Intrastriatal botulinum toxin abolishes pathologic rotational behaviour and induces axonal varicosities in the 6-OHDA rat model of Parkinson's disease

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Intrastriatal botulinum toxin abolishes pathologic rotational behaviour and induces
axonal varicosities in the 6-OHDA rat model of Parkinson's disease
Andreas Wree
a,1
, Eilhard Mix
b,1
, Alexander Hawlitschka
a
, Veronica Antipova
a
, Martin Witt
a,c
,
Oliver Schmitt
a
, Reiner Benecke
b,
⁎
a
Department of Anatomy, University of Rostock, Gertrudenstr. 9, 18057 Rostock, Germany
b
Department of Neurology, University of Rostock, Gehlsheimer Str. 20, 18147 Rostock, Germany
c
Department of Anatomy, University of Technology, Dresden, Germany
abstractarticle info
Article history:
Received 9 July 2010
Revised 14 September 2010
Accepted 23 September 2010
Available online 16 October 2010
Keywords:
Botulinum neurotoxin A
6-Hydroxy dopamine
Striatum
Motor function
Apomorphine-induced rotation
Axonal varicosities
Central pathophysiological pathways of basal ganglia dysfunction imply a disturbed interaction of
dopaminergic and cholinergic circuits. In Parkinson's disease (PD) imbalanced cholinergic hyperactivity
prevails in the striatum. Interruption of acetylcholine (ACh) release in the striatum by locally injected
botulinum neurotoxin A (BoNT-A) has been studied in the rat 6-hydroxydopamine (6-OHDA) model of PD
(hemi-PD). The hemi-PD was induced by injection of 6-OHDA into the right medial forebrain bundle. Motor
dysfunction provoked by apomorphine-induced contralateral rotation was completely reversed for more than
3 months by ipsilateral intrastriatal application of 1–2 ng BoNT-A. Interestingly, BoNT-A injected alone into
the right striatum of naïve rats caused a slight transient ipsilateral apomorphine-induced rotation, which
lasted only for about one month. Immunohistochemically, large axonal swellings appeared within the
striatum injected with BoNT-A, which we tentatively named BoNT-A-induced varicosities. They contained
either choline acetyltransferase or tyrosine hydroxylase. These findings suggest a selective inhibition of
evoked release of ACh by locally applied BoNT-A. Intrastriatal application of BoNT-A may antagonize localized
relative functional disinhibited hypercholinergic activity in neurodegenerative diseases such as PD avoiding
side effects of systemic anti-cholinergic treatment.
© 2010 Elsevier Inc. All rights reserved.
Introduction
A hallmark of Parkinson's disease (PD) is diminished dopaminergic
signaling in the striatum (caudate putamen, CPu), which leads to
increased release of acetylcholine (ACh) by disinhibited tonically
active interneurons with the consequence of a disturbed network
function and consecutive motor dysfunction. Current therapeutic
strategies are based on two major approaches in order to correct the
disturbed circuits of involuntary movement control, i.e. stimulation of
dopamine (DA) receptors on GABAergic medium spiny neurons and
inhibition of hypercholinergic activity of tonically active interneurons
(Day et al., 2006; Obeso et al., 2008). However, both approaches are
compromised by adverse side effects due to systemic drug application
(Whitney, 2007). In the pre-L-DOPA era, the first drugs that turned
out to have clinically significant anti-Parkinson activity were anti-
cholinergics such as atropine and atropine-like derivatives (Duvoisin,
1967; Kaplan et al., 1954; Ordenstein, 1868). Today some of these
compounds are still commonly used in clinical practice, e.g. the
piperidine derivative Biperiden (Akineton®). Also, the PD-like side
effects of anti-psychotics are typically treated with anti-cholinergic
drugs, especially in younger patients (Ekdawi and Fowke, 1966;
Mamo et al., 1999). However, the most serious problem with these
systemically administered anti-cholinergics remains the occurrence
of well-known troublesome peripheral and central side effects, such
as mydriasis, paresis of accommodation (blurred vision), reduced
salivation (dry mouth), parotitis, dry eyes, muscular pain, loss of
power, alteration of voice, dysphagia, regurgitation, constipation,
urinary retention, prostatism, tachycardia, fever in warm weather,
hallucinations, memory problems and confusion. To avoid this
disadvantage we tested local anti-cholinergic treatment applying
botulinum neurotoxin A (BoNT-A) into the CPu of hemiparkinsonian
(hemi-PD) rats, a well established 6-hydroxydopamine (6-OHDA)-
induced animal model of PD (Meredith et al., 2008). In this model, it
has been shown that intraperitoneally applied atropine antagonizes
the profound PD-typical akinesia (Schallert et al., 1978). In combina-
tion with L-DOPA it supported the alleviation of excessive bracing
reactions and suppressed pathological circling of the PD rats (Schallert
et al., 1979). In our study, intrastriatal application of BoNT-A caused
complete long-term abolition of pathological apomorphine-induced
Neurobiology of Disease 41 (2011) 291–298
⁎Corresponding author. Fax: +49 381 494 9512.
E-mail address: reiner.benecke@med.uni-rostock.de (R. Benecke).
1
These authors contributed equally to this work.
Available online on ScienceDirect (www.sciencedirect.com).
