Long-Term rAAV-Mediated Gene Transfer of GDNF in the Rat
Parkinson’s Model: Intrastriatal But Not Intranigral Transduction
Promotes Functional Regeneration in the Lesioned
Deniz Kirik, Carl Rosenblad, Anders Bjo ¨rklund, and Ronald J. Mandel
Wallenberg Neuroscience Center, Department of Physiological Sciences, Division of Neurobiology, Lund University,
223 62 Lund, Sweden
Previous studies have used recombinant adeno-associated vi-
ral (rAAV) vectors to deliver glial cell line-derived neurotrophic
factor (GDNF) in the substantia nigra to protect the nigral
dopamine (DA) neurons from 6-hydroxydopamine-induced
damage. However, no regeneration or functional recovery was
observed in these experiments. Here, we have used an rAAV-
GDNF vector to express GDNF long-term (6 months) in either
the nigral DA neurons themselves, in the striatal target cells, or
in both of these structures. The results demonstrate that both
nigral and striatal transduction provide significant protection of
nigral DA neurons against the toxin-induced degeneration.
However, only the rats receiving rAAV-GDNF in the striatum
displayed behavioral recovery, accompanied by significant re-
innervation of the lesioned striatum, which developed gradually
over the first 4–5 months after the lesion. GDNF transgene
expression was maintained at high levels throughout this pe-
riod. These results provide evidence that rAAV is a highly
efficient vector system for long-term expression of therapeutic
proteins in the nigrostriatal system.
Key words: Parkinson’s disease; 6-hydroxydopamine; glial
cell line-derived neurotrophic factor; gene transfer; tyrosine
hydroxylase; stepping; paw use; sensorimotor behavior; cell
Glial cell line-derived neurotrophic factor (GDNF) has been dem-
onstrated to be a potent factor for protection of nigral dopamine
(DA) neurons against toxin-induced degeneration in vivo (Bjo ¨rk-
lund et al., 1997; Gash et al., 1998; Bohn, 1999). Injections of
GDNF close to substantia nigra can substantially protect nigral DA
neurons from the acute toxin-induced degeneration, provided that
it is administered before onset of cell loss (Beck et al., 1995; Sauer
et al., 1995; Winkler et al., 1996; Lu and Hagg, 1997; Sullivan et al.,
1998; Rosenblad et al., 2000b). However, survival of the DA cell
bodies alone, in the absence of a functional striatal DA innerva-
tion, is not sufficient for preservation of intact motor performance
(Winkler et al., 1996; Rosenblad et al., 2000b; D. Kirik, C. Rosen-
blad, and A. Bjo ¨rklund, unpublished observations). Thus, in the
intrastriatal 6-hydroxydopamine (6-OHDA) lesion model, long-
term delivery of GDNF to the striatum may be necessary to obtain
efficient axonal regeneration and functional recovery.
In vivo gene transfer has been explored as a relatively nonin-
vasive method to deliver long-term GDNF to the brain using
adenoviral, lentiviral, or adeno-associated viral (AAV) vectors
(Bilang-Bleuel et al., 1997; Choi-Lundberg et al., 1997, 1998;
Mandel et al., 1997; Connor et al., 1999; Mandel et al., 1999;
Deglon et al., 2000; Rosenblad et al., 2000a). Recombinant AAV
(rAAV)-based vectors are particularly interesting for gene trans-
fer to the nervous system in that they are able to transduce
postmitotic neurons and support long-term transgene expression
in the brain (Kaplitt et al., 1994; Peel et al., 1997; Bartlett et al.,
1998; Klein et al., 1998; Mandel et al., 1998; Leff et al., 1999; Lo
et al., 1999; Szczypka et al., 1999). There have been no reports of
neurotoxicity or immune reactions in response to rAAV injec-
tions, presumably because of the lack of expression of any viral
proteins after transduction with this vector (Muzyczka, 1992;
Kaplitt et al., 1994). In the nigrostriatal system, rAAV vectors
have been shown to be effective in transducing neurons in both
striatum and substantia nigra, and high levels of transgene expres-
sion have been maintained for at least 3–6 months (Kaplitt et al.,
1994; Klein et al., 1998; Leff et al., 1999; Lo et al., 1999; Szczypka
et al., 1999). The particular affinity of the rAAV vectors for the
nigral DA neurons and their targets raises the possibility that
rAAV-mediated gene transfer can be used to compare the effects
of long-term GDNF expression within the nigral DA neurons
with the effects induced by long-term GDNF delivery in the
striatum acting on the lesioned nigrostriatal afferents.
We report here that protection of nigral DA neurons against
6-OHDA-induced damage can be achieved by rAAV-GDNF
transduction of either substantia nigra or striatum, but that long-
term functional recovery and regeneration of the lesioned nigro-
striatal projection in the intrastriatal 6-OHDA lesion model is
obtained only when GDNF is expressed over an extended time
period in the striatum alone. This suggests that paracrine rather
than autocrine mechanisms are important for functional regener-
ation in the lesioned nigrostriatal DA system.
Received Jan. 18, 2000; revised March 17, 2000; accepted March 27, 2000.
The work was supported by grants from the Swedish Medical Research Council
(National Gene Therapy Program 99XG-13285) and the Parkinson’s Disease Foun-
dation. We thank Cell Genesys Inc. for the generous gift of the rAAV vectors used
in these experiments, and Alicja Flasch, Kerstin Fogelstro ¨m, and Ulla Jarl for
excellent technical support. Richard O. Snyder and Brian A. Donahue produced the
vectors described herein.
Correspondence should be addressed to Deniz Kirik, Wallenberg Neuroscience
Center, Department of Physiological Sciences, Lund University, So ¨lvegatan 17,
223 62 Lund, Sweden. E-mail: firstname.lastname@example.org.
Dr. Mandel’s current address: Gene Therapy Center, Department of Neuro-
science, University of Florida College of Medicine, P.O. Box 100244, Gainesville,
Copyright © 2000 Society for Neuroscience 0270-6474/00/204686-15$15.00/0
The Journal of Neuroscience, June 15, 2000, 20(12):4686–4700
MATERIALS AND METHODS
Recombinant AAV vector production. The rAAV-CMV-GFP and rAAV-
MD-GDNF vectors were prepared as described previously (McCown et
al., 1996; Mandel et al., 1997). Briefly, rAAV vectors were prepared
according to Snyder et al. (1996) with modifications as in Mandel et al.
(1997). Subconfluent 293 cells were cotransfected with the vector plasmid
and the AAV helper plasmid using the calcium phosphate method. Cells
were then infected with Adenovirus Ad5 dl312 at an MOI of 2, and the
infection was allowed to proceed for 60–72 hr. Cells were harvested and
three freeze/thaw cycles were performed to lyse the cells. The cell lysate
was fractionated by ammonium sulfate precipitation, and the rAAV
virions were isolated on two sequential continuous CsCl gradients. The
final particle titer of the rAAV-CMV-GFP was 1.4 ? 1012viral particles
per milliliter, and the rAAV-MD-GDNF was 1.0 ? 1012viral particles
per milliliter as estimated by dot blot analysis.
Subjects. A total of 58 young female Sprague Dawley rats (B&K
Universal, Stockholm, Sweden) were housed four to five in a cage with
free access to rat chow and water during a 12 hr light/dark cycle. The
housing and treatment of the animals were performed according to rules
set by the Ethical Committee for Use of Laboratory Animals at Lund
Surgical procedures. For all of the surgical procedures described herein,
animals received equithesin anesthesia (3 ml/kg, i.p.) before surgery.
