Wedekind et al. - page 1 of 35
Intrastriatal administration of botulinum neurotoxin A normalizes striatal D2R binding
and reduces striatal D1R binding in male hemiparkinsonian rats
Franziska Wedekind1, Angela Oskamp1, Markus Lang2, Alexander Hawlitschka3, Karl
Zilles4,5, Andreas Wree3*, Andreas Bauer1,6*
1
Institute of Neuroscience and Medicine, INM-2, Research Center Jülich, Jülich, Germany
2
Institute of Neuroscience and Medicine, INM-5, Research Center Jülich, Jülich, Germany
3
Institute of Anatomy, Rostock University Medical Center, Rostock, Germany
4
Institute of Neuroscience and Medicine, INM-1, Research Center Jülich, Jülich, Germany
5
Department of Psychiatry, Psychotherapy and Psychosomatics, RWTH Aachen, Aachen,
and JARA - Translational Brain Medicine, Aachen, Germany
6
Department of Neurology, Medical Faculty, Heinrich-Heine-University Düsseldorf,
Düsseldorf, Germany
* Andreas Wree and Andreas Bauer contributed equally to this article.
Keywords: Parkinson disease, 6-hydroxydopamine, botulinum neurotoxin A, dopamine
receptors, positron emission tomography in vitro receptor autoradiography
Correspondence: Franziska Wedekind, Institute of Neuroscience and Medicine, INM-2,
Research Center Jülich, Leo-Brandt-Straße, 52425 Jülich, Germany. Email: f.wedekind@fzjuelich.de
Wedekind et al. - page 2 of 35
Abstract
Cerebral administration of botulinum neurotoxin A (BoNT-A) has been shown to improve
disease-specific motor behavior in a rat model of Parkinson disease (PD). Since the
dopaminergic system of the basal ganglia fundamentally contributes to motor function, we
investigated the impact of BoNT-A on striatal dopamine receptor expression using in vitro
and in vivo imaging techniques (positron emission tomography and quantitative
autoradiography, respectively).
17 male Wistar rats were unilaterally lesioned with 6-hydoxydopamine (6-OHDA) and
assigned to two treatment groups seven weeks later: 10 rats were treated ipsilaterally with an
intrastriatal injection of 1 ng BoNT-A, while the others received vehicle (n = 7). All animals
were tested for asymmetric motor behavior (apomorphine-induced rotations and forelimb
usage) and for striatal expression of dopamine receptors and transporters (D1R, D2R and
DAT). The striatal D2R availability was also quantified longitudinally (1.5, 3, and 5 months
after intervention) in 5 animals per treatment group.
The 6-OHDA lesion alone induced a unilateral PD-like phenotype and a 13% increase of
striatal D2R. BoNT-A treatment reduced the asymmetry in both apomorphine-induced
rotational behavior and D2R expression, with the latter returning to normal values 5 months
after intervention. D1R expression was significantly reduced while DAT concentrations
showed no alteration. Independent of the treatment, higher interhemispheric symmetry in
raclopride binding to D2R was generally associated with reduced forelimb akinesia. Our
findings indicate that striatal BoNT-A treatment diminishes motor impairment and induces
changes in D1 and D2 binding site density in the 6-OHDA rat model of PD.
Wedekind et al. - page 3 of 35
Significance
The pharmacological treatment of Parkinson disease (PD) is characterized by complex and
disabling side effects. The reduction of dominant striatal acetylcholine by cerebral application
of botulinum neurotoxin A (BoNT-A) proved to be promising in preclinical PD research. We
demonstrate that intrastriatal BoNT-A administration normalizes D2 receptor binding in
hemiparkinsonian rats, in correlation to behavioral improvements. Additionally, striatal D1
receptor binding is reduced, while presynaptic dopamine transporter expression is not
affected. Our longitudinal investigation on the dopamine system points to a complex pattern
of reorganization and provides evidence for BoNT-A as a potential alternative or amendment
to dopaminergic pharmacotherapy in PD.
Wedekind et al. - page 4 of 35
1) Introduction
Parkinson disease (PD) is caused by degeneration of neuronal, particularly dopaminergic cells
in the substantia nigra pars compacta (Dauer and Przedborski, 2003). The loss of
dopaminergic input interferes with the overall transmitter availibility in the striatum leading to
an imbalance of transmitter systems including glutamate, gamma-aminobutyric acid, and
acetylcholine (ACh) (Calabresi et al., 2006). The strong branching of striatal cholinergic
interneurons (ChIs), the large population of medium spiny neurons (MSNs), and the high
expression of dopamine receptors underline the particular importance of the cholinergic and
dopaminergic transmitter system (Lim et al., 2014). They modulate each other directly via
metabotropic muscarinic receptors (on MSNs) and dopaminergic receptors (on ChIs) and
indirectly via autoreceptor activation and altered glutamatergic and GABAergic transmission
(Kljakic et al., 2017). In the case of dopamine depletion as observed in PD, reduced
cholinergic autoreceptor function contribute to the abundance in ACh (Ding et al., 2006).