0969-9961/$ –see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.nbd.2010.09.017
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rotation, which indicates a compensatory effect to the dopaminergic
destruction. Simultaneously, large axonal swellings were detected
immunohistochemically both at the light and electron microscopic
levels in the BoNT-A-injected CPu, which were positively stained for
either the cholinergic marker enzyme choline acetyltransferase
(ChAT) or the dopaminergic marker enzyme tyrosine hydroxylase
(TH). We tentatively named these swellings BoNT-A-induced vari-
cosities (BiVs). The findings for the first time describe morphological
changes in response to intrastriatal BoNT-A-application and their
functional consequence. Thereby, our study serves as a proof-of-
principle for a novel type of intervention with local hypercholinergic
activity in the central nervous system (CNS), which may contribute to
new therapeutic strategies for diseases with local over-activity of
cholinergic neurons like in PD.
Materials and methods
Animals
Adult male Wistar rats (strain Crl:WI BR, Charles River Wiga,
Sulzfeld, Germany) aged about 3 months were used and housed in
standard cages at 22 °C ± 2 °C under a 12 h light/dark cycle, with free
access to tap water and a standard diet.
Stereotactic intervention of four animal groups
Hemi-PD was induced in adult male Wistar rats by injection of 6-
OHDA into the right MFB. Rats weighing 280–300 g were anaesthetized
by intraperitoneal injection of ketamine (50 mg/kg body weight) and
xylazine (4 mg/kg body weight) for stereotactic surgery. Four experi-
mental groups were constituted: (1) 6-OHDA-lesioned animals receiv-
ing BoNT-A (6-OHDA-lesion+BoNT-A group), (2) 6-OHDA-lesioned
animals receiving BoNT-A-vehicle substance (6-OHDA-lesion+ sham-
BoNT-A group), (3) sham-6-OHDA-lesioned animals receiving BoNT-A-
vehicle substance (sham-6-OHDA-lesion+ sham-BoNT-A group), and
(4) naïve animals receiving BoNT-A (BoNT-A only group).
Lesions were made in the right MFB by injection of 4 μlof6-OHDA
(24 μg) dissolved in 0.1 M citrate buffer and delivered over 4 min each via
a26gauge5μl Hamilton syringe. The coordinates with reference to
bregma were: anterior–posterior= −2.3 mm, lateral= −1.5 mm and
ventral = −9.0 mm, respectively (Paxinos and Watson, 2007). Successful
lesions were evaluated with apomorphine-induced rotations 28 days
after surgery. Six weeks after 6-OHDA-lesioning animals received
injections of 2×1 μl BoNT-A solution (Lot #13028A1A, List, Campbell,
USA, purchased via Quadratech, Surrey, UK) containing a total of 100 pg,
1 ng or 2 ng BoNT-A into the right CPu delivered over 4 min for each 1 μl
aliquot (6-OHDA-lesion + BoNT-A group). The coordinates with reference
to bregma were: anterior = + 1.3/−0.4 mm, lateral = −2.6/−3.6 mm
and ventral= −5.5 mm, respectively. The first control group received
BoNT-A-vehicle solution containing phosphate-buffered saline with 0.1%
bovine serum albumin (PBS–BSA 0.1%) instead of BoNT-A (6-OHDA-
lesion+ sham-BoNT-A group). The remaining two animal groups
received either only BoNT-A solution without previous lesioning (BoNT-
A only group) or both vehicle solutions instead of 6-OHDA and BoNT-A,
respectively (sham-6-OHDA-lesion + sham-BoNT-A group).
Apomorphine-induced rotation test
The DA receptor agonist apomorphine stimulates the supersensitive
DRD2 (and to a lesser degree DRD1) receptors on the injured DA
depleted hemisphere more than the normal DRD2 and DRD1 receptors
on the intact side causing a net rotation away from the side ofthe lesion,
i.e. anti-clockwise (Ungerstedt et al., 1969). In our study apomorphine
was applied subcutaneously at a concentration of 0.25 mg/kg body
weight in normal saline. Rotations were measured using a self-
constructed automated rotometry device over 40 min modified accord-
ing to Ungerstedt and Arbuthnott (1970). They were defined as
complete 360° ipsilateral turns and reported as net differences between
the two directions per minute. The rotation test was performed 28 days
after 6-OHDA-lesioning and at different time points after BoNT-A or
vehicle application (see Results section and Fig. 1).
Histochemistry
For analysis of the consequences of BoNT-A-treatment on the
morphology and neurotransmitter expression of affected brain regions
investigations were performed applying the following stainings or
immunohistochemical reactions: (1) cresyl violet according to Nissl (for
vital neurons), (2) ChAT (for cholinergic neurons), (3) TH (for
dopaminergic neurons), and (4) glutamic acid decarboxylase-67
(GAD-67) (for GABAergic neurons). In detail, after finishing of rotation
tests animals were killed with an overdose of ketamine/xylazine and
perfused with 3.7% paraformaldehyde solution for fixation. The fixed
brains were cryoprotected, frozen and stored at −80 °C. Coronal brain
sections of 30 μm thickness were prepared by cryo-cutting and
consecutive sections were stained according to Nissl or immunostained
for ChAT, THand GAD-67, respectively, using the avidin–biotin complex
immunoperoxidase method (Vector Laboratories, Burlingame, CA,
USA). The following primary antibodies were applied: polyclonal goat
anti-ChAT affinity purified antibody (Millipore, Schalbach, Germany),
monoclonal mouse anti-TH antibody (clone TH2, Sigma-Aldrich, St.
