Anterograde Axonal Transport of AAV2-GDNF in Rat Basal Ganglia

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Anterograde Axonal Transport of AAV2-GDNF in Rat Basal Ganglia Agnieszka Ciesielska1, Gabriele Mittermeyer1, Piotr Hadaczek1, Adrian P Kells1, John Forsayeth1 and Krystof S Bankiewicz1 Department of Neurological Surgery, University of California, San Francisco, California, USA

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We elucidated the effects of parkinsonian degeneration on trafficking of AAV2-GDNF in the nigro-striatum (nigro-ST) of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. Vector infused into striatum (ST) was transported to substantia nigra (SN), both pars compacta (SNc), and pars reticulata (SNr). In the lesioned hemisphere, glial cell line– derived neurotrophic factor (GDNF) immunoreactivity was only found in SNr consistent with elimination of SNc dopaminergic (DA) neurons by 6-OHDA. Further analysis showed that striatal delivery of AAV2-GDNF resulted in GDNF expression in globus pallidus (GP), entopeduncular nucleus (EPN), and subthalamic nucleus (STN) in both lesioned and unlesioned hemispheres. Injection of vector into SN, covering both SNc and SNr, resulted in striatal expression of GDNF in the unlesioned hemisphere but not in the lesioned hemisphere. No expression was seen in GP or EPN. We conclude that adeno-associated virus serotype 2 (AAV2) is transported throughout the nigro-ST exclusively by anterograde transport. This transport phenomenon directs GDNF expression throughout the basal ganglia in regions that are adversely affected in ­Parkinson’s disease (PD) in addition to SNc. Delivery of vector to SN, however, does not direct expression of GDNF in ST, EPN, or GP. On this basis, we believe that striatal delivery of AAV2-GDNF is the preferred course of action for trophic rescue of DA function. Received 27 August 2010; accepted 18 October 2010; published online 23 November 2010. doi:10.1038/mt.2010.248

Introduction Parkinson’s disease (PD) is characterized by the progressive degeneration of midbrain dopaminergic (DA) neurons of substantia nigra pars compacta (SNc), leading to the pathological DA deficiency in the striatum (ST). However, dysfunction of the other subcortical structures connected to the basal ganglia is also observed.1 Currently available antiparkinsonian treatments, although effective in the early phase of PD, are unable to arrest the degenerative process. Neurotrophic factors may promote restoration of function of DA neurons and protect them from ongoing degeneration. Glial cell line–derived neurotrophic ­factor (GDNF) is a potent trophic factor for nigral DA neurons, an

observation that led to the investigation of GDNF therapy, either by ­recombinant protein infusion2,3 or by gene therapy,4–7 to change the natural course of PD. The pre-eminent vector used for gene therapy of PD in humans has been adeno-associated virus serotype 2 (AAV2), which has amassed a significant safety profile in a number of phase 1 or 2 trials.8–10 Convection-enhanced delivery of AAV2 has proven to be an effective way to distribute AAV2 broadly into ST. This approach to AAV2-GDNF gene transfer has been shown to result in widespread, durable GDNF expression in nonhuman primate (NHP) studies,6,7,11 suggestive of the therapeutic potential of AAV2-GDNF for the treatment of PD. These observations also extend to neurturin, a GDNF homolog that recapitulates many of the biological effects of GDNF in gene transfer experiments.12 One of the critical, and possibly limiting, factors in the AAV2-GDNF gene transfer approach in the treatment of PD is altered trafficking of vector in the degenerated PD brain. The putamen and/or substantia nigra (SN) are the anatomical sites in the brain targeted for AAV2-GDNF delivery,6,7,11,13 although it is unclear whether nigral delivery alone is efficacious in terms of regeneration.11,14 Both of these regions are critical parts of the basal ganglia circuitry and are axonally connected with relevant input and output nuclei.15 It has been postulated that, after transduction of striatal neurons with AAV2-GDNF, retrograde axonal transport (along nigrostriatal fibers) is responsible for delivery of GDNF into DA cells in SNc, where GDNF receptors are located.16 Therefore, progressive degeneration of the nigrostriatal pathway could impair axonal transport of GDNF from putamen into SNc, thus significantly limiting the therapeutic effects of GDNF. The purpose of this study, therefore, was to elucidate the effects of nigral degeneration on the distribution of striatally infused AAV2-GDNF. By comparing transport of vector in the intact versus the severely 6-hydroxydopamine (6-OHDA)–lesioned nigrostriatum (nigro-ST) of the rat, we show that AAV2-GDNF is transported throughout the basal ganglia primarily by anterograde transport. Our findings support the concept of putaminal delivery of AAV2-GDNF in order to distribute vector to other basal ganglia components, such as globus pallidus (GP) and subthalamic nucleus (STN), that are also adversely affected in PD in addition to SN and ST.17 Our findings are supportive of our planned phase 1 clinical trial of putaminal AAV2-GDNF in PD patients.