Therefore, the increased activation of metabotropic acetylcholine receptors (AChRs)
(accompanied by elevated GABAergic und changed glutamatergic receptor activation) alters
the signal transduction of MSNs as the main striatal output, which is already less modulated
by dopamine. Besides strategies to compensate dopamine deficits, attempts have been made
to restore the transmitter balance – for instance, by antagonizing ACh. As an example, it was
recently shown that the ablation of ChIs, the main source of striatal ACh (Oldenburg and
Ding, 2011), diminished the risk of induced dyskinesia in L-DOPA-treated hemiparkinson
mice (Won et al., 2014).
A promising strategy to functionally antagonize striatal cholinergic neurotransmission is local
application of botulinum neurotoxin A (BoNT-A), which blocks the release from presynaptic
terminals via degradation of the synaptosomal–associated proteins of 25 kDa (Dong et al.,
2006; Verderio et al., 2006). BoNT-A is already in use, particularly in symptomatic therapies
Wedekind et al. - page 5 of 35
of drooling in PD (Egevad et al., 2014) and in disorders of cholinergic hyperactivity (e.g.
dystonia, hyperhidrosis, strabismus, blepharospasm) (Lim and Seet, 2010).
We have previously investigated a potential therapeutic application of BoNT-A in the 6hydroxydopamine (6-OHDA) model of PD (Wree et al., 2011). In this model, unilateral
injection of 6-OHDA into the medial forebrain bundle (MFB) leads to rapid degeneration of
nigrostriatal neurons (Meredith et al., 2008), asymmetry in forelimb use (Schallert et al.,
2000), and drug-induced rotations (Ungerstedt and Arbuthnott, 1970). On the molecular level,
the loss of nigrostriatal dopamine input causes an imbalance of dopaminergic and cholinergic
neurotransmission in the caudate-putamen (CPu). The degeneration of nigrostriatal terminals
is also reflected in reduced concentrations of proteins associated with dopamine production
and transport (tyrosine hydroxylase and dopamine transporters [DATs] (Sun et al., 2011)),
whereas long-lasting impacts of 6-OHDA on proteins related to ACh is not yet clear. For
instance, while Ma et al. (2014) reported a decrease in striatal choline acetyltransferase
concentration following 6-OHDA infusions, no changes could be observed by Pezzi et al.
(2005). We used the 6-OHDA model to test the efficacy of a single intrastriatal administration
of BoNT-A. We found significant improvements in motor impairment for up to 6 months
after BoNT-A application (Wree et al., 2011). Histologically, a remarkable number of
varicosities were observed at neuronal processes, while the overall quantity of striatal
neurons, ChIs, and tyrosine hydroxylase levels remained unchanged (Itakura et al., 2014;
Mehlan et al., 2016; Wree et al., 2011). The number of varicosities declined over time, while
remaining varicosities exhibited permanently elevated volumes (Mehlan et al., 2016). There
were no changes in cognitive abilities or striatal volume at the BoNT-A injection site
(Antipova et al., 2013; Holzmann et al., 2012). The present study focuses on the molecular
changes that underlie the observed improved motor behavior. Downregulation of
pathologically increased dopamine D2 receptor (D2R) concentrations is often considered as
Wedekind et al. - page 6 of 35
hint for a promising treatment strategy. We hypothesize that the positive impact of BoNT-A
on motor impairments in the unilateral 6-OHDA rat model will be mirrored by more balanced
dopamine receptor concentrations (especially for D2R) in the CPu, since motor actions are
regulated by basal ganglia circuits in which striatal output function is strongly modulated by
dopamine. Moreover, we assume that the long-lasting impact on behavior improvement will
also be accompanied similarly over time by changes in dopamine receptor concentrations. We
designed a longitudinal behavioral and imaging study to investigate components of the
dopaminergic system. 6-OHDA rats were treated either with BoNT-A or vehicle and
subsequently examined with regard to apomorphine-induced rotation behavior, forelimb use,
and dopamine D1 receptor (D1R), D2R and DAT availability with longitudinal positron
emission tomography (PET) and quantitative in vitro autoradiography.
Wedekind et al. - page 7 of 35
2) Materials and Methods
2.1) Animals
Seventeen adult male Wistar rats (Charles River WIGA, Germany, RRID: RGD_737929)
with an age of around 2 months were housed under standard conditions (22 °C ± 2 °C; 12 h
light/dark cycle) with free access to food and water. The animals were randomly assigned to
two treatment groups (Figure 1):
1. Sham treatment (n = 7): All animals underwent unilateral lesioning, intrastriatal vehicle
administration, behavioral testing and in vitro examinations (histology and autoradiography).
A subset of 5 rats were additionally studied by repeated PET.
2. BoNT-A treatment (n = 10): All animals underwent unilateral lesioning, intrastriatal BoNTA
administration,
behavioral
testing
and
in
vitro
examinations
(histology
and
autoradiography). A subset of 5 rats were additionally studied by repeated PET.
All procedures were conducted according to protocols approved by the local animal welfare
committees (LALLF M-V Rostock and LANUV NRW Recklinghausen, Germany, no.
