apomorphine gra, although not causing any significant effects on the side ip- injection in rats with unilateral nigrostriatal lesions.All rats with silateralto the lesion,resulted in a significantdecrease in GAD67 unilateral nigrostriatal lesionsselected for this study displayed mRNA levels per neuronand a decreasein the numberof labeled robust contraversive circling (>200 turns in 30 min) in response neuronsin the contralateralentopeduncular nucleus (Fig. 8, Table to apomorphinegiven 8 d after the nigrostriatal lesion (Fig. 4). 2), in agreementwith previous data from our laboratory (Sogh- Rats with combinedlesions did not exhibit any appreciablero- omonian and Chesselet, 1992). Confirming the decreasein tational behavior following injection of apomorphine (Fig. 4). GAD67 gene expression,there was a significant decreasein the Similarly, control rats which received only stereotaxicinjections medianof the frequency distribution of the level of labeling and of salinedid not rotate significantly following peripheral admin- a shift to the left in the histogram compared to controls (Fig. istration of apomorphine(Fig. 4). This finding is consistentwith 9B,D; Table 2). The contralateraldecrease in GAD67 mRNA (Fig. the report that subthalamicnucleus ablation markedly reduces 8, Table 2) was blocked in rats with lesionsof both the subtha- dopamineDl and D2 agonist-inducedrotation in rats with a lamic nucleusand substantianigra. Analysis of frequency distri- unilateral nigrostriatal lesion (Anderson et al., 1992). butions of the level of labeling per neuronconfirmed the lack of effects of the combinedlesion on GAD67 mRNA levels in the GAD67 gene expressionin the globus pallidus (external entopeduncularnucleus (Fig. 9B,H, Table 2). pallidurn) Although somatostatinmRNA is only detected at low levels Sections from the pallidum were processedfor detection of in a few neurons of the entopeduncularnucleus and adjacent GAD67 mRNA at the single cell level by in situ hybridization lateral hypothalamusin control rats, nigrostriatal lesionshave histochemistry. Measurementsof GAD mRNA in the pallidum, been previously shown to induce a massiveincrease in the level a brain region which contains both GABAergic inputs and out- of expressionof somatostatinmRNA in theseregions (Sogho- puts, provides unique information on output neurons. Indeed, monian and Chesselet,1991). These findings were confirmed in both inputs and outputs of the pallidum contain GAD and the present study (Fig. 10). Subthalamic lesionsalone had no GABA, whereas only output neurons contain GAD mRNA in effect on somatostatinmRNA expressionin the ipsilateral en- The Journal of Neuroscience, October 1995, 75(10) 6569

lpsilateral Contralateral “1 A CONT ‘1 B CONT

Figure 7. Histograms of frequency distributions of labeling for GAD67 mRNA in the globus pallidus of rats ipsilateral (left panels) or contralateral (right panels) to a sham lesion (A, B; CONT), a unilateral 6-hydroxydopamine lesion of the substantia nigra (C, D; SN), a unilateral kainic acid lesion of the subthalamic nucleus (I?, F; STNJ, or a combination of the two lesions (G, H; SNLSTN) as described in the Mate- rials and Methods section. Quantifica- tion of silver grains over individual neurons was done with the MORPHON image analysis system on sections processed for emulsion autoradiography. Data include values from 50 neurons analyzed per rat (n = S/group) from either the ipsilateral or the contralateral globus pallidus. Note the shift to the I2 G SNlSTN ‘? H SNISTN right in the histogram in C compared IO ‘0 to A illustrating an increase in the level 1 I of labeling for GAD67 mRNA in the ipsilateral globus pallidus following a unilateral substantia nigra lesion. Also note the shift to the left in the histogram in E compared to A illustrating the decrease in the level of GAD67 gene expression in the globus pallidus following a unilateral subthalamic nucleus lesion. The medians of the frequency distributions are presented in Pixels per neuron Table 2. topeduncular nucleus but induced an increase in somatostatin the striatum(Fig. 11). Confirming previous reports by numerous mRNA in the contralateral entopeduncular nucleus (Fig. 10). groups (for review, Graybiel, 1990; Angulo and McEwen, The contralateral increase in somatostatin mRNA observed fol- 1994), 6-OHDA lesions of the substantianigra resulted in a lowing the unilateral subthalamicnucleus lesion was reduced, marked increasein enkephalin gene expressionin the striatum but not abolishedin animalswith combinedlesions (Fig. 10). In ipsilateral to the lesion (Fig. 11A). The levels of substanceP contrast, the ipsilateral increasewas no longer significant in an- mRNA in 6-OHDA lesionedrats were not significantly different imals with combinedlesions (Fig. 10). from levels in control (saline-injected)rats. However, there was a significant decreasein the ipsilateral striatum when compared Enkephalin and substanceP gene expressionin the striatum comparedto the contralateral sideof 6-OHDA lesionedrats (Fig. In order to assessthe effects of the lesionson striatal neurons 11B). Neither the increase in enkephalin nor the decreasein projecting to the pallidum, the level of mRNAs encoding the substanceP mRNA were observed in the striatum of rats which neuropeptidesenkephalin and substanceP were measuredin stri- received both subthalamicnucleus and substantianigra lesions atal sectionswith film autoradiography. Enkephalin and sub- (Fig. 11). stance P are colocalized with GABA in efferent neurons projecting to the globus pallidus and entopeduncularnucleus, re- Discussion spectively (Graybiel, 1990). Lesionsof the subthalamicnucleus This study showsthat unilateral lesionsof the subthalamicnu- did not alter the level of enkephalin or substanceP mRNA in cleus have widespreadeffects on alterationsin gene expression 6570 Delfs et al. l Widespread Effects of Subthalamic Nucleus Lesions in Rats