Correspondence: Krystof S Bankiewicz, Department of Neurological Surgery, University of California, San Francisco, California, 94103-0555, USA. E-mail: [email protected]

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a

4 weeks

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Axonal Transport of GDNF

25 weeks

a

Anatomical schematic

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GAD 65/67

SNc VTA

SNr 1 mm

MT

c

ST

ST

SN

SN

Control

Lesioned

c

GDNF

d

TH

e

Merge GAD/GDNF

f

Merge GAD/GDNF/TH

Figure 2  Glial cell line–derived neurotrophic factor (GDNF), glutamic acid dehydrogenase (GAD) and tyrosine hydroxylase (TH) staining of substantia nigra (compacta and reticulata) after bilateral AAV2GDNF injection to the striatum. (a) Brain slice schematic taken from the rat brain atlas18 corresponds to substantia nigra pars reticulata (SNr) and compacta (SNc), ventral tegmental area (VTA) and medial terminal nucleus (MT) - posterior to bregma: −5.20 mm. (b) GAD65/67-staining after color replacement (red to blue) with Adobe Photoshop. (c) GDNF and (d) TH staining of coronal sections without any color correction. Panel e depicts merging of b and c. Panel f depicts merging of b,c, and e. For details see Materials and Methods.

Figure 1 Transport of glial cell line–derived neurotrophic factor (GDNF) throughout the nonlesioned and lesioned nigrostriatal pathway after injection of AAV2-GDNF to the striatum (ST). GDNF-immunostaining of the coronal sections after bilateral striatal injection of AAV2-GDNF into unilateral 6-hydroxydopamine lesioned rats at (a) 4 weeks and (b) 25 weeks after striatal AAV2-GDNF. GDNF staining was widely distributed throughout most of both hemispheres (top images) and in the substantia nigra (SN). GDNF staining in lesioned SN, however, was less prominent. Strong GDNF expression was seen within SNr in both hemispheres (c) high magnification, bottom images). On the lesioned side, little GDNF signal was seen in substantia nigra pars compacta (SNc). Approximate anatomical coordinates are + 2.0 anterior from bregma (ST level) and –5.20 posterior from bregma (SN).

Results Bilateral striatal injection of AAV2-GDNF After bilateral injection of AAV2-GDNF into the ST of naive animals, robust GDNF staining was revealed throughout the ST and in the SN, both SNc and SN reticulata (SNr) (data not shown) in agreement with previously published studies by our group6,11 and by others.18 Transport of AAV2 to distal locations in brain has been reported to be mediated by both retrograde19–21 and anterograde mechanisms.6,7,11,22–24 The purpose of this study, therefore, was to determine the relative importance of retrograde and anterograde transport in the distribution of AAV2 throughout the basal ganglia, particularly the SNc, in the lesioned nigro-ST. Transport of AAV2 to SNc could proceed via retrograde transport along nigro-ST projections, or in an anterograde direction via direct GABA-ergic projections to SNr and indirect projections to SNc.25 In our experimental paradigm, we infused AAV2-GDNF bilaterally into either the ST or SN of Molecular Therapy vol. 19 no. 5 may 2011