LALLF M-V/TSD/7221.3-1.2-046/10 (1.1-019/08))
2.2) Unilateral lesioning and treatment
To produce unilateral degeneration of dopaminergic neurons and hemiparkinsonian
symptoms, rats weighing 280 ± 12 g (n = 16; one animal’s record was lost) were lesioned by
injection of 6-OHDA (Sigma-Aldrich, Taufkirchen, Germany; product number H4381) into
the right MFB: after intraperitoneal injection of ketamine (50 mg/kg body weight) and
xylazine (4 mg/kg body weight) for anesthesia, 4 µl of 6-OHDA (24 µg) dissolved in 0.1 M
citrate buffer was delivered over 4 min via a 26-gauge 5-µl Hamilton syringe with the
following coordinates referenced from bregma: anterior-posterior = -2.3 mm, lateral = -1.5
mm and ventral = -9.0 mm (Paxinos and Watson, 2007).
Wedekind et al. - page 8 of 35
Seven weeks later, rats (356 ± 30 g) received either 1 ng BoNT-A (n = 10) or vehicle (n = 7)
into the right CPu; after ketamine-xylazine anesthesia, rats were treated with two individual
injections of 1-µl volume containing either BoNT-A (List, USA, purchased via Quadratech,
UK) or 0.1% bovine serum albumin in phosphate-buffered saline (“vehicle”). Each
intrastriatal injection was delivered over 4 min and according to the following coordinates
(with reference to bregma): anterior = 1.3/-0.4 mm, lateral = -2.6/-3.6 mm and ventral = -5.5
mm (Paxinos and Watson, 2007).
2.3) Behavioral testing
After successful unilateral 6-OHDA-induced dopamine depletion, animals tend to turn
contralaterally to the lesioned side after dopaminergic stimulation (Ungerstedt and
Arbuthnott, 1970). Here, 2 weeks before treatment (thus, 5 weeks after 6-OHDA lesioning)
and 2 weeks after treatment, all animals were tested on rotational behavior (net contraversive
turns per minute) after subcutaneous injection of 0.25 mg/kg body weight apomorphine
(Teclapharm, Lüneburg, Germany). Rotations were measured over 40 min, using a selfconstructed automated rotometry device (modified according to Ungerstedt and Arbuthnott
(1970)). Rotations were only analyzed in case of complete 360° turns in the left or right
direction. All rats were also tested for non-drug-induced forelimb use in the same period (2
weeks before and after treatment) by applying the cylinder test (Schallert et al., 2000): In
brief, animals were placed into a transparent cylinder of 20-cm diameter and 30-cm height
and videotaped (Sony), with the recordings being analyzed for the number of forelimb-to-wall
contacts by a person blinded for animal condition. Out of 30 subsequent contacts, the quotient
of left-to-right (L/R) forelimb use was calculated (around 1 = well-balanced use, < 1 =
asymmetric use).
2.4) In vivo [11C]raclopride PET
Wedekind et al. - page 9 of 35
D2R availability was assessed by longitudinal [11C]raclopride PET in a subset of animals at
1.5, 3, and 5 months after treatment. Two animals (one BoNT-A and one sham-treated rat)
were measured simultaneously: Each rat was anesthetized with around 1.6% isoflurane
(ReboPharm, Bocholt, Germany) vaporized in 1.25 L/min oxygen gas and placed in a supine
position (opposite each other, with the heads aligned, but radial shifted to the center of field of
view) in the INVEON dedicated PET scanner (Siemens, Knoxville, TN, USA). Anesthesia
duration was longer in the first scan session (F1.22,9.78 = 8.15; n = 10 per session; p = .014 with
simple contrasts F1,8 ≥ 8.43; p ≤ .02), but not different between groups at any scan session.
Attenuation correction was performed based on a
57
Co-transmission scan (20 min). Animals
received an intravenous injection of [11C]raclopride (over 1 min), either at the beginning of
emission scan (first rat) or 3 min later (second rat). Alternating the groups for being injected
first, times of injection (Ti) did not differ between sham and BoNT-A-treated animals and
took place at 11:28 a.m. ± 0:22 (10 rats; 3 time-points per rat). [11C]raclopride was prepared
as described previously (Schott et al., 2008) with a radiochemical purity > 95% and a mean
specific activity (SA) = 73 ± 34 GBq/µmol (n = 15) at the end of synthesis. SA did not differ
between groups, but was higher at the second scan session (59.85 ± 5.40 GBq/µmol [55.68,
64.03] vs. 30.12 ± 20.43 GBq/µmol [14.37, 45.88] at the first session and 27.02 ± 6.95
GBq/µmol [21.66, 32.38] at the last session with F1.15,9.17 = 14.79; n = 10 per session; p = .003
and repeated contrasts F1,8 ≥ 16.10; p ≤ .004). More PET parameters are listed in Table 1.