After anesthesia, the animals were placed into stereotaxic frames (Kopf
Instruments, Tujunga, CA). All injections were made using a continuous
infusion system (Carnegie Medicin, Stockholm, Sweden) that was at-
tached to a 10 ?l Hamilton microsyringe fitted with a glass micropipette
(outer diameter 60–80 ?m). The anterior-posterior (AP) and medial-
lateral (ML) stereotaxic coordinates were calculated from bregma, and
the dorso-ventral (DV) coordinates were calculated from the dural sur-
face. A burr hole was drilled in the skull at the calculated coordinates. At
the position of the entry of the glass pipette, a small cut in the dura was
made using a 28 gauge stainless steel hypodermic needle.
Injection of AAV vectors. The animals received injections of rAAV
(either rAAV-MD-rGDNF or rAAV-CMV-GFP) suspended in PBS
into the striatum (3 ?l per site, 9 ?l in total), substantia nigra (2 ?l per
site, 4 ?l in total), or both. Coordinates are shown in Table 1. The
injection rate was 1 ?l/min in the striatum and 0.5 ?l/min in the nigra.
During intrastriatal injections the glass pipette was slowly retracted 1 mm
every minute. One minute after the cessation of the infusion, the mi-
cropipette was retracted 1 mm further and left in place for an additional
2 min before it was slowly retracted from the brain. Experimental groups
used in this experiment will be referred to in the text as follows: (1)
rAAV-GDNF injected into SN (n ? 10) is referred to as “SN group”; (2)
rAAV-GDNF injected into STR (n ? 11) is referred to as “STR group”;
(3) rAAV-GDNF injected into SN and STR (n ? 11) is referred to as
“SN?STR group”; (4) two control groups consisting of rAAV-GFP into
substantia nigra and striatum (GFP control group, n ? 6) or non-vector-
injected lesion-only group (n ? 5). These two control groups were
indistinguishable with regard to all variables tested and were therefore
combined into one “control group” (n ? 11) for all analyses and figures;
(5) rAAV-GDNF injected into SN (n ? 5), STR (n ? 5), or SN?STR
(n ? 5). These animals were killed at 4 weeks after the virus injection to
determine the tissue levels of GDNF protein and dopamine and its
metabolites; (6) rAAV-GFP injected into SN (n ? 4). These animals
were killed at 4 weeks after the injection and were processed for GFP
6-OHDA lesions. Four weeks after virus injections, all animals, except
those in groups 5 and 6, received unilateral stereotaxic injections of a
total of 28 ?g of 6-OHDA (calculated as free base; Sigma, St. Louis,
MO) dissolved in ascorbate-saline (0.05%) divided into four 7 ?g depos-
its in the right striatum. The injection rate was 1 ?l/min, and the
micropipette was left in place for an additional 2 min before it was slowly
retracted. The coordinates used (see Table 1) were based on the findings
of our previous experiment (Kirik et al., 1998).
Rotational behavior. All rotational testing was performed in automated
rotometer bowls (Ungerstedt and Arbuthnott, 1970). Spontaneous rotation
was monitored over 30 min to determine whether the animals displayed
asymmetric behavior. The data are presented as total number of full 360°
rotations in ipsilateral and contralateral directions. The activity of the
animals was assessed by calculating the total number of rotations regardless
of the direction. Drug-induced rotational asymmetry was assessed using
apomorphine-HCl (Research Biochemicals Incorporated; 0.25 mg/kg, s.c.),
SKF-82958 HCl (full D1 dopamine agonist, Research Biochemicals Incor-
porated; 0.1 mg/kg, s.c.), and D-amphetamine sulfate (Apoteksbolaget; 1.0
or 2.5 mg/kg, i.p.). Rotations were monitored for 40, 60, and 90 min for
apomorphine, SKF-82958, and D-amphetamine, respectively. All drug-
induced rotational asymmetry scores are expressed as full 360° rotations
per minute, with ipsilateral rotations assigned a positive value.
Forelimb akinesia (stepping test). The animals were tested for forelimb
akinesia in a stepping test (Schallert et al., 1992) as described by Olsson
et al. (1995). The test was performed twice daily on four consecutive days
with the mean of the data taken from the last 3 d constituting the final
Cylinder test. This test, which is a modification of a motor test of
forelimb asymmetry described first by Schallert and Lindner (1990), was
performed as described by Schallert and Tillerson (1999). Briefly, during
video recording, the animal is allowed to move freely in a clear glass
cylinder until it has performed 10 rears during which it places at least one
paw on the cylinder wall. Mirrors are placed behind the cylinder so that
the video camera can have visual access to all paw placements around the
cylinder. An observer blinded to animal identities viewed the videotapes
and counted the number of left and right forepaw contacts to the walls of
the cylinder from a minimum of 20 contacts. The data are presented
contralateral (left) forepaw contacts as percentage of total.
Staircase (paw reaching) test. A modified version of the staircase test
described by Montoya et al. (1991) was used. The animals received food
pellets in their home cages just before a 2 d food deprivation period,
leading to ?15% loss in body weight. The animals were then placed into
Plexiglas test boxes and were tested for 15 min on 7 consecutive days. At
this point, the standard central platform (27 mm) was replaced with a
wider platform (34 mm) to make the task more difficult, and the animals
were tested for an additional 5 d. After each test the number of pellets
taken from the stairs and the number of misses were counted separately.
The difference between the pellets taken and misses constitutes the total
number of successful retrievals, which serves as the dependent variable
for statistics and graphical representation.
Prelesion behavioral testing. To be able to monitor the effects of GDNF
overexpression in the intact rats (Horger et al., 1998), the animals were
tested before 6-OHDA lesion on the stepping test and the spontaneous
and amphetamine-induced rotational asymmetry tests. The stepping test
was performed during the 3rd week after the injection of the virus. After
the stepping test was completed, spontaneous rotation was monitored
over a 30 min period, which was followed by the amphetamine-induced
Table 1. Stereotaxic coordinates for the rAAV and 6-OHDA injections
7 ?g free base in 2 ?l per siteStriatumNigra
AP and ML are mm from bregma; DV is mm from dura.
aIntrastriatal injections start at DV ?5.5 from the dura, and the micropipette is pulled up to ?4.5 and ?3.5 during injection.
Kirik et al. • AAV-GDNF Promotes Motor Recovery in a PD ModelJ. Neurosci., June 15, 2000, 20(12):4686–4700 4687
rotation test. The animals in each group were divided into two subgroups
and received either 1.0 or 2.5 mg/kg D-amphetamine sulfate intraperi-
toneally over 2 weeks with the subgroups receiving alternate doses in a
crossover design (Cochran and Cox, 1957).
Post-lesion behavioral testing. Amphetamine-induced rotational behav-
ior was monitored at 4, 7, 10, 13, 16, and 22 weeks after the lesion.
Stepping tests were performed during the 3rd, 7th, 14th, 18th, and 22nd
week after the lesion. Before the first and after the last post-lesion
stepping tests, the animals were videotaped in the cylinder test. Starting
at the 20th week after lesion, the animals were tested in the staircase test
as described above (for details, see Fig. 1). Apomorphine-induced rota-
tion was monitored at 3 and 18 weeks after lesion. Four days after each
apomorphine administration, animals received subcutaneous injections
of SKF-82958. The two control groups (the GFP control group and the
lesion-only group) did not differ from each other in any of the tests, and
therefore their data were pooled for further analysis.
Tissue levels of GDNF and dopamine and its metabolites. To determine the
level of GDNF protein, DA, and DA metabolites from the same samples,
at the time of 6-OHDA lesion, 15 rats received rAAV-MD-GDNF into SN
(n ? 5), STR (n ? 5), and SN/STR (n ? 5) as described above. Four weeks
after vector injection, the virus-injected animals and five naive control
animals were injected with the l-aromatic amino acid decarboxylase inhib-
itor NSD-1015 (Sigma, 100 mg/kg, i.p.) 30 min before they were killed.