Louis, MO, USA) and monoclonal mouse anti-GAD-67 antibody
(Chemicon International, Millipore, Temecula, CA, USA). Endogenous
peroxidase was blocked with 3% H
2
O
2
in 0.1 M PBS for 15 min followed
by three washings with PBS for 5 min. Nonspecific binding of proteins
was blocked by incubation with 3% rabbit serum (for ChAT staining) or
with 3% goat serum and 1.5% horse serum (for TH and GAD-67 staining)
at room temperature for 60 min. Sections were then incubated (1) in
10 μg/ml of primary antibody overnight at 4 °C. After subsequent
washing with PBS, sections were (2) incubated with either biotinylated
rabbit anti-goat IgG (for ChAT staining) or with biotinylated horseanti-
mouse IgG (for TH and GAD-67 staining) overnight at 4 °C before color
development with diaminobenzidine and ammonium nickel sulfate for
enhancement.
Image analysis of immunohistochemical stainings
For quantification of immunohistochemical findings an optical grid
was applied and at least 1000 total cells per CPu section were counted
manually for ChAT-stained cells (see Fig. 2). In both ChAT- and TH-
stained sections of BoNT-A-injected CPu peculiar axonal varicosities,
intentionally named BoNT-induced varicosities (BiVs), were found.
They were counted in areas of equal size and localization at both sides
of the brain thereby comparing the density of BiVs between the
lesioned and the intact sides. For quantitation of BiVs, sections were
digitized at a resolution of 0.23 μm using the virtual slide scanner
Mirax (Zeiss, Jena, Germany) (Mikula et al., 2007). Virtual slides were
converted to a multiresolution image pyramid to allow the interactive
definition of the region of interest (ROI), i.e. the CPu complex as
illustrated by a TH-stained section in Fig. 3. BiVs were segmented
using a multiscale (gray level classes) multifeature (roundness, local
BiV staining intensity, and BiV area) approach implemented in Matlab
(Mathworks). Due to the large amount of data (Gigapixel images) a
parallelization was performed on Linux 64Bit systems. The result of a
typical segmentation is shown in Figs. 3E and F. The segmented BiVs
were mapped in 2D (Fig. 3C) to judge the spatial distribution of BiVs.
The local density of BiVs was color coded (Fig. 3D).
In the ChAT-stained sections the evaluation is more complex due to
quantification of ChAT-positive neurons and BiVs (Figs. 2A–C). Due to
Gauss-distributed noise anisotropic noise filtering need to be performed
for successful segmentation of nerve fibres (Figs. 3B and C). The total
number of neurons was estimated using the optical fractionator in
292 A. Wree et al. / Neurobiology of Disease 41 (2011) 291–298

Stereoinvestigator 8.0 using the BX51 (Olympus, Hamburg, Germany)
three axes motorized videomicroscope. In each case (serial sections of an
animal) the coefficientoferrorwassmallerthanorequalto0.05.
Visualization of immunofluorescent stainings was performed on the
confocal laser scanning microscope Eclipse E400 (Nikon, Düsseldorf,
Germany).
Immunoelectron microscopy
For ultrastructural analysis of BiVs, striatal sections were immu-
noreacted with antibodies against TH and ChAT, embedded in epoxy
resin and evaluated with a transmission electron microscope. In
detail, animals were perfused with PBS containing 3.7% paraformal-
dehyde and 0.05% to 0.1% glutaraldehyde (pH 7.2). Brains were
dissected out after 1 h and postfixed overnight, cryoprotected and
processed as described for light microscopy with the following
exceptions: 50–60 μmfloating sections were cut and collected in
0.1 M PBS and treated with 3% H
2
O
2
for 15 min. No permeabilizing
agents were used. All following steps were performed in a 12-well cell
culture plate. Incubation times of primary antibodies were extended
to 2 days. Secondary antibodies were biotinylated and visualized with
standard ABC/DAB methods. No Nickel amplification was performed.
After the DAB reaction the sections were washed in PBS and postfixed
in cacodylate buffer (pH 7.6) containing 2.5% glutaraldehyde
overnight, then washed and postfixed in 1% osmium tetroxide for
30 min. After dehydration in a series of graded ethanols and
infiltration with a mixture of 100% ethanol/Epon 812 resin (v/v) and
pure Epon overnight, the sections were embedded between two Aclar
sheets (Ted Pella, Redding, CA, USA), fixed between glass slides and
polymerized at 60 °C for 24 h. After light microscopic evaluation of the
immunoreaction, regions of interest were cut out, one Aclar sheet
removed, and the specimen glued on an Epon cylinder. Ultrathin
sections were mounted on Formvar-coated slot copper grids con-
trasted with 2% aqueous uranyl acetate (8 min) and lead citrate
(2 min) and analyzed with a Zeiss EM 906 transmission electron
microscope at 80 kV (Zeiss, Oberkochen, Germany).
Statistics
Data of time-kinetics of the apomorphine-induced rotation test
were compared by one way analysis of variance (ANOVA) for
Gaussian distributed values. Significant results were derived from
the post-hoc test according to Holm–Sidak (Figs. 1A and B). Non-
Gaussian distributed values were compared by the Kruskal–Wallis
test (Figs. 1C and D). For comparison of the rotations of animals
treated with BoNT-A with the corresponding rotations of vehicle-
treated animals 2-tailed Student's t-test for unpaired observations
was applied. Likewise the results of quantitative immunohistochem-
istry were compared by 2-tailed Student's t-tests for unpaired
observations. The level of significance was set at pb0.05 for all
statistical analyses.
Ethics
All animal experiments were approved by the local Animal
Research Committee of the state Mecklenburg-Western Pomerania
(LALLF M-V/TSD/7221.3-1.1-019/08).