rats unilaterally lesioned with 6-OHDA. We then allowed rats to survive for either 4 or 25 weeks in order to reveal any possible transport effects related to the fact that AAV2 is known to be a slowly uncoating virus.26 However, because there were no significant differences between ST-to-SN transport at 4 and 25 weeks (Figure 1a,b), all subsequent experiments were of 4 weeks in duration after vector infusion. First, we injected AAV2-GDNF bilaterally into the ST of unilaterally lesioned rats (n = 8). Robust and stable GDNF expression was obtained in both striata in all rats (Figure 1a,b). Concomitant GDNF expression was also seen in SN of both hemispheres. However, more detailed inspection of SN in control and lesioned hemispheres revealed that, whereas both SNc and SNr were GDNF-positive on the unlesioned side (Figure 1c, left panels), only SNr contained GDNF-positive cells on the lesioned side (Figure  1c, right panels). Elimination of SNc-to-ST projections in the parkinsonian brain ablates this effect. We conclude from this experiment that direct retrograde transport of vector from ST to SNc might occur along SN-to-ST projections in the normal brain or alternatively along indirect pathways in an anterograde direction.

AAV2-GDNF transport to SNr Neurons in the SNr are primarily GABA-ergic. In order to confirm that the GABA-ergic neurotransmission system is responsible for the axonal transport of GDNF from ST to SNr, we used image-­processing to superimpose images precisely to produce a composite resembling a double-staining. Serial sections through SN were stained with an antibody specific for GAD65/67, as well as with antibodies against GDNF and tyrosine hydroxylase (TH). 923

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Axonal Transport of GDNF

a

External globus pallidus

Control

Lesioned

a

GDNF

b

TH

ST

ST

SN

SN

Bregma ~ −1.30 mm

b

Entopeduncular nucleus

Bregma ~ −3.50 mm

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Subthalamic nucleus

Bregma ~ −3.80 mm

Figure 3 Distribution of glial cell line–derived neurotrophic factor (GDNF) after striatal AAV2-GDNF injection. (a) The external globus pallidus (GP) contained GDNF-positive cells (single arrow) and GDNFpositive fibers in both the unlesioned (left panels) and lesioned (right panels) hemisphere. (b) Strong intracellular (single arrow) and extracellular GDNF signal was also detected in entopeduncular nucleus (EPN) again in both hemispheres. (c) GDNF was also present in the subthalamic nucleus (STN) similar in both lesioned and unlesioned STN.

Single staining revealed that, in the nigral region (Figure 2a schematic), a dense network of glutamic acid dehydrogenase (GAD)immunoreactive fibers was localized to the SNr (Figure  2b). Figure  2b also shows that the intensity of GAD staining in SNr was similar in both hemispheres. After merging and adjusting both images, the first showing GAD signal from SN (Figure 2b) and the second showing GDNF signal from the same area on the adjacent section (Figure 2c), we found that GAD staining from SNr (blue) overlapped well with GDNF staining (brown) (Figure  2d). After adding the third image of TH staining (Figure 2e), GDNF staining in the lesioned SN was seen in SNr (Figure 2f). In contrast, in the unlesioned side, GDNF staining was found in both SNr and SNc. Data obtained from the control hemisphere suggested that transport of AAV2-GDNF from the ST to the SNc could be mediated by either anterograde or retrograde axonal transport. However, data obtained on the 6-OHDA-lesioned side strongly indicate the primacy of anterograde axonal transport to SN from ST. The expression of GDNF in the SNr is likely mediated by anterograde transport of AAV2-GDNF by direct striatal GABAergic projections to SNr. We asked, therefore, whether similar indirect GABA-ergic projections could mediate vector transport to SNc. As shown in Figure  3, infusion of AAV2-GDNF into ST resulted in GDNF expression not only in external GP (Figure  3a),entopeduncular nucleus (EPN) (Figure 3b), components of the direct GABA-ergic pathway, but also in STN, part of the indirect pathway (Figure 3c). This pattern of expression was independent of 6-OHDA lesioning of SNc. 924