PET data were acquired in list mode for 70 min, normalized and scatter corrected. A dynamic
histogram sequence of 6× 10 s, 3× 20 s, 8× 60 s, 4× 300 s and 3× 600 s (24 frames, starting at
Ti (Alexoff et al., 2004) was applied. Images were reconstructed 2D as filtered backprojection and with a voxel size of 0.7764 x 0.7764 x 0.796 mm3 (matrix size 128 x 128 x
159). Volumes of the first 17 frames (first 10 min post injection, p. i.) and of all 24 frames
were integrated for the coherent registration to the Schiffer’s rat brain template, implemented
Wedekind et al. - page 10 of 35
in PMOD software 3.4 (PMOD Technologies, Zurich, Switzerland). The resultant
transformation was applied to all individual frames producing reasonable volumes of interests
such as 43.55 mm3 (CPu) and 196.98 mm3 (cerebellum) (Mengler et al., 2014). Regional
time-activity curves (TACs) were calculated and striatal binding potentials (BPNDs), defined
as ratio of specifically bound and nondisplaceable radioligand in tissue at equilibrium, were
generated separately for each CPu and for all three consecutive investigations. Modeling
based on the simplified reference tissue model (SRTM), with cerebellum as reference and
Poisson weighting on measures (Lammertsma and Hume, 1996). For comparison, BPNDs
derived from the late phase (starting at 20 min p. i.), with fixed tissue-to-plasma clearance (k2’
= 0.24/min) and referenced to the cerebellum, were determined via Logan reference modeling
(Lettfuss et al., 2012).
2.5) Tissue preparation for in vitro studies
All rats were sacrificed by decapitation under isoflurane anesthesia 5 months after treatment
(rats which received a PET scan were decapitated immediately after the last scan). Each brain
was dissected quickly (5 ± 1 min), snap frozen in 2-methylbutane (Sigma-Aldrich,
Taufkirchen, Germany; product number 320404), stored at -80 °C and cut into 20 µm coronal
cryostat slices of CPu and substantia nigra. The sections (thaw-mounted onto silane-coated
slides) were stored at -80 °C until in vitro experiments were performed.
2.6) In vitro [3H]SCH23390, [3H]raclopride and [3H]mazindol autoradiography
For D1R binding, striatal sections were pre-treated with 50 mM Tris-HCl buffer (pH 7.4)
containing 5 mM KCl, 2 mM CaCl2 (Merck, Darmstadt, Germany; product number 102382),
1 mM MgCl2 (Merck, Darmstadt, Germany; product number 105832) and 120 mM NaCl
(Merck, Darmstadt, Germany; product number 106404) for 20 min. Afterwards, binding sites
were labeled with 1.67 nM [3H]SCH23390 (SA = 2978.5 GBq/mmol) in buffer supplemented
Wedekind et al. - page 11 of 35
with 1 µM mianserin (90 min). For determination of non-specific binding, adjacent slices
were incubated likewise in parallel, but in the presence of the D1R antagonist SKF 83566 (1
µM). Finally, sections were washed in ice-cold buffer (2x 10 min) and quickly dipped into
ice-cold deionized water.
In a similar manner, striatal slices were pre-incubated in 50 mM Tris-HCl buffer (pH 7.4)
containing 0.1% ascorbic acid and 150 mM NaCl (Merck, Darmstadt, Germany; product
number 106404) for 20 min, followed by incubation in buffer supplemented with 0.57 nM
[3H]raclopride (SA = 2753 GBq/mmol) for 45 min to label D2R. Adjacent sections were
investigated in the same way, but in the presence of the D2R antagonist (+)-butaclamol (1
µM) for determination of non-specific binding. Slides were rinsed in ice-cold buffer (6x 1
min) and quickly dipped into ice-cold deionized water.
For DAT binding, striatal slices were incubated in 50 mM Tris-HCl buffer (pH 7.9) with 120
mM NaCl (Merck, Darmstadt, Germany; product number 106404) and 5 mM KCl (30 min)
and with 300 mM NaCl, 5 mM KCl, 300 nM desipramine and 3.98 nM [3H]mazindol (SA =
765.9 GBq/mmol) for 60 min. Sections were washed in buffer (2x 3 min) and quickly dipped
in deionized water. All steps were performed on ice and in parallel with the dopamine
reuptake inhibitor nomifensine (100 µM during main incubation) to determine non-specific
binding on adjacent slices.
After binding procedure, slides were dried under a cold ventilator stream and exposed
together with [3H] standards of known radioactivity to [3H]-sensitive films (Carestream
Health Inc., Rochester, NJ, USA) (12 - 15 weeks). Following film processing and
digitalization, grey values were non-linearly transformed into concentrations of radioactivity
as described previously (Zilles et al., 2004). Receptor and transporter concentrations (fmol/mg
protein) were calculated using the corresponding SA, ligand concentration and dissociation
constant (KD; 1.67 nM, 0.55 nM and 4 nM for [3H]SCH23390, [3H]raclopride and
Wedekind et al. - page 12 of 35
[3H]mazindol, respectively). Binding was calculated and displayed in color-coded linearized
images using Axiovision (Zeiss, Göttingen, Germany, RRID: SCR_002677) and Matlab
(Mathworks, Aachen, Germany, RRID: SCR_001622). Per brain, dorsal left and right CPu
were defined as regions of interest (ROI) in four to five sections (including one adjacent
section with non-specific binding), based on a standard rat atlas (Paxinos and Watson, 2004)
and cell body staining. Binding was averaged for each hemisphere and calculated as specific
binding, which was finally averaged for each treatment.
Since [3H]SCH23390 and [3H]raclopride also bind to other receptor subtypes (e.g. to D5R and
to D3R, respectively), binding experiments were optimized for D1R and D2R: they were
performed with radioligand concentrations around the corresponding KDs, which were
determined in saturation experiments. In addition, specific binding was calculated by
subtracting unspecific binding (<13% and 43% of the total binding in receptor and transporter
binding assays) from total binding. Accordingly, the data presented in this study reflect D1R
and D2R concentrations rather than D1/5R and D2/3R binding sites.