Results
In order to determine the effective and tolerated dose of BoNT-A to
be applied intrastriatally, we first injected BoNT-A into naïve rat
Fig. 1. Apomorphine-induced rotation of hemi-PD rats treated with intrastriatal BoNT-A. In order to induce a toxic model of PD, Wistar rats received 6-OHDA injections into the right
MFB, which leads to an apomorphine-induced asymmetric rotation away from the site of lesion, i.e. anti-clockwise, of about 9 rotations per minute (positive values). Six weeks after
6-OHDA-application rats received injections of 1 ng or 2 ng BoNT-A (A, B) or vehicle (C) into the ipsilateral CPu (0 d). (A, B) BoNT-A abrogated the apomorphine-induced rotation
completely for 3 months. Subsequently, the pathologic rotation was slowly restored, but even after 6 months there was still a significant reduction seen. (C) Vehicle treatment had a
slight non-significant reducing effect up to 12 months. (D) For control, naïve rats were injected with vehicle substance of 6-OHDA solution (0.1 M citrate buffer) into the right MFB
and subsequently with vehicle substance of BoNT-A (PBS–BSA 0.1%) into the ipsilateral CPu. This double-sham treatment had no effect on apomorphine-induced rotation. All results
are presented as mean values ±SD. Asterisks indicate significant changes in comparison to initial values (0 d) according to one way ANOVA and post-hoc Holm–Sidak test (A, B) and
Kruskal–Wallis test (C, D), respectively. P-values: * b0.01, ** b0.001.
293A. Wree et al. / Neurobiology of Disease 41 (2011) 291–298

striata and estimated their apomorphine-induced rotational behav-
iour. As 100 pg BoNT-A was the minimum dose revealing a tendency
of asymmetric apomorphine-induced rotation and 5 ng BoNT-A was
occasionally lethal to the animals, we used doses of 100 pg–2ng
BoNT-A for subsequent experiments in rats with and without prior
lesioning of the substantia nigra pars compacta by 6-OHDA. Effects on
motor function and on striatal neuronal morphology were measured
over a time period of up to twelve months.
Effect of instrastriatally applied BoNT-A on apomorphine-induced
rotation in hemi-PD rats
6-OHDA injections into the right medial forebrain bundle (MFB)
caused apomorphine-induced rotations away from the lesion site, i.e.
anti-clockwise (positive values between 8 and 10 per min, 0 d in
Figs. 1A–C), a hallmark of hemi-PD. When the rats six weeks after 6-
OHDA application received injections of 1 ng or 2 ng BoNT-A into the
ipsilateral CPu, this treatment abolished the apomorphine-induced
rotations completely for about 3 months. After this time the reducing
effect slowly declined (Figs. 1A and B). BoNT-A-vehicle caused no
significant changes (Fig. 1C). Comparison of apomorphine-induced
rotations of BoNT-A-treated 6-OHDA-lesioned rats with the
corresponding vehicle-treated 6-OHDA-lesioned rats revealed that
the suppressive BoNT-A effect was significant up to 6 months for 1 ng
BoNT-A (p-values according to Student's t-test: BoNT-A versus vehicle
at 1 month: 0.0001, at 3 months: 0.0025, at 6 months: 0.0239, at
9 months: 0.3472 and at 12 months: 0.8187). Additional control
experiments were performed by sham 6-OHDA-lesioning with
injection of the vehicle (0.1 M citrate buffer) into the right MFB
followed by sham BoNT-A-treatment with injection of the vehicle
substance PBS–BSA 0.1% into the ipsilateral CPu. This double-sham
treatment did not cause significant apomorphine-induced rotations
during the 12 month observation period (Fig. 1D).
If BoNT-A alone was applied to the CPu of naïve rats at doses of 1–
2 ng, apomorphine induced 2–3 rotations per minute towards the
injection site as soon as 2 weeks after toxin application (data not
shown). However, this effect was transient and vanished after
2 months. 100 pg BoNT-A and vehicle solution had no significant
effect.
In order to test the effect of intrastriatal BoNT-A application on
non-drug induced sensorimotor function in hemi-PD rats we analyzed
the forelimb-use asymmetry by applying the cylinder test described
by Schallert and Tillerson (2000). A tendency of improvement of the
asymmetric forelimb usage by BoNT-A was seen over the time period
of up to 12 months with significance at the high dose of 2 ng, whereas
sham lesion and sham BoNT-A application did not affect the
asymmetric forelimb usage significantly (see Supplement 1).
Effect of BoNT-A on CPu morphology
Concerning the morphological consequences of intrastriatal BoNT-
A-application the total number of ChAT-positive neurons was not
different between BoNT-A-treated and untreated CPu, neither after
injection of 100 pg BoNT-A (ipsilateral: 22,233 ±1036; contralateral:
19,846±4039; n =3) nor after injection of 1 ng BoNT-A (ipsilateral:
20,500±2076; contralateral: 20,799 ± 2979; n = 4). Preliminary
counts performed 6 months after BoNT-A-application confirmed this
finding (data not shown).