Figure 4 Transport of glial cell line–derived neurotrophic factor (GDNF) throughout the nonlesioned and lesioned nigrostriatal pathway after injection of AAV2-GDNF to the substantia nigra. (a) GDNF staining is widely distributed throughout infused nigral region in both unlesioned and lesioned hemisphere (bottom image). Striatal GDNF expression (diffuse pattern due to secretion of the protein) was seen in the unlesioned striatum but not in the lesioned striatum (top image). These data indicate that nigral injection of AAV2-GDNF does not result in striatal expression of GDNF. Approximate anatomical coordinates are + 2.0 anterior from bregma [striatum (ST) level] and –5.20 posterior from bregma [substantia nigra (SN) level].18 (b) Note the asymmetrical tyrosine hydroxylase (TH) staining at the level of ST (top image) and SN (bottom image) after 6-hydroxydopamine lesion.

Bilateral nigral injection of AAV2-GDNF If the primary mechanism by which AAV2 is distributed in the brain is anterograde transport, it follows that infusion of AAV2GDNF into SN should result in ST expression only when the SNc projections are intact but not when they have been destroyed by 6-OHDA intoxication, or severely damaged as in PD. As shown in Figure  4, nigral injection of AAV2-GDNF generated abundant GDNF expression in ST (left hemisphere). In contrast, complete lesion of the SNc (right hemisphere) abrogated striatal GDNF expression. Densitometric quantification of GDNF staining throughout the ST showed that, on the lesioned side, striatal GDNF density was drastically reduced to 5.5 ± 1.59% of that on the unlesioned side. It is worth noting that 6-OHDA lesion does not eliminate striatal innervation of SNr (Figure 3). Thus, if there were significant retrograde transport of vector from SN to ST, GDNF should have been in evidence in the lesioned ST. The fact that this did not occur lead us to conclude that AAV2 is almost exclusively anterograde in line with previous studies in NHPs.24 In addition, no significant GDNF staining was found in EPN and GP (data not shown), either in lesioned or unlesioned hemispheres, indicating again that no significant retrograde transport occurred after nigral vector injection.

Discussion We are developing AAV2-GDNF-based gene transfer for treatment of PD. Selection of the correct anatomical targets for our approach is thus critically important. Our preclinical NHP studies www.moleculartherapy.org vol. 19 no. 5 may 2011