DOPA decarboxylase inhibition results in accumulation of DOPA that
would otherwise be converted to DA, thus providing an opportunity to
determine in vivo measure of striatal tyrosine hydroxylase (TH) activity.
The animals were then deeply anesthetized with sodium pentobarbital and
decapitated. The brains were rapidly removed, and the corpora striata were
dissected dorsal to the anterior commissure and freed from cortex and
septal nuclei. The dissected tissue was then chopped into small pieces,
mixed, and divided equally into two halves and frozen separately (one part
for GDNF ELISA, and the other for DOPA, DA, and DOPAC measure-
ments). A 2- to 3-mm-diameter punch centered on the substantia nigra pars
compacta on each side was taken from a 3 mm thick coronal slice that
contained the rAAV vector injection site.
For determination of tissue GDNF protein levels, tissue from the
striatum and nigra were sonicated (Vibra Cell Sonics and Materials Inc.,
Danbury, CT) in a homogenization buffer (150 mM NaCl, 50 mM
HEPES, pH 7.4, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride,
and 0.6 ?M leupeptin) at a tissue concentration of 50 mg/ml (wet weight
per volume). Tissue levels of GDNF were determined from tissue ho-
mogenates by ELISA using a commercial kit, according to supplier’s
recommendations (G3240; Promega, Madison, WI). The standard curves
for determining GDNF levels in tissue samples were undertaken using
rat GDNF protein (a generous gift of Dr. H. Phillips, Genentech, Inc.).
For determination of DOPA, DA, and DOPAC levels, the striatal
pieces were immediately frozen in liquid nitrogen and stored until
assayed by a combined radioenzymatic method, as described by Schmidt
et al. (1982).
Perfusion and tissue processing. After the behavioral testing was com-
pleted (24 weeks post-lesion), the animals were deeply anesthetized with
pentobarbital and perfused through the ascending aorta with 50 ml of
isotonic saline, followed by 250 ml of ice-cold 4% paraformaldehyde in
0.1 M phosphate buffer (PB), pH 7.4. Brains were removed and post-fixed
for 2 hr in the same solution and then transferred to 20% sucrose in 0.1
M PB before sectioning on a freezing-stage microtome at 40 ?m.
Immunohistochemistry. Standard immunohistochemical procedures
were used as described previously (Sternberger et al., 1970). For TH
immunohistochemistry, the sections were preincubated with 5% normal
horse serum (NHS) and then incubated overnight at room temperature
with a 1:2000 dilution of mouse anti-TH antibody (Chemicon, Temecula,
CA) in 2% NHS. For GDNF immunohistochemistry, the sections were
preincubated with 5% NHS and then incubated overnight at room
temperature with a 1:1000 dilution of goat anti-GDNF antibody (R&D
systems) in 2% NHS. GFR?-1 immunohistochemistry was performed by
preincubation with 5% normal swine serum (NSS) and followed by
incubation overnight at 4°C with a 1:500 dilution of rabbit anti-GFR?-1
antibody (a kind gift from Dr. C. Ibanez, Karolinska Institute, Stock-
holm) in 2% NSS. Appropriate secondary antibodies directed against the
species in which the primary antibody was raised were used in all cases.
In all staining protocols, incubation with the secondary antibody was
followed by incubation with avidin–biotin–peroxidase complex (ABC,
Vector Laboratories, Burlingame, CA). The reactions were visualized
using 3,3-diaminobenzidine as a chromogen. Sections were mounted on
chrome–alum-coated slides, dehydrated in ascending alcohol concentra-
tions, cleared in xylene, and coverslipped in Depex.
Nigral cell counts. The unbiased stereological estimation of the total
number of the cells in substantia nigra was made using the optical
fractionator, as described in detail previously (Kirik et al., 1998). This
sampling technique is not affected by tissue volume changes and does not
require reference volume determinations (West et al., 1991). The sec-
tions used for counting covered the entire substantia nigra, from the
rostral tip of the pars compacta back to the caudal end of the pars
reticulata. This yielded 9–11 sections in a series. Sampling was per-
formed using the Olympus C.A.S.T.-Grid system (Olympus Denmark
A/S, Albertslund, Denmark). A counting frame (5445 ?m2) was placed
randomly on the first counting area and systematically moved through all
counting areas until the entire delineated area was sampled. Actual
counting was performed using a 100? oil objective (NA 1.4). Guard
volumes (5 ?m from the top and 5–8 ?m from the bottom of the section)
were excluded from both surfaces to avoid the problem of lost caps, and
only the profiles that came into focus within the counting volume (with
a depth of 10 ?m) were counted. The total number of neurons was
calculated according to the optical fractionator formula [for more details,
see West et al. (1991)]. The coefficient of error (CE) attributable to the
estimation was calculated according to Gundersen and Jensen (1987). CE
?0.10 was accepted.
Striatal fiber density measurements. The optical densities of the TH-
immunoreactive fibers in the striatum were measured using the NIH 1.62
Image program on a Macintosh 9500 computer connected to a digital
camera (ProgRes) and a constant illumination table. For each animal the
optical density was measured at seven rostocaudal levels over the whole
striatum according to the atlas of Paxinos and Watson (1998): (1) AP,
?1.6; (2) AP, ?1.0; (3) AP, ?0.2; (4) AP, ?0.3; (5) AP, ?0.9; (6) AP,
?1.4; and (7) AP, ?2.1 relative to bregma. To estimate the specific TH
staining density, the optical density readings were corrected for nonspe-
cific background density, as measured from a completely denervated part
of the striatum.
Significant differences between different treatments were assessed using
parametric analysis of variance (ANOVA). Individual contrasts among
means were used only when there was a significant interaction term
within the ANOVA, in the form of simple-main effects analysis using
Systat 5.2.1 (Kirk, 1968). Post hoc testing between the groups consisted
of Tukey honestly significant difference (HSD). The Greenhouse-Geisser
Epsilon test, to determine homogeneity of variance among groups sub-
jected to ANOVAs, yielded nonsignificant results for all contrasts ( p ?
0.05). The staircase data were subjected to a logarithmic transformation
to control the variability attributable to the poor performance of some
animals early in the acquisition phase of the learning. Significance was
accepted at the 95% probability level.
The study was designed to determine whether long-term overex-
pression of GDNF in striatum and/or substantia nigra by rAAV-
mediated gene transfer may provide protection of the nigrostria-
tal pathway and promote regeneration and functional recovery in
the intrastriatal 6-OHDA lesion model. The rAAV-GDNF vector
was injected unilaterally into striatum (STR group, n ? 11) or
substantia nigra (SN group, n ? 10) or both structures (SN?STR
group, n ? 11). Rats injected with an rAAV encoding green
fluorescent protein (GFP, n ? 6) and a group of uninjected rats
(n ? 5) served as control. Vector injections were made 4 weeks
before the intrastriatal 6-OHDA injection to allow sufficient time
for the GDNF gene to be fully expressed (Mandel et al., 1997,
1999). To induce consistent long-lasting deficits in motor behav-
ior and substantial DA cell loss, a four-site intrastriatal 6-OHDA
lesion was applied (Kirik et al., 1998). The 6-OHDA-lesioned
animals were allowed to survive for 6 months during which time
4688 J. Neurosci., June 15, 2000, 20(12):4686–4700Kirik et al. • AAV-GDNF Promotes Motor Recovery in a PD Model
they were repeatedly tested on a battery of drug-induced and
spontaneous motor behaviors (Fig. 1).
GDNF expression in intact animals
GDNF expression at the time of the lesion was assessed by
ELISA in tissue samples obtained from a separate group of
nonlesioned animals (n ? 14) that were killed 4 weeks after
vector injection (Table 2). Detectable levels of GDNF protein
were measured in the transduced nigra and striatum in all three
rAAV-GDNF-injected groups (0.22–2.28 ng/mg tissue, 4- to 35-
fold above baseline; effect of side F(1,22)? 16.1, p ? 0.0001).