Table 2. Medians of the frequency distributions of labeling and number of neurons labeled for GAD67 mRNA after lesions

CONT SN STN SN/STN Mediansa Ipsilateral GP 78.2 + 1.6 119.4 ? 4.2” 62.4 f 1.4* 72.1 k 3.9 Contralateral GP 77.8 2 2.6 79.6 ? 1.8 80.4 2 2.1 73.2 5 2.2 Ipsilateral EP 67.4 2 1.8 68.4 2 2.8 63.4 2 1.6 71.0 2 2.8 Contralateral EP 65.8 2 3.3 52.0 + 0.9* 66.0 2 1.7 69.8 -t 1.7 Number of labeled neuronsb Ipsilateral GP 66.2 5 6.8 65.0 + 10.2 67.1 t 4.2 58.9 2 3.7 Contralateral GP 63.2 k 5.3 64.8 z 6.9 68.9 2 7.3 60.9 -c 4.5 Ipsilateral EP 21.1 5 3.9 24.6 + 1.7 18.5 2 2.8 21.6 + 3.1 Contralateral EP 27.4 ? 7.9 11.7 + 2.1* 16.7 + 1.6 17.6 t 3.2 *p < 0.05 when compared to the corresponding side of control animals using ANOVA and Dunnett’s post hoc comparison. u Medians of frequency distributions of labeling for GAD67 mRNA in the globus pallidus (GP) and entopeduncular nucleus (EP) both ipsilateral and contralateral to unilateral lesions of the substantia nigra (SN), subthalamic nucleus (STN), or a combination of the two lesions (SNBTN). Frequency distributions of levels of labeling were constructed for individual animals and their medians were determined. Data are mean + SEM of values obtained for five rats per group. Absolute values cannot be compared between the GP and EP because sections were processed in separate experiments. h Number of labeled neurons per mm2 were measured using camera lucida drawings. induced by unilateral lesions of the nigrostriatal pathway. The the data reveal that unilateral subthalamic lesions in otherwise data suggest that the subthalamic nucleus may contribute in intact rats produce strong ipsiversive rotational behavior in re- more ways than previously thought to the functional conse- sponse to apomorphine but have only restricted effects on the quences of dopamine depletion in the basal ganglia. In addition, levels of mRNAs examined.