The most striking novel morphological finding of our study was
the appearance of regularly occurring axonal swellings in the BoNT-A-
infiltrated areas, which we named BoNT-A-induced varicosities
(BiVs). These structures were found selectively at any injection site
Fig. 2. Immunohistochemical staining of the rat CPu for ChAT one month after ipsilateral intrastriatal application of 2 ng BoNT-A. (A) 6 ChAT-positive neurons are visible. Arrows
are pointing to ChAT-positive axonal swellings (BiVs). (B) Segmentation of nerve fibres (blue), neurons (red) and BiVs (green) with a magnification in (C). Scale bars: in A 30 μm,
in B 20 μm, in C 10 μm. (D and E) Quantitative analysis of BiVs in CPu sections. (D) Density [n/mm³] of BiVs in the CPu detected in TH and ChAT immunohistochemistry after injection
of 2 ng or 100 pg of BoNT-A is dose-dependently increased as confirmed by Student's t-tests (* =p b0.05). (E) Projection areas of BiVs were not different between the CPu treated
with 2 ng and 100 pg BoNT-A. Error bars are indicating SEM.
294 A. Wree et al. / Neurobiology of Disease 41 (2011) 291–298

of BoNT-A, i.e. in the ipsilateral, but not in the contralateral CPu of 6-
OHDA-lesioned animals, as well as in the BoNT-A-treated CPu of naïve
rats, but not in its opposite site. The size of the BiVs was
heterogeneous ranking between 2 and 9 μm in diameter when
investigating the histochemically stained CPu sections of BoNT-A
treated hemi-PD or naïve rats. BiVs were reactive either for ChAT
(Figs. 2A–C and 4A and B) or for TH (Figs. 3 and 4A), but never double-
stained for both marker enzymes (Figs. 4A and B) and never stained
for TH in the dopaminergic deafferented CPu of 6-OHDA-lesioned
animals (Fig. 4B). Quantitative evaluation revealed the following
(Figs. 2D and E): The density of ChAT-positive BiVs [n/mm
3
] was
higher in animals treated with 2 ng (11,836± 2404) than in animals
treated with 100 pg (7989 ±1488) BoNT-A. For TH-positive BiVs this
dose-dependent difference was even higher (2 ng: 15,188± 3157;
100 pg: 4607± 1409). In contrast, the mean BiV area was not
significantly different between animals treated with 2 ng and
100 pg BoNT-A, neither for ChAT-positive nor for TH-positive BiVs
(ChAT-positive BiVs: 2 ng: 6.34 ± 0.34 μm
2
, 100 pg: 6.73 ±0.29 μm
2
;
TH-positive BiVs: 2 ng: 6.95±1.02 μm
2
, 100 pg: 8.82 ±0.72 μm
2
).
GABAergic BiVs were not detected (data not shown).
TH-positive BiVs in naïve rats injected with BoNT-A were further
investigated by electron microscopy. Ultrastructurally, these struc-
tures appear as large axonal dilations filled with TH-immunoreactive
vesicles and some scattered mitochondria (Figs. 4D and E). Synapses,
however, were not positively identified so far.
Discussion
For detection of functional effects of intrastriatal BoNT-A in the rat
hemi-PD model we chose the apomorphine-induced rotation test.
This test provides a sensitive and rapid behavioural correlate of
striatal damage and has been used to evaluate the potential efficacy of
therapeutic trials with tissue and/or cell grafts in different conditions
of striatal degeneration (Emerich et al., 1996; Nikkhah et al., 1994;
Norman et al., 1988). As a first step, the effective and tolerated BoNT-A
dose was determined. In the literature an effective dose range of
BoNT-A between 4 ng and 150 ng was reported for injections into
skeletal muscles in humans (Borodic et al., 1996) and between 135 pg
and 1875 pg for injections into whisker pad muscles (135 pg), corpus
vitreous (1.875 ng), superior colliculus (225 pg) and hippocampus
(300 pg), respectively, in Sprague Dawley rats (Antonucci et al.,
2008). We found effective doses of 1 ng–2 ng BoNT-A for the
intrastriatal application, whereas higher doses turned out to be
toxic. Therefore, the dose most suitable for therapeutic intervention in
Fig. 3. Immunostaining for quantification of BiVs in the CPu one month after unilateral application of 2 ng BoNT-A. (A) TH-stained coronal brain section. The injection site is in the
right CPu (arrow) and the evaluated region of interest (ROI) is shown in B. The segmented BiVs are mapped in C. In D the concentric distribution increase of BiVs is visualized. The
scale displays the number of BiVs around a central BiV. (E) 1024 ×1024 large image tile of the 10,000 ×20,000 large image of the ROI (B) with segmented BiVs. (F) Magnification from
E showing details of the segmentation quality. Scale bars: in A 1 mm, in E 50 μm, in F 10 μm.
295A. Wree et al. / Neurobiology of Disease 41 (2011) 291–298

the rat PD model appears to be around 1 ng and this dose is also
recommended to start with in other conditions of local cholinergic
hyperactivity in the CNS.
Functional effect of intrastriatally applied BoNT-A
In the 6-OHDA-lesioned hemi-PD rats the dopaminergic input into
the right CPu is destroyed leading to a right side DA receptor
supersensitivity mainly involving DRD2 receptors (Kaasinen et al.,
2000). Thereby apomorphine acts in the right CPu as an enhancer of
the inhibitory action with the consequence of anti-clockwise rotation.