GDNF levels were ?10-fold higher in the animals that had
received vector injections in the nigra alone (the SN group), or in
both nigra and striatum (the SN?STR group), than in the
striatum-injected animals (the STR group) (effect of group F(2,22)
? 73.4, p ? 0.0001). The high level of GDNF in the striatum in
the SN group, moreover, suggests that the GDNF protein had
been transported from the nigra to the striatum in the animals
that had received vector injections in the SN.
In vivo TH enzyme activity (measured as the rate of L-DOPA
accumulation after inhibition of the decarboxylase enzyme) and
DA turnover (estimated as the DOPAC/DA ratio) were mea-
sured bilaterally in the striatum in the same group of animals. As
shown in Table 3, there was a two- to threefold increase in DA
turnover on the vector-injected side in all rAAV-GDNF-injected
groups, whereas a significant increase in TH activity was detected
only in the combined SN?STR group.
The impact of GDNF overexpression on the behavior of the
intact animals was assessed during the third week after vector
injection, i.e., during the week preceding the 6-OHDA lesion. The
rAAV-GDNF-injected animals in the SN and SN?STR groups
showed a significant spontaneous contralateral turning bias (i.e., in
the direction away from the vector-injected hemisphere) (Fig. 2A),
as well as an increased general locomotor activity (total rotations in
both directions) compared with the controls (F(3,39)? 6.18, p ?
0.001). Although a twofold increase in contralateral rotations was
observed in the STR group versus the control group, this asymme-
try did not reach statistical significance. These differences were
further accentuated after challenge with D-amphetamine (Fig. 2B);
i.e., all three rAAV-GDNF-injected groups showed strong
amphetamine-induced contralateral rotation, and they were signif-
icantly more active in the test, as indicated by an increase in the
total number of turns during the 90 min test (F(3,39)? 9.284, p ?
0.0001). On the other hand, the rAAV-GDNF-treated animals
displayed normal forelimb stepping behavior before the 6-OHDA
lesion (F(3,78)? 0.15, p ? 0.93) (Fig. 4E,F, pre-lesion values).
3 weeks after the vector injection, the animals were tested for forelimb akinesia using the stepping test ( pre step), followed by spontaneous motor
asymmetry (spon. rot.) and D-amphetamine-induced motor asymmetry ( pre amph I–II). 6-OHDA was injected into four sites in the striatum at a dose
of 7 ?g per site at week 0. Post-lesion motor impairment was evaluated using repeated drug-induced (amph I–VI, apo I–II, and SKF-82958 I–II; shown
above the time line) and spontaneous tests (step I–V, cylinder and staircase tests; below the time line). The animals were perfused for immunohisto-
chemical analysis at 24 weeks after lesion, as described in Materials and Methods.
Schedule of surgeries and testing. The animals received injections of the viral vectors 4 weeks before the 6-OHDA lesion (?4). Beginning
Table 2. GDNF protein levels (ng/mg tissue) measured by ELISA in punches from striatum and the
substantia nigra region at 4 weeks after rAAV-GDNF injection
SN group (n ? 5)
STR group (n ? 5)
SN ? STR group (n ? 4)
0.07 ? 0.02
0.02 ? 0.01
0.05 ? 0.04
1.58 ? 0.31a
0.22 ? 0.09a
2.28 ? 0.35a
0.09 ? 0.02
0.05 ? 0.03
0.10 ? 0.04
1.41 ? 0.49a
0.11 ? 0.03
1.00 ? 0.11a
Means ? SEM.
aDifferent from noninjected side p ? 0.05 (ANOVA followed by post hoc Tukey HSD).
Table 3. DOPA levels and DOPAC/DA ratios in the striatum in
non-lesioned control and rAAV-GDNF-injected animals, 4 weeks
Experimental groupDOPA (fmol/mg tissue)DOPAC/DA
Control (n ? 5)
SN group (n ? 5)
STR group (n ? 4)
SN ? STR group (n ? 4)
759.2 ? 44.8
538.4 ? 211.9
975.3 ? 89.8
1905.3 ? 345.9b
0.021 ? 0.003
0.048 ? 0.008a
0.046 ? 0.004a
0.065 ? 0.014a
All animals were pretreated with NSD-1015 (100 mg/kg) 30 min before they were
ap ? 0.05, different from control.
bp ? 0.05, different from all other groups.
Kirik et al. • AAV-GDNF Promotes Motor Recovery in a PD ModelJ. Neurosci., June 15, 2000, 20(12):4686–4700 4689
Taken together, these data indicate that the level of overex-
pression of GDNF obtained in the rAAV-GDNF-injected ani-
mals was sufficient to induce a marked functional upregulation in
the intact nigrostriatal DA neurons and that this effect was most
pronounced in the SN?STR group. Moreover, the contralateral
turning bias (spontaneously and after amphetamine challenge)
was strongest in those groups that displayed the highest level of
striatal GDNF and weakest in those groups with lower GDNF
levels (Table 2).
Reversal of lesion-induced functional impairments by
rAAV-mediated GDNF delivery
Beginning 3 weeks after the intrastriatal 6-OHDA lesion, the
animals were tested on a battery of drug-induced and spontane-
ous motor tests (Fig. 1). In the tests performed 3–5 weeks after
the lesion, all vector-injected groups showed a similar degree of
impairment both in the drug-induced rotation tests (Fig. 3, Table
4) and in the spontaneous motor tests (forelimb akinesia and
paw-use tests) (Fig. 4). These data indicate that the acute impact
of this large 6-OHDA lesion was the same in all four experimen-
Over the subsequent weeks, significant functional recovery was
observed in the STR group, both in amphetamine-induced rota-
tion (Fig. 3) and in the animals’ ability to use their forelimbs
(cylinder and staircase tests) (Fig. 4A–D). In contrast, neither the
SN nor the SN?STR groups showed any significant recovery in
any of the behavioral tests. The improvement in the STR group,
as demonstrated by reduced amphetamine-induced rotational
behavior, developed gradually over time until 23 weeks after the
6-OHDA lesion. At 23 weeks, the asymmetry was completely
abolished in 8 of the 11 rats in the STR group. These fully
recovered animals had also recovered normal forelimb use in the
cylinder test (Fig. 4B). In contrast, rotation in response to the
mixed D1/D2 agonist apomorphine or the D1 agonist SKF-82958
was unaffected at all time points (Table 4).
To further characterize the ability of the rAAV-GDNF-treated
rats to use their affected forelimb, two different versions of a
staircase test were performed. The first 7 d of testing was per-
formed using a platform of a standard width (Fig. 4C, narrow
platform). During this initial period of the test, the STR group
was able to successfully retrieve more pellets with their contralat-
control groups displayed no side bias, as indicated by the lack of a difference between their number of rotations in either direction versus the ipsilateral
direction (Tukey HSD post hoc test p ? 0.995). In contrast, SN and STR/SN vector-injected groups spontaneously rotated more contralateral to the
vector injection (closed bars) as compared with the ipsilateral side (open bars, significant main effect of group F(3,78)? 6.8; p ? 0.0004, Tukey HSD post
hoc, SN p ? 0.0001, STR/SN p ? 0.0001). Asterisks indicate significantly increased contralateral versus ipsilateral rotations ( p ? 0.05). Pound signs (#)
indicate significantly greater rotational rate versus controls ( p ? 0.05). B, Pre-lesion amphetamine rotation. Similarly to the spontaneous rotational data,
control animals displayed no asymmetrical behavior in response to 2.5 mg/kg amphetamine. However, all rAAV-GDNF-injected animals displayed
highly significant asymmetries in the direction contralateral to the vector injection (closed bars). Asterisks indicate significantly higher contralateral
rotational rates as compared with ipsilateral rotational rates ( p ? 0.05). Pound signs (#) indicate greater contralateral rotational rates compared with
the control group ( p ? 0.05). Note the different scales in A and B.