Effects of unilateral subthalamic lesions q ipsilateral The rotational behavior induced by apomorphine in rats with H contralateral unilateral lesionsof the subthalamicnucleus confirmed previous data (Kafetzopoulos and Papadopoulos,1983). In the present study, however, the dose and injection volume of kainic acid were fivefold lower to avoid damageto adjacentareas, indicating that the effect was specifically due to the subthalamicnucleus lesion. Peripheral administrationof apomorphineincreases the firing rate of subthalamic nucleus neurons in intact rats (Thompsonand Walters, 1993). After unilateral lesions,this increasedactivity would only occur in the intact subthalamicnucleus, and the resulting asymmetry could be responsiblefor the rotational behavior. The motor pathways mediating this effect remain to be determined. In contrast to unilateral nigrostriatal lesions, lesions of the subthalamicnucleus had minimal effects on gene expressionin the basal ganglia. However, the small decreasein GAD67 mRNA levels observed in the ipsilateral globus pallidus is of CONT SN STN SN/STN interest becauseit suggeststhat excitatory subthalamicinputs Location of lesion (Parent and Hazrati, 1995) contribute to maintaining levels of GAD67 mRNA in this region. This result is not in contradiction Figure 8. Effect of subthalamic nucleus and substantia nigra lesions on the expression of GAD67 mRNA in the entopeduncular nucleus. with recent findings of Andrkn et al. (1995), who showed in- Rats received either a sham lesion (COlvT), a unilateral 6-hydroxydo- creasedGAD activity in the globus pallidus after lesion of the pamine lesion of the substantia nigra (W), a unilateral kainic acid lesion subthalamicnucleus in the marmoset.Indeed, the enzyme GAD of the subthalamic nucleus (STN), or a combination of the two lesions is present in both afferent terminals and output neuronsin the (.SN/siW) as described in the Materials and Methods section. Sections globus pallidus, whereas,GAD mRNA is only present in the were processed for in situ hybridization histochemistry and emulsion autoradiography. The bars represent the mean number of pixels occu- cell bodies of output neurons(Chesselet et al., 1987, 1993). The pied by silver grains per neuron on the side ipsilateral (open bars) or decreasein GAD67 mRNA levels we observed may be related contralateral to the lesion (hatched bars) and are expressed as percent to changesin patterns of firing activity observed in the globus of control 2 SEM. Asterisksindicate statistically significant differences pallidus after lesions of the subthalamic nucleus (Ryan and (p < 0.05) between absolute values of the corresponding sides of control and lesioned animals (n = 5 per group) with ANOVA and Dunnett’s Sanders,1993). This adds to previous observations that levels post hoc comparison. of GAD67 mRNA are correlated with patterns of electrophysi- The Journal of Neuroscience, October 1995, 15(10) 6571

lpsilateral Contralateral ‘; A CONT 8 1

Figure 9. Histograms of frequency distributions of labeling for GAD67 mRNA in the entopeduncular nucleus ‘1”] F STN of rats ipsilateral (left panels) or contralateral (right panels) to a sham lesion (A, B; COiVT), a unilateral 6-hydroxydopamine lesion of the substantia nigra (C, D; Slv), a unilateral kainic acid lesion of the subthalamic nucleus (E, F; STN), or a combination of the two lesions (G, F; SN/STN)as described in the Materials and Methods section. Quantification of silver grains over individual neurons was done with the MORPHON image analysis system 10 on sections processed for emulsion au- ‘;’ G SNISTN 9” SNISTN toradiography. Data include values 8. 8 1 from 50 neurons analyzed per rat (n = 7. 5/group) from either the ipsilateral or 6, I I the contralateral entopeduncular nucleus. Note the shift to the left in the histogram in D compared to B illustrating a decrease in the level of labeling for GAD67 in the contralateral entopeduncular nucleus following a unilateral substantia nigra lesion. The medians of the frequency distributions are present- Pixels per neuron ed in Table 2. ological activity in GABAergic neurons (Litwak et al., 1990; 1992). The consequences of subthalamic lesions could be blunt- Segovia et al., 1990; Drengler and Oltmans, 1993). ed in the entopeduncular nucleus because of this anatomical or- Although the subthalamic nucleus also projects to the ento- ganization. peduncular nucleus (Nauta and Cole, 1978; Kita and Kitai, 1987; Parent and Smith, 1987; Takada et al., 1988; Smith et al., 1990; Effects of subthalamic lesionsin rats with unilateral Berendse and Groenewegen, 1991), no significant changes in nigrostriatal lesions GAD67 mRNA were observed in this region after subthalamic All previously observed effects of 6-OHDA lesions on the pa- lesions. Alterations in the entopeduncular nucleus could occur rameters examined (Graybiel, 1990; Soghomonian and Chesse- with a different time course than those in the globus pallidus, let, 1991, 1992; Angulo and McEwen, 1994) were confirmed in as previously observed after pharmacological treatments (Delfs this study, and all these effects were abolished in rats with com- et al., 1995a). However, Blandini and Greenamyre (1995) ob- bined nigrostriatal and subthalamic nucleus lesions. It is unlikely served that cytochrome oxidase and succinate dehydrogenase, that failure to observe the effects produced by the 6-OHDA markers for neuronal activity, were decreased in the ipsilateral lesion alone in animals with combined lesions is due to differ- globus pallidus but not in the entopeduncular nucleus after uni- ences in the location or extent of nigrostriatal lesions between lateral subthalamic lesions. In addition to excitatory inputs from the two groups. Indeed, the histology of the lesions and the level the subthalamic nucleus, the entopeduncular nucleus receives of depletion of dopaminergic terminals were similar in rats with inhibitory inputs from the globus pallidus (Bolam and Smith, and without subthalamic esions. Furthermore, rats with the com- 6572 Delfs et al. *Widespread Effects of Subthalamic Nucleus Lesions in Rats