In PD the well-known hypercholinergic state interferes negatively
with the basal ganglia loops (2), the hyperactive tonic cholinergic
interneurons targeting the GABAergic medium spiny projection
neurons of the CPu. Local application of BoNT-A into the CPu abolishes
ACh release, thus reducing the apomorphine-induced contralateral
rotations. It cannot be excluded that striatal damage, which would
also yield ipsilateral rotations, may antagonize the contralateral
Fig. 4. Immunofluorescence of the rat CPu for ChAT (red) and TH (green) visualized by confocal laser scanning microscopy. (A) Section of the CPu of a naïve rat one month after
application of 2 ng BoNT-A. ChAT-positive cell bodies and projections are clearly separated from TH-positive projections. Both types of projections display several axonal swellings
(BiVs) of heterogeneous size (arrows: TH-positive BiVs, arrowheads: ChAT-positive BiVs). Double-positive BiVs are not detected. (B) CPu of a rat with 6-OHDA-lesion and
subsequent ipsilateral injection of 1 ng BoNT-A. Double staining for ChAT and TH shows ChAT-positive cell bodies and BiVs (arrowheads), but no TH-positive projections.
(C) A corresponding section contralateral to the side shown in B displays no BiVs, but ChAT-positive cell bodies and projections and TH-positive projections of similar density as at
the ipsilateral side of rats treated with BoNT-A only (shown in A). (D–E) Electron microscopy of TH-positive BiVs. (D) A 50 μm thick TH-positive section of a naïve BoNT-A treated rat,
embedded in Epon, exhibiting BiVs (arrows) approx. 5 μm in diameter. Asterisks mark the injection channel, filled with macrophages. The insert marked by a rectangle shows a BiV
similar to that enlarged in (E). A dilated axonal projection of a TH-positive neuron is filled with vesicles (approx. 30 nm) and mitochondria. The axon partly wraps other, non-TH-
reactive axonal processes. Two TH-positive projections of this BiV are arrow-marked. Scale bars: 100 μminA–C, 20 μm in D and 1 μminE.
296 A. Wree et al. / Neurobiology of Disease 41 (2011) 291–298

circling. However, striatal neuron loss by BoNT-A application is
unlikely, since the number of cholinergic neurons is not reduced in the
CPu after BoNT-A treatment. Analysis of the asymmetric forelimb
usage argues for a beneficial effect of intrastriatal BoNT-A application
on non-drug induced motor function, too, although the effect was
slight and extended studies with different non-drug induced tests are
needed in order to evaluate its therapeutic potential.
Surprisingly, the rotation-reducing effect of BoNT-A in the hemi-
PD rats lasts for at least half a year. This is interesting, since thereby
the compensatory BoNT-A effect in the CPu lasts longer than the
conventional inhibitory BoNT-A effect on peripheral muscle activity,
which is usually terminated to about 3 months due to intermittent
axonal sprouting and reformation of affected transport proteins
(Benecke and Dressler, 2007; de Paiva et al., 1999). Of course, the
regeneration time in the periphery depends also on the injection site
and size of affected structures. It is somewhat longer than 3 months,
when small muscles or perspiratory and salivary glands are injected
(Heckmann et al., 2005; Reid et al., 2008). However, there may also be
more differences between the mechanisms of peripheral and central
BoNT effects. In this context, the novel morphological finding of
axonal swellings that we intentionally called BiVs could be of
relevance.
Effect of BoNT-A on striatal morphology
In any BoNT-A-infiltrated areas, whether they contained only
cholinergic terminals, i.e. in 6-OHDA-lesioned rats, or whether they
contained both cholinergic and dopaminergic terminals, i.e. in naïve
rats, BiVs appeared along the neuronal axons. At first glance, they
resemble classical boutons as previously described, e.g., for cortical
neurons (de Paola et al., 2006). Concerning the mechanism of their
formation, BoNT-A binds via its heavy chain component with low
affinity to gangliosides and with high affinity to the synaptic vesicle
protein SV2 (Dong et al., 2006; Mahrhold et al., 2006) of presynaptic
membranes. As a next step receptor-mediated endocytosis occurs
followed by release of the light chain of BoNT-A from the acidified
endosome into the cytoplasm due to reduction of a disulfide bond. At
the vesicular membrane the light chain due to its Zn
2+
-dependent
metalloprotease activity cleaves the SNARE target SNAP25. This
prevents the fusion of neurotransmitter containing vesicles with the
presynaptic plasma membrane, the prerequisite of neurotransmitter
secretion into the synaptic cleft, and may lead to accumulation of
several vesicles, which subsequently become confluent and form the
varicosity-like structures. Of interest, at the motor endplate ACh is
predominantly released via full vesicle fusion, whereas in the CNS a
transient “kiss-and-run”mechanism may prevail under physiological
conditions (Brunger et al., 2009), which are impaired in the presence
of BoNTs. Details of the differences of the mechanisms of action of
BoNT-A in the CNS and at the motor endplate are currently not fully
understood. Their disclosure remains a task of future investigations.
The selectivity of BoNT binding to nerve cell terminals determines
the selectivity of neurotransmitter blockade by BoNTs with preference
of cholinergic terminals. However, it is well known that depending on
their concentration BoNTs can inhibit the evoked release of a variety
of neurotransmitters including DA, norepinephrine, serotonin, gluta-
mate, GABA and glycine (Ashton and Dolly, 1988; Bigalke et al., 1981;
Pearce et al., 1997). In the femtomolar range BoNT-A blocks the
neuromuscular junction, whereas in the picomolar range it affects
almost all transmitter systems (Bigalke et al., 1985). Therefore, it is
not surprising that we found in addition to BiVs containing the marker
enzyme of cholinergic neurons ChAT also BiVs containing the marker
enzyme of dopaminergic neurons TH. The exact mechanism by which
the BiVs are generated is unknown. Sequential electron micrographs
will be required in order to get deeper insight in the early events of
BiV formation after BoNT-A-application.