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were designed to address the influence of the degenerated nigrostriatal pathways on putaminal AAV2-GDNF therapy. Therefore, the purpose of this study was to understand how AAV2-GDNF (including expressed GDNF protein) is transported through the parkinsonian basal ganglia. In the unlesioned rat nigro-ST, the infusion of vector into either ST or SN resulted in robust expression in both locations, as well as in parts of the indirect pathway (GP, EPN). In contrast, injection of vector into the lesioned nigro-ST resulted in a much more restricted pattern of transduction. Striatal infusion of vector gave expression only in the SNr and parts of the indirect pathway. We interpret the SNr data to be indicative of direct anterograde transport of vector via GABAergic projections. Of course, elimination of the SNc by 6-OHDA lesioning also eliminates the possibility of SNc transduction. However, injection of AAV2-GDNF into the lesioned SN did not result in transduction of ST even though the SNr was still intact, demonstrating the almost complete lack of retrograde transport of AAV2-GDNF and/or GDNF protein in the basal ganglia. This finding is also consistent with the lack of significant GDNF staining in EPN and GP after SN injection of vector. The present rodent data is consistent with our previous studies of intracranial delivery of AAV2 vector in NHP6,7,11,27–30 and a phase 1 clinical study in PD patients.31 Collectively, our data suggest anterograde axonal transport of AAV2 and/or transgene product. For example, AAV2-AADC and AAV-TK delivery into the putamen of NHP resulted in detection of transgene in the GP and STN, but not cortex, strongly indicating anterograde transport only.32–34 Similarly, anterograde axonal transport is indicated in animals that received AAV2-GDNF6,7,11 or AAV2-hASM into the thalamus,35 where transgene was detected mainly in the cortex.24,35 In PD patients treated with putaminal AAV2-AADC, FMT PET detected robust AADC expression in the putamen, but failed to detect any AADC in the cortex, suggesting lack of retrograde axonal transport in humans,36 consistent with results obtained in MPTP-lesioned Rhesus macaques.6 Our finding that transport of AAV2-GDNF in basal ganglia is exclusively mediated by anterograde axonal transport has important therapeutic indications. First, it suggests that the degenerated nigro-ST should not inhibit GDNF trafficking to the SN when AAV2-GDNF is delivered into the putamen of PD patients, offering the possibility of regeneration of remaining DA neurons in SNc. Second, anterograde axonal trafficking of GDNF via direct and indirect striato-nigral pathways should deliver GDNF into other parts of the basal ganglia circuitry involved in PD, thereby offering potential therapeutic efficacy unrestricted to just putamen and SNc but including pallidus and other basal ganglia structures also adversely affected by loss of DA innervation in PD.17 Third, there appears to be little rationale for exposing PD patients to unnecessary risks associated with AAV2-GDNF administration into the midbrain region that may lead to severe adverse effects such as bleeding or weight loss.18,37 Finally, because AAV2 is transported anterogradely but not retrogradely, AAV2-GDNF delivery into the SN in PD patients is not expected to reach regions of the basal ganglia that are adversely affected by PD and may respond to GDNF therapy. In summary, the present study suggests that our current understanding of AAV2-GDNF transport within the parkinsonian basal ganglia supports a striatal but not nigral delivery in the clinical development of AAV2-GDNF for the treatment of PD. Molecular Therapy vol. 19 no. 5 may 2011

Axonal Transport of GDNF

Materials and Methods Animals. Male Sprague–Dawley rats (9–10 weeks old) weighing 250–300 g were purchased from Charles River Laboratory (Wilmington, MA). All animals were caged in groups of three in a room with 12:12 hours light/ dark cycle with lights on at 5:00 am and the temperature and humidity of the animal room were maintained at 19–21 °C and 50–60%, respectively. All animals had free access to food and water. All surgical procedures were conducted in accordance with regulations of the Institutional Animal Care and Use Committee of the University of California, San Francisco. Lesion with 6-OHDA. All animals were anesthetized with peritoneal injec-

tions of ketamine (5 mg/kg), and they were then maintained under 2% isoflurane for the duration of the surgery in a small-animal stereotactic frame (David Kopf Instruments, Tujunga, CA). The skull was exposed and a burr-hole was created to permit insertion of a cannula for injection of 6-OHDA into the medial forebrain bundle. Animals received a stereotactic injection of 4 μl of 2 mg/ml 6-OHDA in 0.9% saline, infused at a rate of 0.5 μl/minute for 10 minutes into the medial forebrain bundle in the right hemisphere at the following coordinates: antero-posterior –2.2 mm, mediolateral 1.5 mm relative to bregma, and ventro-dorsal –8.0 mm below the dura.38 A customized, stepped silica cannula was used for the infusion as previously described.39 To avoid reflux of 6-OHDA, the cannula was left in place for 2 minutes after infusion. The skin incision was closed and subcutaneous buprenorphine (Buprenex; 0.01 g/kg) was administered postoperatively as an analgesic. Animals were allowed to recover before returning them to the animal housing facility. Behavioral measurement. Four weeks after 6-OHDA infusion, the rota-