300 and somatostatinmRNAs previously observed in the entope- 0 ipsilateral duncular nucleus after unilateral nigrostriatal lesions (Sogho-’ c q contralateral monian and Chesselet,1991, 1992). Blockade of the ipsilateral increasein somatostatinmRNA in the entopeduncularnucleus after combinedlesions suggests that this effect may be mediated by increasedactivity in output neurons from the subthalamic * nucleusas well. Evidence that unilateral subthalamiclesions also blocked contralateral effects of dopaminergiclesions is not sur- * prising in view of recent evidence that subthalamiclesions in the marmosetinduce bilateral changesin a variety of neurotransmitter-related enzymesin the basalganglia (Andren et al., 1995). Unilateral dopaminedepletion or pharmacologicalmanipulations of the substantianigra have also been shown to induce contralateral effects on neurotransmittersand metabolismin basalganglia (Cheramy et al., 1981; Savaki et al., 1984; Zetterstrom et al., 1986; Normand et al., 1988; Robinson and Whishaw, 1988; Lindefors and Ungerstedt, 1990; Savaki et al., 1992). In addition, we have recently observed that unilateral nigrostriatal le- CONT SN STN SN/STN sionsinduce a bilateral increasein GAD67 mRNA in the retic- Location of lesion ular thalamic nucleus (Delfs et al., 199513).The neuronal pathways mediating the decrease in GAD67 mRNA in the Figure 10. Effect of subthalamic nucleus and substantia nigra lesions contralateral entopeduncular nucleus after 6-OHDA lesions, on the expression of somatostatin mRNA in the entopeduncular nucleus. Rats received either a sham lesion (CONT), a unilateral 6-hydroxydo- however, remain ill defined(Soghomonian and Chesselet,1992). pamine lesion of the substantia nigra (SN), a unilateral kainic acid lesion Becausechanges in basal ganglia output resulting from dopa- of the subthalamic nucleus (STN), or a combination of the two lesions mine depletion ultimately affect cortical neurons,it is possible (SNLSTN) as described in the Materials and Methods section. Sections that contralateral effects of unilateral dopaminergiclesions are were processed for in situ hybridization histochemistry and emulsion mediatedby bilateral corticostriatal projections (McGeorge and autoradiography. The bars represent the mean number of pixels occupied by silver grains per neuron on the side ipsilateral (open bars) or Faull, 1989). contralateral to the lesion (hatched bars) and are expressed as percent Recent data suggestthat corticostriatal pathways may also be of control 2 SEM. Asterisks indicate statistically significant differences important for the effects of dopaminedepletion in the striatum. @ < 0.05) between absolute values of the corresponding sides of con- Electrophysiological and binding studiesprovide indirect evi- trol and lesioned animals (n = 5 per group) with ANOVA and Dunnett’s post hoc comparison. dencefor increasedrelease of glutamatefrom corticostriatal neurons in the striatum of dopamine-denervatedrats (Calabresiet al., 1993; Wtillner et al., 1994). Furthermore, Campbell and bined lesionsshowed no rotational behavior, whereasrats with Bjorklund (1994) have shown that knife-cut transectionsof the subthalamic nucleus lesionsalone exhibited strong ipsiversive cerebral cortex prevent enkephalin mRNA increasesin the stria- rotations. Thus, a nigrostriatal lesion sufficient to counteract the turn of rats with unilateral nigrostriatal lesions.A contribution rotational behavior resulting from the subthalamiclesion was of the corticostriatal pathways may explain why changesin en- present in each rat with combinedlesions. kephalin and substanceP mRNA levels in the striatum were Both spontaneousactivity and burst firing increasein the sub- blocked in animalswith combinedlesions. Indeed, it is unlikely thalamic nucleus after dopaminedepletion (FCger et al., 1991; that this effect was due to the lesion of neuronsprojecting from Robledo and Feger, 1991; Hollerman and Grace, 1992; Bergman the subthalamicnucleus to the striatum becausethis pathway is et al., 1994). Furthermore,NMDA receptorsare down-regulated sparseand restricted to the rostroventral striatum(Kita and Kitai, in the globus pallidus after 6-OHDA lesionssuggesting an over- 1987), whereaschanges in enkephalin and substanceP mRNAs active glutamatergicinput, most likely from the subthalamicnu- were observed in the entire dorsal striatum. Similarly, theseef- cleus (Porter et al., 1994). The absenceof changesin GAD67 fects are probably not mediatedby projections from the globus mRNA in the globus pallidus after combined lesionssuggests pallidus becauseblockade of haloperidol-inducedincreases in that increasedneuronal activity in the subthalamicnucleus trig- GAD67 mRNA in the globus pallidus by scopolaminedoes not gers the increasein GAD67 mRNA levels observed after uni- abolishenkephalin increases in the striatum (Delfs et al., 1995a). lateral nigrostriatal lesions(Kincaid et al., 1992; Soghomonian Stimulation of dopamine receptors in the striatum modulates and Chesselet,1992). It has been proposedthat increasedfiring neuronal responsesto glutamate, the neurotransmitterof corti- in the subthalamicnucleus after dopaminedepletion resultsfrom costriatal neurons (Cepeda et al., 1993). Therefore, direct loss decreasedinhibition by afferents from the globus pallidus (Albin of stimulation of striatal dopaminereceptors and changesin ac- et al., 1989; Robledo and Feger, 1991; Bergman et al., 1994). tivity of corticostriatal neuronsindirectly mediatedby the sub- This is unlikely, however, in view of evidence of increasedburst thalamic nucleus may concur to induce changesin striatal gene firing activity and GAD mRNA expressionin the globus palli- expressionafter nigrostriatal lesions. dus. A reduced stimulation of dopamine receptors by endoge- In conclusion, the results lead one to reconsiderthe relative nousdopamine normally presentin collateralsof the nigrostriatal contribution of dopaminedepletion within the striatum, and of pathway (Bjorklund and Lindvall, 1984; Parry et al., 1994) polysynaptic pathways activated by the subthalamicnucleus, to could insteadtrigger the increasedactivity of subthalamicneu- the molecular effects observed after dopamine depletion. The rons (Campbellet al., 1985; Flores et al., 1993). pivotal role of the subthalamicnucleus may explain the striking The presentresults confirm the complex alterationsin GAD67 beneficial effects of lesioning subthalamicneurons or antago- The Journal of Neuroscience, October 1995, 75(10) 6573