Spontaneous motor asymmetry in intact animals was assessed 3 weeks after the vector injection. A, Pre-lesion spontaneous rotation. The
weeks after the striatal 6-OHDA lesion, all groups showed clear ipsilat-
eral rotation that was equivalent among the groups ( p ? 0.5, simple main
effects). However, as early as 7 weeks after the 6-OHDA lesion, the STR
vector-injected group began to display reduced amphetamine-induced
rotations. From 10 weeks and onward, the STR group was significantly
different from all other groups (main effect of group; F(3,39)? 3.464, p ?
0.03). Asterisks indicate a significant difference from earlier time points
within the STR group, and the pound sign (#) denotes a significant
difference from all other groups ( p ? 0.05 by simple main effects). All
values are means ? SEM.
Post-lesion amphetamine-induced motor asymmetry. Four
4690 J. Neurosci., June 15, 2000, 20(12):4686–4700 Kirik et al. • AAV-GDNF Promotes Motor Recovery in a PD Model
eral paw than all other groups over the entire test period (Fig.
4C). After asymptotic performance was reached, the standard
platform was replaced by a wider platform to make the test more
difficult for an additional 5 d. During the second 5 d testing period
(Fig. 4C, days 8–12), the STR group again performed better than
all other groups. In addition, in the second more difficult test the
SN group tended to perform worse than the other groups and
performed significantly worse than the STR group on all days
(simple main effects, p ? 0.025 on all days) and the remaining
groups on two of the days (Fig. 4C). In contrast to the improve-
ments observed in staircase and cylinder tests, contralateral (left)
forelimb use in the stepping test was severely impaired in all
groups and remained impaired throughout the experiment in all
groups (Fig. 4E,F).
Preservation of the integrity of the nigrostriatal
pathway in rAAV-GDNF-treated animals
The protection of the nigral DA neurons and the preservation of
the nigrostriatal projection was evaluated by TH immunohisto-
chemistry. Four coronal levels were chosen for illustration of the
status of the nigrostriatal pathway: the central striatum (Fig. 5),
caudal striatum and globus pallidus (GP) (Fig. 6), medial fore-
brain bundle (MFB) (Fig. 7), and substantia nigra (Fig. 8).
In the control group, the four-site intrastriatal 6-OHDA injec-
tion induced an extensive loss of TH-positive neurons (?85%) in
the substantia nigra pars compacta (Fig. 8B, and inset) as com-
pared with the intact hemisphere (Fig. 8A). There was a similar
loss of TH innervation in large portions of the striatum, with the
exception of some spared fibers that innervated the most medial
and ventral striatum (Fig. 5, compare A, B; Fig. 6, compare A, B).
The extensive striatal denervation was associated with near-
complete absence of TH-positive preterminal axons in the GP
(Fig. 6, compare A, B) and was also apparent in the internal
capsule and the MFB (Fig. 7, compare A, B).
rAAV-mediated GDNF expression had significant neuropro-
tective effects on TH-positive cells in the substantia nigra pars
compacta in all three rAAV-GDNF-treated groups (Fig. 8, inset).
The protection was near complete (91.2%) in the SN group (Fig.
8C), 56.8% in the STR group (Fig. 8D), and 78.6% in the
SN?STR group (Fig. 8E). All animals in the SN and SN?STR
groups displayed extensive sprouting of TH-positive fibers dorsal
and rostral to the nigra, surrounding the vector injection site,
leading to disorganization of fibers at the level of MFB (Fig. 7C,
E). This extensive sprouting response also extended into the
subthalamic nucleus, the entopeduncular nucleus (EP), and the
ventral thalamus, but failed to reach the striatum (Figs. 5C, 6C).
In the STR group, substantial TH-positive sprouting was ob-
served within the GP that also innervated the adjacent areas of
the striatum (Fig. 6D). The sprouting fibers predominantly inner-
vated the caudal and ventral parts of the striatum extending
dorsally to the central striatum, whereas the dorsal and lateral
striatal regions remained denervated (Fig. 5D). This sprouting
response was much less pronounced in the SN?STR group and
completely absent in the SN group. In addition to this sprouting
response in the STR group, the TH-positive fibers leaving the
substantia nigra and entering the MFB were relatively normal in
appearance (Fig. 7, compare A, D) compared with all the other
rAAV-GDNF-treated groups (Fig. 7, compare D, C, E). At the
level of the GP (Fig. 6D), in addition to the disorganized densely
stained sprouting TH-positive fibers, TH-positive fibers with nor-
mal appearance and organization could also be observed in the
myelinated fiber bundles of the internal capsule. This subpopu-
lation of normal striatofugal TH-positive fibers was not observed
in the other experimental groups (Fig. 6B,C, E). These observa-
tions suggest that in the STR group, lesion-induced retrograde
axon degeneration did not progress caudally past the GP.
Densitometry measurements (Fig. 5, inset) showed a significant
increase of striatal TH innervation in the STR group compared
with all other treatment groups, from ?10% of normal in the
most denervated caudal regions of the lesioned controls to ?25–
30% of normal in the rAAV-GDNF-injected animals.
Expression of GFP in the nigra and striatum
One of the control groups, killed at 6 months after injection, was
injected with the rAAV-GFP vector to serve as a control for
transduction. Immunohistochemistry for GFP confirmed the ear-
lier observation (Klein et al., 1998) that the transduction effi-
ciency of the rAAV vector is relatively high in substantia nigra
and relatively lower in striatum in comparison (Fig. 9, compare A,
D). In the striatum, GFP-positive cells were observed adjacent to
the injection site to a width of ?300 ?m (Fig. 9A,B), and at least
90% of these cells had a neuron-like morphology that appeared to
be both medium-sized spiny and aspiny neurons commonly found
in striatum (Fig. 9B). Surprisingly, although the transduction in
the striatum was confined to a small area, many of the GP
neurons, ?1 mm distal to the caudal injection site of the vector,
were also GFP positive (Fig. 9A,C).
In the nonlesioned animals injected with the rAAV-GFP vector
(Fig. 9D,E), many GFP-positive neurons were observed in the
substantia nigra, most prominently in the pars compacta. The vast
majority of the transduced cells were neuronal in appearance, and
the transduction extended ?1.5 mm medial-lateral from the in-
jection site within the substantia nigra. Of the GFP immunore-
active cells within the pars compacta, most were morphologically
indistinguishable from normal nigral DA neurons (Fig. 9E). The
observed nigral DA neuron and pallidal tropism of rAAV trans-
duction is consistent with a higher degree of FGFr-1 expression
[FGFr-1 is the putative receptor for rAAV internalization (Qing
et al., 1999)] in these anatomical regions (Matsuo et al., 1994). In
the 6-OHDA-lesioned animals that received the injection of the
same vector, only a few GFP-expressing cells were found in pars
compacta. In adjacent midbrain areas, however, similar numbers
Table 4. Apomorphine (0.25 mg/kg) and SKF-82958 (0.1 mg/kg) induced rotation (full turns/min)
5 weeks 18 weeks5 weeks 18 weeks
Control (n ? 5)
SN group (n ? 5)
STR group (n ? 4)
SN ? STR group (n ? 4)
?6.2 ? 1.4
?8.9 ? 0.9
?9.7 ? 1.3
?9.1 ? 1.2
?6.2 ? 0.9
?9.8 ? 0.9
?8.3 ? 0.9
?10.3 ? 1.0
?10.9 ? 2.0
?13.0 ? 2.0
?12.2 ? 2.5
?13.7 ? 1.9
?7.1 ? 2.1
?11.6 ? 1.7
?6.1 ? 1.7
?12.6 ? 2.0
Mean ? SEM at 5 and 18 weeks after the 6-OHDA lesion.