Nonradioactive chemicals and tritiated radioligands were purchased from Sigma-Aldrich
(Taufkirchen, Germany; product numbers T2913, P9333, M2525, S110, A5960, D033,
D3900, N1530), if not noted otherwise, and Perkin Elmer (Rodgau, Germany; product
numbers NET-930, NET975, NET816), respectively.
2.7) Histology
For optimal ROI definition, histology was performed on corresponding brain sections, which
were fixated in neutral-buffered formalin-solution (30 min), washed in deionized water (2x 5
min) and stained with cresyl violet (30 min) for Nissl substance. Additionally, cell bodies or
myelin were stained in alternating formalin-fixated sections following the protocols of Merker
and Gallyas, respectively (Gallyas, 1979; Merker, 1983). In brief, after several pre-treatment
Wedekind et al. - page 13 of 35
and washing steps, slides were transferred to developer solutions containing silver nitrate (70
min for cell body or 30 min for myelin staining, respectively). Sections were washed, fixated
in a T-max fixation solution (Kodak, Germany; product number 5089198) and washed again.
All sections were dehydrated in ascending solutions of propanol (70% - 100%), cleared in
XEM-200 (2x 10 min; Vogel, Germany, purchased via Diatec, Bamberg, Germany; product
number 1-019614), mounted in DPX (Sigma-Aldrich, Taufkirchen, Germany; product number
44581) and dried until digital processing using Axiovision (Zeiss, Göttingen, Germany,
RRID: SCR_002677).
Pretreatment and washing steps in silver-based cell body and myelin stainings were as
follows:
Pretreatment: overnight incubation in formic acid and hydrogen peroxide (cell bodies) or in
pyridine and acetic anhydride (Sigma-Aldrich; Taufkirchen, Germany; product number
822278) (myelin), respectively. Additional incubation in ammoniacal silver nitrate for myelin
staining only (30 min).
Washing: rinse in demineralized water. Before and after incubation in the developer solutions,
washing was performed with 1% acetic acid.
Chemicals were obtained from Merck (Darmstadt, Germany; product numbers 104002,
105235, 1015120, 1009966, 100264, 108597, 109728, 100062) if not noted otherwise.
2.8) Statistics
Values are expressed as mean ± standard deviation (SD) and statistical analyses were
performed using IBM SPSS Statistics 22 (SPSS Inc., Chicago, IL, USA, RRID:
SCR_002865) with p < .05 being considered significant. Where appropriate, 95% confidence
intervals are displayed in squared parentheses. Gaussian distribution was checked inspecting
skewness and kurtosis (raw data and zvalues) and the Shapiro-Wilk normality test.
Wedekind et al. - page 14 of 35
Homogeneity of within-variances was revised with Levene test and Hartley Fmax < critical
value. Data on forelimb usage was compared between groups with 1-way analysis of
covariance (ANCOVA) using the pretreatment scores as covariate, with regression slopes and
covariate (unpaired 2-tailed Student t test) being equal across groups. Data on rotational
behaviour displayed unequal regression slopes (“treatment*covariate” F1,13 = 9.51; n = 7 – 10;
p = .009), hence a general linear model (GLM) analysis was reported. Absolute data from
binding experiments were compared between groups by a mixed repeated measure analysis of
variance (RM-ANOVA with ‘hemisphere’ as within-factor for autoradiographic data, or
‘hemisphere’ and ‘scan’ as within-factors for PET data, respectively). Homogeneity of
between-subject variance was tested and corrected according to Mauchly sphericity test. RMANOVA was followed either by contrasts (scan parameters) or by 2-tailed Student t tests with
Bonferroni-correction (binding data), respectively. The p values were multiplied by 3 due to
paired (within-subject comparison in each group) and unpaired (between-group comparison of
contralateral hemispheres) Student t testing. Binding data, expressed as percentage of
contralateral binding, was compared by unpaired 2-tailed Student t test for each scan session
and target separately. Parametric analyses of total correlation (thus neglecting the separation
into treatment groups) are performed, reporting Pearson r.
Wedekind et al. - page 15 of 35
3) Results
Effects of intrastriatal BoNT-A treatment on 6-OHDA rats were investigated longitudinally
with behavioral testing and PET, and invitro with autoradiography and histological
techniques. The time course of experimental procedures is depicted in Figure 1.
3.1) Unilateral 6-OHDA lesion
All investigated animals (n = 17) underwent unilateral dopamine depletion by application of 4
µl 6-OHDA into the right MFB. Individual lesion effects were verified both in vivo by
assessing apomorphine-induced rotations and spontaneous forelimb-usage 5 weeks after
lesioning and in vitro by DAT autoradiography. It is to note that tyrosine hydroxylase
quantification was not feasible in non-fixated samples and thus outcome of behavioral tests
and quantification of striatal DAT were used to determine nigrostriatal degeneration (Heuer et
al., 2012; Liu et al., 2014; Molinet-Dronda et al., 2015).