Since intrastriatal BoNT-A-application did not substantially affect
the number of ChAT-positive neurons in the BoNT-A-injected CPu,
there is no morphological indication for a non-specific toxic effect of
BoNT-A on this cell population of the BoNT-A-treated CPu. However,
testing of their functional integrity awaits further histochemical and
electrophysiological investigations.
In summary, intrastriatal application of BoNT-A leads to a well-
tolerated long-lasting localized reduction of cholinergic activity within
the CPu. In the 6-OHDA rat model of PD, pharmacologically induced
pathological motor activity is attenuated and spontaneous motor
activity slightly improved. Extended functional test in the rodent and
primate models of PD will show, whether these findings can contribute
to new therapeutic strategies for treatment of neurodegenerative
diseases with localized imbalanced hypercholinergic activity such as
PD. Since detection of TH-positive BiVs argues for an inhibition of
residual intrastriatal DA release by BoNT-A, a comprehensive new
therapeutic concept for PD should combine local application of BoNT-A
by a gentle procedure (Rogawski, 2009) with conventional systemic
application of L-DOPA or DA agonists (Whitney, 2007). Respective
trials in the animal model are under way. In addition, our findings may
enable the development of improved animal models of hypo-
cholinergic disorders such as Huntington's disease and progressive
supranuclear palsy for pathophysiological investigations and pharma-
cological manipulation (Pisani et al., 2007).
Supplementary data to this article can be found online at
doi:10.1016/j.nbd.2010.09.017.
Acknowledgments
We gratefully acknowledge Mrs. Barbara Kuhnke and Mrs. Antje
Schümann,Mrs Doreen Streichert and Mrs Martina Pinkert for excellent
technical assistance and Dr. Carsten Holzmann (University of Rostock)
for performing the statistical analysis of data. We are thankful to Prof.
Gordon W. Arbuthnott (Okinawa, Japan) and Dr. Stefan J.-P. Haas
(University of Rostock) for helpful suggestions and discussions and
critical revision of the manuscript. This work was supported by an
intramural grant of the FORUN program of the University of Rostock
[Project number 889005 to A.H.].
References
Antonucci, F., Rossi, C., Gianfranceschi, L., Rossetto, O., Caleo, M., 2008. Long-distance
retrograde effects of botulinum neurotoxin A. J. Neurosci. 28, 3689–3696.
Ashton, A.C., Dolly, J.O., 1988. Characterization of the inhibitory action of botulinum
neurotoxin type A on the release of several transmitters from rat cerebrocortical
synaptosomes. J. Neurochem. 50, 1808–1816.
Benecke, R., Dressler, D., 2007. Botulinum toxin treatment of axial and cervical dystonia.
Disabil. Rehabil. 29, 1769–1777.
Bigalke, H., Heller, I., Bizzini, B., Habermann, E., 1981. Tetanus toxin and botulinum A
toxin inhibit release and uptake of various transmitters, as studied with particulate
preparations from rat brain and spinal cord. Naunyn Schmiedebergs Arch.
Pharmacol. 316, 244–251.
Bigalke, H., Dreyer, F., Bergey, G., 1985. Botu linum A neurotoxin inhibits non-
cholinergic synaptic transmission in mouse spinal cord neurons in culture. Brain
Res. 360, 318–324.
Borodic, G., Johnson, E., Goodnough, M., Schantz, E., 1996. Botulinum toxin therapy,
immunologic resistance, and problems with available materials. Neurology 46,
26–29.
Brunger, A.T., Rongsheng, J., Breidenbach, M.A., 2009. Interactions between botulinum
neurotoxins and synaptic vesicle proteins. In: Jankovic, J., Albanese, A., Atassi, M.Z.,
Dolly, J.O., Hallett, M., Mayer, N.H. (Eds.), Botulinum Toxin. Therapeutic Clinical
Practice and Science. Saunders Elsevier, Philadelphia, pp. 41–52.
Day, M., Wang, Z., Ding, J., An, X., Ingham, C.A., Shering, A.F., Wokosin, D., Ilijic, E., Sun, Z.,
Sampson, A.R., Mugnaini, E., Deutch, A.Y., Sesack, S.R., Arbuthnott, G.W., Surmeier,
D.J., 2006. Selective elimination of glutamatergic synapses on striatopallidal
neurons in Parkinson disease models. Nat. Neurosci. 9, 251–259.
de Paiva, A., Meunier, F.A., Molgó, J., Aoki, K.R., Dolly, J.O., 1999. Functional repair of
motor endplates after botulinum neurotoxin type A poisoning: biphasic switch of
synaptic activity between nerve sprouts and their parent terminals. Proc. Natl Acad.
Sci. USA 96, 3200–3205.
de Paola, V., Holtmaat, A., Knott, G., Song, S., Wilbrecht, L., Caroni, P., Svoboda, K., 2006.
Cell type-specific structural plasticity of axonal branches and boutons in the adult
neocortex. Neuron 49, 861–875.
297A. Wree et al. / Neurobiology of Disease 41 (2011) 291–298

Dong, M., Yeh, F., Tepp, W.H., Dean, C., Johnson, E.A., Janz, R., Chapman, E.R., 2006. SV2 is
the protein receptor for botulinum neurotoxin A. Science 312, 592–596.
Duvoisin, R.C., 1967. Cholinergic–anticholinergic antagonism in parkinsonism. Arch.
Neurol. 17, 124–136.
Ekdawi, M.Y., Fowke, R., 1966. A controlled trial of anti-Parkinson drugs in drug-
induced Parkinsonism. Br. J. Psychiatry 112, 633–666.