Kirik et al. • AAV-GDNF Promotes Motor Recovery in a PD ModelJ. Neurosci., June 15, 2000, 20(12):4686–4700 4691
of transduced cells were observed as in the unlesioned control
animals, which is consistent with the fact that both dopaminergic
and nondopaminergic neurons outside the substantia nigra (e.g.,
the A8 cell group) are spared by the 6-OHDA lesion (data not
shown). In one of the GFP-transduced intact rats, GFP-positive
fibers could be traced from the substantia nigra, along the length
of the nigrostriatal pathway, into the striatum where they were
seen to ramify into patches of fine GFP-positive terminals (Fig.
9F,G). This pattern of GFP immunoreactivity after a nigral GFP
transduction suggests that the GFP protein was anterogradely
6-OHDA lesion. There was a highly significant ipsilateral side bias present in all groups at this time point, as indicated by only 10–15% of contralateral
limb use in this test (F(1,76)? 0.408, p ? 0.52). B, The animals were tested again in the cylinder test 23 weeks after the 6-OHDA lesion. At this time
point, there was a general improvement across all groups (effect of time p ? 0.001). In the STR group the improvement was more pronounced, but this
trend did not reach significance (group effect p ? 0.07). However, considering only the animals from the STR group that displayed full compensation
in the amphetamine-induced rotation test (8/11 animals, STR-comp), these animals used their contralateral paw to contact the sides of the cylinder at
near normal levels (?45%), and they performed significantly better than the uncompensated animals and the controls (F(1,39)? 17.5, p ? 0.0001). C,
Staircase (skilled limb use) test, data from the contralateral paw. The first period of testing (day 1–7) was performed using the standard narrow platform.
The groups performed significantly differently from one another (F(3,39)? 5.4, p ? 0.003). The groups improved over the course of the testing (effect
of training; F(6,234)? 46.5, p ? 0.0001), but the rate of learning did not differ between the groups (time ? group interaction, F(18,234)? 1.6, p ? 0.058).
However, the STR group was able to successfully retrieve significantly more pellets than the other three groups on all days (asterisks; simple main effects,
p ?0.02), whereas the control, SN, and SN?STR groups did not perform differently from one another (simple main effects, p ? 0.1 on each individual
day). Beginning on day 8 the test was made more difficult by using a wider platform. In this part of the test, the groups differed from each other in their
ability to successfully retrieve pellets (F(3,39)? 3.2, p ? 0.035). The STR group again performed significantly better than all the other groups (asterisks,
simple main effects, p ? 0.05, except on day 8 and day 10, where 0.08 ? p ? 0.05). The SN group performed significantly worse than the controls and
the SN?STR on day 8 and day 12 (†, simple main effects, p ? 0.05), whereas the control group and the SN?STR groups did not perform differently
at any time point ( p ? 0.4 on all days). D, Staircase (skilled limb use) test, data from the ipsilateral paw. Performance with the ipsilateral paw was
significantly better than that of the contralateral paw for all four groups ( p ? 0.0001). E, F, Performance of the contralateral (E) and the ipsilateral
forelimb (F) in the stepping test. In the pre-lesion testing ( gray shaded column) there was no difference between the groups on either side (F(3,78)? 0.3,
p ? 0.82). The lesion severely affected the number of steps on the contralateral side in all groups (effect of side; F(1,78)? 543.9, p ? 0.0001), whereas
the performance on the ipsilateral side was unaffected. No improvement was observed over time in any of the groups in this test. The legend in the bottom
right corner refers to the symbols representing each group and applies to C–F. All values are means ? SEM.
Spontaneous behavior. A, Spontaneous limb use was evaluated using the cylinder test (Schallert and Tillerson, 1999) 3 weeks after the
4692 J. Neurosci., June 15, 2000, 20(12):4686–4700Kirik et al. • AAV-GDNF Promotes Motor Recovery in a PD Model
transported to the terminals in striatum, which is in agreement
with the high levels of GDNF protein levels measured in the
striatum in the SN group (Table 2).
In contrast to the 6-OHDA injection sites that appeared as
small circumscribed scars at the 6 month time point, the rAAV
vector injection sites were without signs of any nonspecific dam-
age. This suggests low nonspecific toxicity of rAAV injections;
this issue will be examined in greater detail in a later study.
Distribution of GDNF immunoreactivity in the
Immunohistochemisty was used to visualize the distribution of
the GDNF protein 6 months after the injection of the vector (Fig.
10). The GDNF immunoreactivity observed in the striatum near
the area of transduction was mainly extracellular (with some
exceptions; see below) and was present in neither vector-injected
controls nor the contralateral hemisphere of any brain (data not
shown). In the STR (Fig. 10A) and SN?STR (Fig. 10C) groups,
there was a wide distribution of the GDNF protein within the
striatum, extending into the deep layers of the cortex laterally
(Fig. 10A,C,D,F). Caudally, the area of GDNF immunoreactiv-
ity extended into the GP in both groups (Fig. 10D,F). In general,
the SN?STR group displayed a wider distribution of GDNF
immunoreactivity at all rostrocaudal levels. Strongly immunore-
active cellular profiles were observed in the most ventrolateral
part of the striatum, in the adjacent cortex (Fig. 10C, M) and in
the GP (Fig. 10F,N). Because these strongly GDNF-positive
Note the higher intensity of TH-positive fiber innervation in the STR (D) group. The innervation in the SN (C) and SN?STR (E) groups was not different
from the controls. The scale bar in A represents 500 ?m and applies also to photomicrographs in B–E. The inset shows the density of TH-positive fibers in the
striatum measured at seven rostrocaudal levels, as shown in sketch. The 6-OHDA lesion induced extensive degeneration of the striatal TH-positive innervation
in the control group. There was a highly significant difference in striatal TH-positive fiber density between the groups (F(3,273)? 18.6, p ? 0.0001). Anterior
regions (level I–III) were relatively more spared compared with posterior regions (levels IV–VII) across all groups (F(6,273)? 5.2, p ? 0.0001). However, there
was not a significant level ? group interaction (F(18,273)? 0.60, p ? 0.92); therefore, post hoc tests between groups were performed regardless of level. The STR
group displayed significantly higher TH fiber density across all rostrocaudal levels compared with all other groups (*p ? 0.05; Tukey HSD). All values are
means ? SEM.
TH immunocytochemical staining of the central striatum. The 6-OHDA lesion resulted in degeneration of the TH-positive fibers in the controls (B).
Kirik et al. • AAV-GDNF Promotes Motor Recovery in a PD ModelJ. Neurosci., June 15, 2000, 20(12):4686–4700 4693
cells (Fig. 10C,M) were distal to the vector injection site and these
areas were not transduced in animals receiving identical injec-
tions of the rAAV-GFP vector, it is likely that these cells had
accumulated GDNF released from transduced cells that were
localized closer to the site of vector injection. Using an antibody
to the GFR?-1 receptor (Trupp et al., 1997; Kokaia et al., 1999),
adjacent sections were examined for GFR?-1 staining. Strongly
GFR?-1-immunoreactive cells were found precisely in the areas
corresponding to those containing the GDNF-positive cells, sug-
gesting that the accumulation of GDNF into these cells may be
receptor mediated. The cellular GFR?-1 staining was much
stronger in the vector-injected hemisphere (data not shown).
In striatally transduced animals, GDNF immunoreactivity
could be observed also in more caudal striatal efferent structures,
including the EP (Fig. 10G, I) and the substantia nigra pars
reticulata (Fig. 10J,L), on the injected side. In the pars reticulata,
GDNF-positive cellular profiles were observed (Fig. 10K,L,O).