Behavioral testing showed typical asymmetrical rotational movements (8 ± 2.72 contraversive
turns/min after apomorphine stimulation; n = 17) and forelimb usage (L/R ratio of 0.36 ±
0.36; n = 17). There were no behavioral differences between animals assigned to treatment
and sham groups. After completion of the in vivo investigations, all brains underwent in vitro
DAT autoradiography in order to quantify the lesion effect and the impact of BoNT-A on
presynaptic DAT. All lesioned brains displayed a strong ipsilateral reduction of DAT (90.01%
± 8.89%; n = 17) without any difference between BoNT-A and sham-treated animals.
Furthermore, there was a massive reduction of silver-stained Nissl-positive cell bodies in the
ipsilateral substantia nigra (Figure 2A-C).
3.2) BoNT-A treatment
The 6-OHDA lesioned rats were assigned to two groups with either BoNT-A (n = 10) or sham
(n = 7) treatment. There were no differences between these groups with regard to 6-OHDA
Wedekind et al. - page 16 of 35
lesion effects and body weight. All animals underwent behavioral testing before and after
treatment (Figure 1). Before treatment, 6-OHDA animals showed turns in opposite direction
to the lesioned side and prevalent right forelimb usage. There were no differences between the
treatment groups. After BoNT-A treatment, scores for rotational behavior were significantly
different with regard to reduced asymmetry (1.32 ± 1.05 vs. 6.78 ± 3.72 contraversive
turns/min, GLM with F1,14 = 21.21; n = 7 - 10; p = .0004). In contrast, BoNT-A did not affect
forelimb preference, although differences in L/R usage tend to be diminished (0.62 ± 0.37 vs.
0.24 ± 0.36) (Figure 2C).
3.3) Longitudinal PET studies
Pairs of BoNT-A and sham-treated 6-OHDA rats (n = 5 per group) were simultaneously
investigated with [11C]raclopride PET at 1.5, 3, and 5 months after treatment. Scan parameters
and striatal tracer delivery (referenced to cerebellar influx, R1; data not shown) did not differ
between both groups (Figure 1 and Table 1).
There was a constant trend towards higher [11C]raclopride binding to striatal D2R after 6OHDA-lesioning (17% to 25.7% in ipsilateral CPu; paired Student t tests with t4 ≥ -2.57; n =
5; p = .062 to.114) which tended to return to normal values after BoNT-A administration (8.2% to 1% in ipsilateral CPu) (Figure 3 and Table 2). Mean [11C]raclopride BPND values
were not significantly different (even when they were analyzed separately for each single scan
session). However, percent changes in [11C]raclopride BPNDs became significantly different
between groups at 5 months after treatment (17% in sham-treated vs. -8.2% in BoNT-Atreated animals, unpaired Student t test with t8 = 2.37; n = 5; p = .045). Rats with an ipsilateral
increase in interstriatal D2R binding show higher forelimb asymmetry (Pearson r = -0.84, n =
10, p = .002 for the first scan; r = -0.733, n = 10, p = .016 for the second scan and r = -0.773,
n = 10, p = .009 for the third PET scan; Figure 4).
3.4) In vitro autoradiography
Wedekind et al. - page 17 of 35
Striatal D1R and D2R binding sites were studied with quantitative autoradiography in all
animals 5 months after treatment (Figure 5 and Table 3).
There was a significant decrease in the mean ipsilateral D1R concentrations in BoNT-Atreated rats (mixed 1-way RM-ANOVA with “hemisphere*treatment” F1,15 = 11.51; n = 7 10; p = .004 and Bonferroni-corrected Student t test, p < .0001) in contrast to sham-operated
animals that showed no changes 5 months after treatment. Mean striatal [3H]raclopride
binding to D2R was fairly balanced between hemispheres, while it was increased in the
lesioned CPu of sham-treated animals (“hemisphere*treatment” F1,15 = 11.61; n = 7 - 10; p =
.004 with Bonferroni-adjusted Student t test, p = .039). All sham-treated animals (except for
the one with decreased ipsilateral [11C]raclopride binding in PET and smaller reduction in
DAT concentration) showed increased D2R concentrations.
BoNT-A treatment increased D1R and reduced D2R percentage differences in striatal binding
(unpaired Student t test with t15 = 3.17; n = 7 - 10; p = .006 and t15 = 3.49; n = 7 - 10; p =
.003, respectively). For in vitro raclopride (D2R) data, there was an excellent correlation to in
vivo interhemispheric changes, measured by PET 5 months after treatment (Pearson r =
0.964, n = 10, p < .0001).
Wedekind et al. - page 18 of 35
4) Discussion
In the present study we explored the longitudinal effects of a BoNT-A application in 6-OHDA
lesioned rats on striatal markers of the dopamine system. PET and quantitative in vitro
autoradiography showed that SCH23390 binding to D1R was reduced, while raclopride
binding to D2R returned to normal 5 months after BoNT-A treatment. Treatment also
alleviated turning asymmetry, and individuals with reduced interhemispheric differences in
raclopride binding displayed improved symmetry in forelimb use, although BoNT-A was
without significant effect on this spontaneous behavior. This is to our knowledge the first
study demonstrating that the beneficial effects of BoNT-A on motor behavior are
accompanied by a recovery of striatal D2R binding and a reduction in D1R binding. Our
findings point to a potential treatment strategy in PD that is not primarily dopamine based but
instead initiates a complex pattern of chemical and functional reorganization.