tional behavior of the rats was tested. Apomorphine-induced rotation was monitored as described previously.40 Full body rotations were recorded over a period of 60 minutes and the data are expressed as net full body turns/minute. Those rats exhibiting a rate exceeding 4 rotations/minute in response to 0.05 mg/kg subcutaneous apomorphine (Sigma-Aldrich, St Louis, MO) were selected for injection of AAV2-GDNF vector. AAV2-GDNF injection. The cDNA coding for human GDNF was cloned

into an AAV2 shuttle plasmid, and a recombinant AAV2 carrying GDNF under the control of the cytomegalovirus promoter was generated by a triple transfection technique as previously described.41,42 The titer (genome copies) was determined by quantitative PCR. The final titer of AAV2-GDNF vector used in this study was 1.1 × 1013 vector genomes per ml. Five weeks after the 6-OHDA lesion, intact or lesioned rats received bilateral infusions of AAV2GDNF (10 μl per hemisphere) into the ST or SN at stereotactic coordinates relative to the bregma and dura: (i) for the ST (antero-­posterior +0.8, mediolateral 3.0, dorsoventral −5.0); or (ii) for the SNc (antero-­posterior −5.3; mediolateral 2.5, dorsoventral −7.6).38 Vector infusions were performed with the same intra-cerebral delivery system described above for 6-OHDA infusion (flow rate: 0.5 μl/minute for 20 minutes). Tissue processing and immunohistochemistry

Single-staining protocol: Four or 25 weeks after AAV2-GDNF injections, rats were deeply anesthetized with sodium pentobarbital (90 mg/kg intraperitoneal) and perfused transcranially with ice-cold phosphatebuffered saline (PBS) followed by 60 ml of 4% paraformaldehyde in 0.1 mol/l PBS. The brains were removed, postfixed for 24 hours in 4% paraformaldehyde and then transferred to 30% sucrose in 0.1 mol/l PBS at 4 °C for cryoprotection. The fixed brains were cut into serial 40 μm coronal sections with a freezing microtome (Microm HM 450, Thermo Scientific, Waldorf, Germany). The sections were collected in sequence (20 sets of sections), stored in 24-well plates in cryoprotectant solution (0.5 mol/l phosphate buffer, pH 7.4, 30% glycerol and 30% ethylene glycol) and stored at 4 °C until further processing. For the assessment of AAV2-GDNF transport, whole brain coronal sections were taken at the level of ST, dorsal ST, external GP, EPN, zona incerta, STN, and SN. Free-floating sections for GAD and TH staining