These observations are highly consistent with anterograde trans-
port of striatal rAAV-produced GDNF from the striatum cau-
dally along the striatal efferent pathways.
In animals that received nigral rAAV-GDNF transductions
(SN and SN?STR groups), GDNF immunoreactivity was prom-
inent in and around the substantia nigra (Fig. 10K). In the SN
group, GDNF protein could be observed in the MFB at the level
of the EP (Fig. 10H,I). However, no GDNF immunoreactivity
was observed in the striatum or the GP (Fig. 10B,E) of SN-
The results show that the rAAV vector system can express the
GDNF protein long-term at functionally efficient levels in the
nigrostriatal system, in both the striatum and the substantia nigra.
Transduction was six- to sevenfold higher in the substantia nigra
than in the striatum. In agreement with previous findings (Klein
et al., 1998), the rAAV-GFP vector labeled larger numbers of
cells in the substantia nigra region than in the striatum, and
?90% of the transduced cells were neurons. In the mesenceph-
alon, GFP was expressed preferentially in the substantia nigra
pars compacta, and anterograde transport of the GFP protein was
observed along the axons of the nigrostriatal pathway and in
through the GP (as in A) are lost in the controls (B). Note the sprouting in the STR group within the both GP and striatum (D). In the SN?STR group
a less intense sprouting was observed in the GP (E) and was completely absent in the SN group (C). Arrowheads point to the area shown in high power
adjacent to the individual figures. The scale bar in A represents 500 ?m and applies also to photomicrographs in B–E.
TH immunocytochemical staining of the globus pallidus and the caudal sectors of the lateral striatum. Normal preterminal axons passing
4694 J. Neurosci., June 15, 2000, 20(12):4686–4700 Kirik et al. • AAV-GDNF Promotes Motor Recovery in a PD Model
terminals within the striatum. The transgenic GDNF was widely
distributed extracellularly up to a distance of ?2 mm from the
production site, which is consistent with GDNF being a secreted
protein and able to diffuse widely within the host tissue. More-
over, in the rats receiving injections of rAAV-GDNF in the
striatum, GDNF was effectively transported anterogradely along
the striatonigral pathway to GP, EP, and substantia nigra. An-
terograde transport along the nigrostriatal pathway, from substan-
tia nigra to striatum, is suggested by the ELISA measurements;
the absence of GDNF immunoreactivity in the striatum in the SN
group indicates that staining was completely abolished in the
Functional impact on the intact nigrostriatal
rAAV-GDNF injections in the substantia nigra provide a tool to
express GDNF preferentially within the DA neurons themselves.
In the striatum, by contrast, the vector was expressed in the
neuronal targets of the nigrostriatal pathway. Overexpression of
GDNF by the rAAV-GDNF vector in either site induced func-
tional effects in the intact nigrostriatal DA system, observed as
spontaneous motor asymmetry in animals receiving vector injec-
tions in the nigra, and a high rate of amphetamine-induced
turning in animals injected in either substantia nigra or striatum.
Increased striatal DA turnover was found in all groups, and
increased striatal DA synthesis was seen in the SN?STR group.
These data suggest that long-lasting activation of DA turnover in
the intact nigrostriatal system can be achieved by overexpression
of GDNF in the DA neurons themselves or by GDNF secretion
from cells in the striatal target area. Interestingly, forelimb step-
ping was unaffected in these rats, suggesting that overexpression
of GDNF unilaterally in the intact nigrostriatal system did not
interfere with normal motor function.
Protection of DA neurons against the toxic damage
Significant rescue of nigral DA neurons was obtained with rAAV-
GDNF injection in either substantia nigra or striatum. This effect
was most pronounced in the animals receiving vector injections in
the substantia nigra, and the magnitude of cell protection ap-
peared to match the level of GDNF expressed in the nigral
region, as measured by ELISA at the time of 6-OHDA injection.
In the SN group, we observed near-complete (91%) protection
with a GDNF tissue level of ?1.4 ng/mg (which represents a 15-
to 20-fold increase over the endogenous GDNF concentration)
(Table 2). This suggests that overexpression of GDNF within the
DA neurons themselves is particularly efficient for the rescue of
the nigral cell bodies after 6-OHDA-induced axotomy. In a
previous study (Choi-Lundberg et al., 1997), GDNF was ex-
pressed in the nigral region with an adenoviral vector. In this
case, only partial protection was obtained, despite a tissue level of
GDNF that was severalfold higher than in the present experi-
ment. This difference may be explained by the fact that the
adenoviral vector is expressed mainly outside the DA neurons
and that GDNF in this case is likely to act in a less efficient,
paracrine manner. Indeed, infusion of GDNF protein, over the
substantia nigra at a dose of 2.5–3 ?g/d, gives only 60–70%
placed dorsally in the bundle are lost because of the lesion (B). Note the abnormal sprouting in the SN and SN?STR groups (C, E) and the preservation
of the normal pattern in the STR group (D). Rectangles indicate the area shown in high power adjacent to the individual figures. Scale bar (shown in
A): A–E, 500 ?m.
TH immunocytochemical staining of the MFB. TH-positive axons are observed in bundles in the untreated brain (A). Most of the axons
Kirik et al. • AAV-GDNF Promotes Motor Recovery in a PD ModelJ. Neurosci., June 15, 2000, 20(12):4686–4700 4695
survival in 6-OHDA-lesioned animals (Lu and Hagg, 1997;
Rosenblad et al., 2000b).
Injection of GDNF protein into the striatum is effective in
protecting nigral DA neurons when given before or soon after the
6-OHDA lesion (Kearns et al., 1997; Rosenblad et al., 1999; Kirik,
Rosenblad, and Bjo ¨rklund, unpublished observations). In the
present experiment, rAAV-GDNF injections into the striatum had
a highly significant, but incomplete, protective effect on the DA
cells. This suggests that GDNF delivered at the level of the axon
terminals may be as efficient as delivery at the level of the cell
bodies. However, higher levels and/or a greater spread of GDNF
throughout striatum may be required for complete nigral cell
Regeneration and functional recovery
The acute behavioral impairment seen in all rAAV-GDNF-
injected groups indicates that overexpression of GDNF, at the
levels obtained here, were not sufficient to protect the striatal
DA terminals against the acute toxic damage. In the SN group,
no functional recovery was seen despite near-complete protec-
tion of the DA cell bodies. This is consistent with previous
studies using intranigral injections of GDNF showing that
sparing of nigral DA neurons in the absence of a functional
striatal innervation is insufficient for functional recovery in the
intrastriatal 6-OHDA lesion model (Winkler et al., 1996;
Rosenblad et al., 2000b). Massive sprouting of TH-positive fibers
occurred in and around the MFB, close to the rescued DA cell
bodies in substantia nigra. The meshwork of fibers was seen to
extend rostrally up to the border of the GP, overlapping with the
distribution of the GDNF immunoreactivity. The extent of dener-
vation in the striatum and loss of axons along the pathway were
similar to that of the lesioned controls, suggesting that expression
of GDNF in the nigral cells was unable to direct any axonal
regeneration into the striatal target.
In the STR group, sprouting and regeneration were abundant
in the GP and the caudal and ventral striatum, and the TH-
positive axons along the nigrostriatal pathway were partly pre-
served. From the distribution of sprouting fibers, it appears that
the continuous expression of GDNF had promoted the regrowth
of TH-positive fibers from the axon endings in the GP and
extending into the striatum. Thus, the efficient striatal reinnerva-
tion seen in the STR group is likely to be caused by a combination
of a partial protection of the lesioned nigrostriatal axons followed
by regeneration into the region of high GDNF expression. This is
consistent with the time course of functional recovery in the STR
group, which was delayed in onset and developed progressively
over the first 4–5 months after the lesion, as seen in amphetamine
rotation and the staircase and cylinder tests. The aberrant pal-
lidostriatal TH innervation pattern in the STR group (Fig. 6A?,
D?), coupled with the protracted time course of the recovery,
unequivocally demonstrates remodeling of the nigrostriatal tract
rather than a GDNF-induced reexpression of the TH enzyme in
the nigrostriatal DA system.