A broad range of short and long-term side effects limits the use of anticholinergic medications
in PD (Calabresi et al., 2006; Katzenschlager et al., 2003; Lim et al., 2014). BoNT-A injected
into the ipsilateral striatum of hemiparkinsonian rats reduces hypercholinism directly at the
site of action (Holzmann et al., 2012) and improves motor function for up to 6 months without
inflammatory reactions or side effects on cognitive behavior (Antipova et al., 2013; Wree et
al., 2011). To understand the molecular mechanisms underlying these actions of BoNT-A, we
investigated postsynaptic dopamine receptors which are of crucial importance for basal
ganglia circuitry and connectivity.
Our findings indicate that BoNT-A affects striatal D1 and D2 binding sites similarly, since
both were reduced 5 months after intervention. It is unlikely that this downregulation is
caused by neurotoxic effects as numbers of striatal neurons are unchanged up to 12 months
after treatment (Antipova et al., 2013). Moreover, it appears implausible that alleviation in 6OHDA-induced D2R binding is independent of BoNT-A, because interhemispheric
Wedekind et al. - page 19 of 35
differences have been shown to persist at later stages (Blunt et al., 1992; Sun et al., 2011) and
were also detected in the sham-treated animals here. Likewise, results from PET
investigations on patients with PD and parkinsonian monkeys suggest that the downregulation
of pathologically increased D2R concentrations is most probably caused by long-term
treatment with dopamine agonists rather than by disease progression (Doudet et al., 2002a;
Seeman and Niznik, 1990). In an earlier study it has also been shown that blockade of
muscarinic interactions leads to reduced [11C]raclopride binding in monkeys (Tsukada et al.,
2000), we therefore assume that BoNT-A treatment normalizes striatal raclopride binding by
an inhibition of ACh neurotransmission. Whether this impact of BoNT-A on D2R availability
is time dependent has yet to be clarified, since the variability in our PET data is comparable to
that described in the literature (Lettfuss et al., 2012), but the lesion-induced differences may
be too small to evaluate treatment effects (differences between sham and BoNT-A treatment
did not reach significance in the microPET experiments at 1.5 and 3 months).
Regarding D1R and their striatal expression during PD, data are conflicting, showing either no
or bidirectional changes in untreated and treated conditions (Blunt et al., 1992; Fornaretto et
al., 1993; Guigoni et al., 2005; Hurley and Jenner, 2006). Down-regulation of D1R mRNA
and receptor protein by 6-OHDA intervention was small, was only found in animals with
medium-sized lesions, and unaffected by intrastriatal implantation of dopamine cells (Blunt et
al., 1992; Da Cunha et al., 2008). We therefore hypothesize that, in the present study, the
reduction in D1R concentrations is attributable to BoNT-A instead of 6-OHDA intervention.
On the other hand, no change in striatal D1R expression was observed in mice lacking the
muscarinic M4 AChR (M4 mAChR) (Schmidt et al., 2011), despite the reciprocal involvement
of both transmitter targets in striatonigral signaling (Mao et al., 2016) and transmitter release
(Tzavara et al., 2004). Thus, whether M4 mAChRs are directly involved in the effect of
BoNT-A on striatal D1R needs to be proven.
Wedekind et al. - page 20 of 35
Changes in dopamine receptor concentrations as a function of transmitter concentration
should be addressed by microdialysis and by multireceptor (especially muscarinic receptor)
studies (the latter is currently under investigation within a cooperative study). Furthermore
longer time periods should be included, as BoNT-A exhibits its enzymatic activity up to 10
months in primary rat spinal neurons (Whitemarsh et al., 2014), and BoNT-A-induced
varicosities on cholinergic neurons were observed up to 12 months (Hawlitschka et al., 2013;
Mehlan et al., 2016). Long-lasting effects ≥ 5 months might indicate a persistent disruption of
ACh release by BoNT-A or a delayed adaption of receptor expression to changed ACh levels.