Emerich, D.F., Lindner, M.D., Winn, S.R., Chen, E.Y., Frydel, B.R., Kordower, J.H., 1996.
Implants of encapsulated human CNTF-producing fibroblasts prevent behavioral
deficits and striatal degeneration in a rodent model of Huntington's disease. J.
Neurosci. 16, 5168–5181.
Heckmann, M., Plewig, G., Group, Hyperhidrosis Study, 2005. Low-dose efficacy of
botulinum toxin A for axillary hyperhidrosis: a randomized, side-by-side, open-
label study. Arch. Dermatol. 141, 1255–1259.
Kaasinen, V., Ruottinen, H.M., Någren, K., Lehikoinen, P., Oikonen, V., Rinne, J.O., 2000.
Upregulation of putaminal dopamine D2 receptors in early Parkinson's disease: a
comparative PET study with [11C] raclopride and [11C]N-methylspiperone. J. Nucl.
Med. 41, 65–70.
Kaplan, H.A., Machover, S., Rabiner, A., 1954. A study of the effectiveness of drug
therapy in parkinsonism. J. Nerv. Ment. Dis. 119, 398–411.
Mahrhold, S., Rummel, A., Bigalke, H., Davletov, B., Binz, T., 2006. The synaptic vesicle
protein 2C mediates the uptake of botulinum neurotoxin A into phrenic nerves.
FEBS Lett. 580, 2011–2014.
Mamo, D.C., Sweet, R.A., Keshavan, M.S ., 1999. Managing antipsychotic-induced
parkinsonism. Drug Saf. 20, 269–275.
Meredith, G.E., Sonsalla, P.K., Chesselet, M.F., 2008. Animal models of Parkinson's
disease progression. Acta Neuropathol. 115, 385–398.
Mikula, G., Jaroszewicz, Z., Kolodziejczyk, A., Petelczyc, K., Sypek, M., 2007. Imaging
with extended focal depth by means of lenses with radial and angular modulation.
Opt. Express 15, 9184–9193.
Nikkhah, G., Bentlage, C., Cunningham, M.G., Björklund, A., 1994. Intranigral fetal
dopamine grafts induce behavioral compensation in the rat Parkinson model. J.
Neurosci. 14, 3449–3461.
Norman,A.B., Calderon,S.F., Giordano, M., Sanberg,P.R., 1988. A novelrotational behaviour
model for assessing the restructuring of striatal dopamine effector systems: are
transplants sensitive to peripherally acting drugs? Prog. Brain Res. 78, 61–67.
Obeso, J.A., Rodríguez-Oroz, M.C., Benitez-Temino, B., Blesa, F.J., Guridi, J., Marin,
C., Rodriguez, M., 2008. Functional organization of the basal ganglia:
therapeutic implications for Parkinson's disease. Mov. Disord. 23 (Suppl 3),
S548–S559.
Ordenstein, L., 1868. Sur la paralysie agitante et lasclérose en plaque géneralisée. Thèse
de Paris. Delahaye, Paris.
Paxinos, G., Watson, C., 2007. The Rat Brain in Stereotaxic Coordinates, sixth ed.
Academic Press, San Diego.
Pearce, L.B., First, E.R., MacCallum, R.D., Gupta, A., 1997. Pharmacologic characterization
of botulinum toxin for basic science and medicine. Toxicon 35, 1373–1412.
Pisani, A., Bernardi, G., Ding, J., Surmeier, D.J., 2007. Re-emergence of striatal cholinergic
interneurons in movement disorders. Trends Neurosci. 30, 545–553.
Reid, S.M., Johnstone, B.R., Westbury, C., Rawicki, B., Reddihough, D.S., 2008. Randomized
trial of botulinum toxin injections into the salivary glands to reduce drooling in
children with neurological disorders. Dev. Med. Child Neurol. 50, 123–128.
Rogawski, M.A., 2009. Convection-enhanced delivery in the treatment of epilepsy.
Neurotherapeutics 6, 344–351.
Schallert, T., Tillerson, J.L., 2000. Intervention strategies for degeneration of dopamine
neurons in parkinsonism. Optimizing behavioural assessment of outcome. In:
Emerich, D.F., Dean, R.L., Sandberg, P.R. (Eds.), Central Nervous System Diseases:
Innovative Animal Models from Lab to Clinic. Humana Press, Totowa, N.J., pp.
131–151.
Schallert, T., Whishaw, I.Q., Ramirez, V.D., Teitelbaum, P., 1978. Compulsive, abnormal
walking caused by anticholinergics in akinetic, 6-hydroxydopamine-treated rats.
Science 199, 1461–1463.
Schallert, T., De Ryck, M., Whishaw, I.Q., Ramirez, V.D., Teitelbaum, P., 1979. Excessive
bracing reactions and their control by atropine and L-DOPA in an animal analog of
Parkinsonism. Exp. Neurol. 64, 33–43.
Ungerstedt, U., Arbuthnott, G.W., 1970. Quantitative recording of rotational behavior in
rats after 6-hydroxy-dopamine lesions of the nigrostriatal dopamine system. Brain
Res. 24, 485–493.
Ungerstedt, U., Butcher, L.L., Butcher, S.G., Andén, N.E., Fuxe, K., 1969. Direct chemical
stimulation of dopaminergic mechanisms in the neostriatum of the rat. Brain Res.
14, 461–471.
Whitney, C.M., 2007. Medications for Parkinson's disease. Neurologist 13, 387–388.
298 A. Wree et al. / Neurobiology of Disease 41 (2011) 291–298