The increased striatal TH-positive innervation seen in the STR
group did not occur in the SN?STR group. This difference may
be attributable to the intense local sprouting induced by overex-
pression of GDNF in the nigra that may have prevented regen-
eration of the lesioned axons toward the striatal source of GDNF.
If so, expression of GDNF in the nigral DA neurons themselves
may be detrimental, rather than positive, for the ability of the
lesioned nigrostriatal fibers to regenerate and reinnervate the
compacta in all GDNF vector-injected groups (C–E) compared with the lesioned control (B). Scale bar (shown in A): A–E, 500 ?m. The inset shows the
TH-positive cell numbers in the substantia nigra estimated at 27 weeks after lesion using stereological counting methods as described in Materials and
Methods. In the untreated hemisphere, TH-positive cell numbers were closely similar in all four groups ( p ? 0.99). The 6-OHDA lesion resulted in
?88% reduction in the TH? cells in the control group. There was a significant protection of nigral TH-positive cells in all rAAV-GDNF-treated groups
(ANOVA, followed by post hoc Tukey HSD tests, p ? 0.0001): 91 and 78%, respectively, in the SN and SN?STR groups (Tukey HSD tests, p ? 0.99).
The protection of TH-positive cells in the STR group was significantly less, at 57% (Tukey HSD tests, p ? 0.05), but the TH-positive nigral cell survival
was still highly significant compared with controls. *p ? 0.0001 different from intact side;?p ? 0.0001 different from all other groups. The values inside
the bars indicate percentage TH-positive cells compared with the intact side. All values are means ? SEM.
TH immunocytochemical staining of the cell bodies in the substantia nigra. Note the protection of the TH-positive cell bodies in the pars
4696 J. Neurosci., June 15, 2000, 20(12):4686–4700 Kirik et al. • AAV-GDNF Promotes Motor Recovery in a PD Model
from a single injection site in posterior striatum. GFP-positive cells are apparent in both the striatum and the GP. The box in the striatum (STR) indicates
the area of enlargement shown in B, and the box in the GP indicates the area of enlargement shown in C. Scale bar, 500 ?m. CC, Corpus callosum. B,
GFP-expressing cells with neuronal morphology along the injection tract. Scale bar, 100 ?m. C, GFP-positive neurons in the GP, distal to the injection
site (scale bar as in B). D, rAAV-GFP transduction in the substantia nigra from an intact animal (4 weeks survival). There is transduction throughout
the substantia nigra pars compacta (SNc) as well as dorsally along the needle track (arrows). SNr, Substantia nigra, pars reticulata. The box indicates the
area of enlargement in E. Scale bar, 500 ?m. E, The GFP-positive cells in this higher magnification are morphologically consistent with substantia nigra
pars compacta DA neurons. Scale bar (shown in E): E–G, 50 ?m. F. GFP-positive fibers could be traced along the length of the nigrostriatal pathway
in the same animal. These fibers were observed to ramify into patches of fine GFP-positive terminals in the striatum (G).
Examples of rAAV-GFP transductions in lesioned and unlesioned animals. A, Lesioned control, 6 months survival; rAAV-GFP transduction
Kirik et al. • AAV-GDNF Promotes Motor Recovery in a PD ModelJ. Neurosci., June 15, 2000, 20(12):4686–4700 4697
(C, F, I, L) groups, taken from the same specimen in each group. The most anterior level (A–C) illustrates a central striatal region. Note the diffuse
extracellular staining and immunoreactive cellular profiles in the ventral striatum and the adjacent cortex in the STR and SN?STR groups (A, C). The next
posterior level (D–F) illustrates the GP and caudal striatum. Similarly, in striatally transduced animals GDNF immunoreactivity covers both the striatum
and GP (B, F). GDNF-positive cellular profiles are observed in both the striatum (C, M) and the GP (F, N) of the SN?STR group. In the SN group, both
the central and caudal striatum as well as the GP are devoid of GDNF immunoreactivity (B, E). At the level of the EP (G–I), GDNF immunoreactivity
is observed in the EP in the STR group (G), in the ventral thalamus (Th) in the SN group (H), and in the MFB, Th, and EP in the SN?STR group (I).
Note the staining restricted to the reticulata (SNr) in the STR group (J), and the more widespread staining in the SN and SN?STR groups (K, L). The
box in C indicates the area of enlargement shown in M, the box in F indicates the area of enlargement in N, and the box in L indicates the area of enlargement
shown in O. IC, Internal capsule. Scale bars: A–F, 1 mm; G–L, 750 ?m; M, 50 ?m (applies also to N, O).
GDNF immunoreactivity, 6 months after vector injection, is illustrated at four levels in the STR (A, D, G, J), SN (B, E, H, K), and SN?STR
4698 J. Neurosci., June 15, 2000, 20(12):4686–4700 Kirik et al. • AAV-GDNF Promotes Motor Recovery in a PD Model
denervated striatum. The absence of any significant functional
recovery in the SN?STR group is in line with this interpretation.
In this experiment, three independent biological actions of
GDNF on the nigrostriatal DA neurons have been demonstrated:
first, an upregulation of DA synthesis and turnover in intact nigral
neurons leading to spontaneous and drug-induced asymmetries;
second, a protection of nigral DA cells against toxin-induced cell
death; and third, functional regeneration and reinnervation of the
nigrostriatal pathway. The results demonstrate that the rAAV
vector system can be used to express GDNF at biologically
efficient levels both in the nigrostriatal DA neurons and in their
neuronal targets in the striatum. Although transduction at both of
these sites afforded significant rescue of the lesioned nigral DA
cell bodies, only GDNF expression in the striatum was able to
preserve the integrity of the projecting axons, stimulate reinner-
vation of the denervated striatum, and promote functional recov-
ery in the intrastriatal 6-OHDA lesion model. This suggests that
GDNF acting in an autocrine manner is highly efficient in pre-
serving the integrity of the nigral cell bodies. However, expres-
sion of GDNF within the DA neurons themselves was unable to
sustain the lesioned axons or induce any striatal reinnervation or
functional recovery. Target-derived GDNF expression, by con-
trast, was less efficient in protecting the nigral cell bodies but had
a prominent long-term stimulatory effect on degeneration and
reinnervation of the initially denervated striatum, accompanied
by significant recovery in both drug-induced and spontaneous
motor behaviors. After nigral transduction, extensive sprouting
was observed close to the DA cell bodies. This local sprouting,
however, was detrimental rather than beneficial in that it seemed
to impair function and block striatal regeneration in animals
receiving vector injections in both the substantia nigra and
PD is a degenerative disorder of the dopaminergic nigrostri-
atal system that can progress over years (Fearnley and Lees,
1991; Morrish et al., 1996). In PD, as in the intrastriatal
6-OHDA lesion model, striatal DA innervation appears to
degenerate before the death of the nigral cell bodies (McGeer
et al., 1988; Fearnley and Lees, 1991; Gibb and Lees, 1994).
Indeed, when 70–80% of striatal DA is depleted and PD
symptoms begin to manifest themselves, only ?50% of the DA
cell bodies have been lost (Fearnley and Lees, 1991; Gibb and
Lees, 1994). These data suggest that nigral DA neurons may
survive for some time without an intact functional nigrostriatal
projection. If so, the present data imply that the striatum,
rather than the nigra, should be the primary target for gene
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