A ENKEPHALIN mRNA B SUBSTANCE P mRNA * 200 0 ipsilateral Cl ipsilateral G H contralateral H contralateral

100

0 CONT SN STN SN/STN CONT SN STN SN/STN Location of lesion Figure II. Effect of subthalamic nucleus and substantia nigra lesions on the expression of enkephalin (A) and substance P mRNA (B) in the striatum. Rats received either a sham lesion (COlvr), a unilateral 6-hydroxydopamine lesion of the substantia nigra (Slv), a unilateral kainic acid lesion of the subthalamic nucleus (STiv), or a combination of the two lesions (SN/STN) as described in the Materials and Methods section. Sections were processed for in situ hybridization histochemistry and film autoradiography. The bars represent the mean optical density measured from film autoradiograms on the side ipsilateral to the lesion (open bars) or contralateral to the lesion (hatched bars) and are expressed as percent of control t SEM. Asterisks indicate statistically significant differences (p < 0.05) between absolute values of the corresponding sides of control and lesioned animals (n = 5 per group) with ANOVA and Dunnett’s post hoc comparison. Plus sign (+) indicates a statistically significant difference 0, < 0.05) between the ipsilateral and contralateral side using a paired t test. nizing their effects on symptoms resulting from dopamine de- subthalamic nucleus. II. Neuronal activity in the MPTP model of pletion in animal models and patients with Parkinson’sdisease narkinsonism. J Neurophvsiol 72:507-520. Bj&klund A, Lindvall O* (1984) Dopamine-containing systems in the (Bergman et al., 1990; Aziz et al., 1991; Brotchie et al., 1991; CNS. In: Handbook of clinical neuroanatomy, Vol 3 (BjBrklund A, Klockgether et al., 1991; Greenamyre et al., 1994; Limousin et H&felt T, eds), pp 55-122. Amsterdam: Elsevier. al., 1995). 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