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Axonal Transport of GDNF

were washed three times in PBS only, and in PBS with 0.1% Tween 20 for GDNF staining. Washes with PBS or PBST were performed between each immunohistochemical step. After a final rinse, endogenous peroxidase activity was quenched for 30 minutes in 1% hydrogen peroxide in PBS. For TH and GAD staining, blocking was achieved with Background Sniper (Biocare Medical, Concord, CA) for 45 minutes at room temperature. Blocking for GDNF staining used 20% normal horse serum in PBS for 60 minutes at room temperature. Thereafter, sections were incubated overnight with a monoclonal mouse anti-GAD65/67 IgG (1:10,000 dilution; Millipore Bioscience Research Reagents, Temecula, CA) or a polyclonal goat antihuman GDNF IgG (1:300 dilution; R&D Systems, Minneapolis, MN). For TH staining, sections were incubated for ~48 hours with a monoclonal mouse anti-TH IgG (1:5,000 dilution; Millipore Bioscience Research Reagents). All antibodies were dissolved in DaVinci diluent (Biocare Medical). After three rinses in PBS for 5 minutes each at room temperature, sections for TH and GAD staining were incubated in Mach 2 anti-mouse horseradish peroxidase (HRP) polymer (Biocare Medical), and those for GDNF staining in anti-goat HRP polymer (Biocare Medical) for 1 hour at room temperature. The activity of HRP was visualized with commercially available kits: for GDNF–3,3′-diaminobenzidine peroxide substrate; for TH–3,3′-diaminobenzidine with nickel and for GAD Vector NovaRED peroxide substrate (Vector Laboratories, Burlingame, CA). Immunostained sections for TH and GDNF were mounted on gelatinized slides, dehydrated in alcohol and xylene and cover-slipped with Cytoseal (Richard-Allan Scientific, Kalamazoo, MI). In contrast, the mounted GAD-stained sections were dried in 37 °C and cover-slipped in the absence of alcohol and xylene with Vectra Mount (Vector Laboratories). Double-staining protocol for TH/GDNF and TH/GAD: Double immunohistochemical staining for TH/GDNF and TH/GAD was performed with cocktails of the same primary antibodies as described in the singlestaining protocol. After three rinses in Tris-buffered saline at pH 8.0 with 0.1% Tween 20, sections were incubated in anti-goat HRP polymer (Biocare Medical) for GDNF and anti-mouse Mach 2 alkaline phosphatase polymer (Biocare Medical) for TH or GAD. The activity of HRP was visualized by 3,3′-diaminobenzidine peroxidase substrate. Thereafter, sections were rinsed three times in Tris-buffered saline at pH 8.0 with 0.1% Tween 20, and then the GAD or TH antigenic sides were visualized on the sections with Vulcan Fast Red Chromogen Kit 2 (Biocare Medical) according to the manufacturer’s instructions. To achieve a sufficiently red color for the second staining, the sections were inspected under a Stereomaster zoom microscope (Fisher Scientific, Fremont, CA). The sections were mounted on gelatinized slides, dried at 37 °C and cover-slipped with EcoMount (Biocare Medical). No significant nonspecific signal was generated from the secondary antibodies, as determined by control experiments where the primary antibody was omitted from the immunohistochemical procedure. All sections were examined and digitally photographed on a Zeiss Axioskop microscope (Carl Zeiss, Thornwood, NY). The multi-immunolabeling combined with Adobe Photoshop imaging: The colocalization of GAD- and GDNF-staining at the level of SN reticulata was revealed by Adobe Photoshop 6 software. From each rat, three consecutive sections centered at the level of the medial terminal nucleus (MTN) and chromogenically stained with anti-GDNF (brown), anti-GAD (red) and anti-TH (black) respectively. Images of GAD staining were rendered in false color to aid discrimination and individual immunochemistry (GAD/GDNF/TH) stains were superimposed and made visible by manipulation of each image’s opacity to 30%. Densitometry of GDNF staining: From each rat, the optical density of the GDNF-positive staining in the ST and SN was determined bilaterally from three coronal striatal sections through the ST: AP = +1.0, +0.2, −0.40 and the SN: AP = −4.80, −5.30, −5.80. Images of these coronal sections were captured on an Epson Perfection V700 Photo high-resolution digital scanner.. In the first step, the images were first converted to gray-scale, then ST or SN were outlined on the computer screen and finally the pixel density was measured over the defined cross-sectional areas via Image J

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software v1.40. For a given ST or SN, a line was drawn around the whole structure to define the area-of-interest. For nonspecific background correction, the value was normalized by subtracting the pixel density of a region of cortex (negative for GDNF) in the same section. The mean labeling for each area was calculated from three adjacent brain sections from the same rat. Finally, the density of GDNF staining areas (ST or SN) in the 6-OHDA-lesioned hemisphere was expressed as a percentage of the density of GDNF staining in the same areas in the contralateral hemisphere. Statistical analysis. Analyses are expressed as mean ± SD The data,

c­ alculated as a ratio of the unlesioned (contralateral) side to lesioned ­(ipsilateral) side, were evaluated for significance by the Mann–Whitney U-test for nonparametric samples (Statistica 6.0 software). Statistical ­significance was defined as P < 0.05.

ACKNOWLEDGMENTS This study was supported by a grant to KSB from NIH-NINDS.

References

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