Preclinical studies on rodents illustrate that unbalanced limb use and turning behavior are
reduced when abating the predominance of ACh by nigrostriatal intervention (Maurice et al.,
2015; Won et al., 2014). Blockade of M4 mAChR, located on D1 MSNs (Lim et al., 2014),
alleviates the akinetic dysfunction in a 6-OHDA lesion model (Ztaou et al., 2016). AChinduced changes in electrophysiology of D2 MSNs (Day et al., 2008) provoke PD-like
akinesia in mice (Kondabolu et al., 2016; Maurice et al., 2015). In the present study, we
detected dopamine receptor changes after BoNT-A treatment, along with alleviated turning
asymmetry described in the literature (Antipova et al., 2013; Wree et al., 2011). We
confirmed that BoNT-A dosage was insufficient to improve forelimb deficits (Wree et al.,
2011), but individual forelimb akinesia correlated to interhemispheric differences in
raclopride binding during the entire time course. These findings raise the question of whether
there is a causal link between ACh levels, behavioral alterations, and changes in dopamine
receptor binding. Although the link between forelimb asymmetry and imbalance in striatal
dopamine receptor concentration is not yet clear, former studies have shown that turning
behavior of 6-OHDA lesioned rats was linked to changes in receptor expression. An increase
in D2R concentration was detected in contraversive turning rats, and a decrease in D1R
expression was found in ipsiversive turning rats 1 week after 6-OHDA lesion (Da Cunha et
Wedekind et al. - page 21 of 35
al., 2008). Doudet et al. (2002a) showed that a D2R increase is only present in symptomatic,
while D1R downregulation was detected in both, symptomatic and asymptomatic MPTPinduced, untreated parkinsonian monkeys (Doudet et al., 2002b). Blunt et al. (1992) report
that in rats a 6-OHDA-induced D2R upregulation was reversed after intrastriatal implantation
of fetal dopamine cells, while apomorphine-induced asymmetry in rotation was not
completely normalized. Zhou et al. (1996) showed that direct inhibition of striatal D2R
mRNA in 6-OHDA lesioned mice temporarily alleviates drug-induced imbalanced turning
response independent of mAChR stimulation. Based on a complex underlying circuitry, we
assume that blocked ACh release improves asymmetric rotational behavior in part via M4
mAChR on D1 MSNs, possibly by increasing the number of ipsiversive turns, which in turn
might be mirrored in reduced D1R concentrations. It would be desirable to investigate BoNTA effects on dopamine receptors along behavioral assessment, to illuminate the exact
contribution of both receptor subtypes to long-term behavioral improvements. It should be
noted that although the unilateral 6-OHDA model cannot be transferred directly to PD (PD is
not 1-sided, PD symptoms occur already with smaller nigrostriatal damage, beneficial effects
in the model might be restricted in their efficacy in patients,…), it remains reasonable to
obtain an initial estimation of the antiparkinsonian potency of new drugs because of the direct,
quantifiable link between nigrostriatal neuron loss and behavior (Przedborski, 2017).
In conclusion, our data support previous reports on behavioral improvements after BoNT-A
treatment and indicate that BoNT-A leads to receptor-dependent effects on the nigrostriatal
dopamine system in the 6-OHDA lesion model of PD. D2R levels, which were ipsilaterally
increased by 13% after 6-OHDA lesion, returned to normal at 5 months after striatal BoNT-A
treatment. D1Rs were significantly reduced, while DAT showed no alteration. Rotational
behavior was alleviated, and individual weak asymmetric forelimb use was correlated with
reduced interhemispheric differences in raclopride binding to D2R. These findings on the
Wedekind et al. - page 22 of 35
dopamine receptor system point to a complex pattern of reorganization following striatal
BoNT-A application and provide evidence for BoNT-A potentials in the treatment of PD.
However, further experiments are required to provide direct causal evidence for receptor
changes in association to behavioral alterations.
Wedekind et al. - page 23 of 35
Acknowledgements
The authors thank U. Haase, S. Krause, T. Kroll, S. Lehmann, M. Vögeling and F. Winzer for
excellent technical assistance, S. Beer for PET scanner quality assurance, and D. Nabbi, D.
Schneider, S. Laskowski, and D. Elmenhorst for helpful discussions. N. Kornadt-Beck, T.
Juraschek, S. Holz and N. Hartwigsen are gratefully acknowledged for their support in animal
housing.
Conflict of interest
The authors declare no potential conflicts of interests, including financial, personal, or other
relationships, that could inappropriately influence or be perceived to influence the work
presented here.
Author contributions
A.B., A.W., K.Z., and F.W. designed and planned the project. F.W. performed binding
experiments and analyzed data; and prepared the manuscript. A.W. and A.H. performed
lesioning, BoNT-A treatment, and behavioral testing. A.O. assisted with binding experiments,
and M.L. performed [11C]raclopride syntheses. F.W., A.W., K.Z., and A.B. wrote the
manuscript.
Wedekind et al. - page 24 of 35
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Figure Legends
Figure 1 Study design. Male Wistar rats with unilateral 6-OHDA injection into the right MFB
received either 1 ng BoNT-A (n = 10) or vehicle (n = 7) into the ipsilateral CPu. Two weeks
before and after treatment, animals were tested for motor performance by determining
apomorphine (APO)-induced rotational behavior and forelimb use. D2R availability was
longitudinally quantified with PET and [11C]raclopride at 1.5, 3, and 5 months after treatment.
Finally, D1R, D2R and DAT autoradiography and histological stainings for cell bodies and
myelin were performed in vitro subsequent to the last PET scan.
Figure 2 Unilateral 6-OHDA lesion. Unilateral 6-OHDA lesioning was proven by asymmetry
in striatal DAT concentration, nigral cell body number, and motor behavior. (A) Striatal
ipsilateral (ipsi) DAT concentration is reduced compared with contralateral (contra) and
comparable between groups. Representative, color-coded coronal images show the total
binding (upper row) and the non-specific binding (lower row) with the scale assigning colors
to DAT concentrations in picomoles per milligram protein. Error bars denote SD; filled and
open symbols indicate BoNT-A and sham treatment, respectively. Iindividual changes are
displayed in the insert on the right side. Significance indications: **** p < .0001 (Bonferronicorrected paired Student t test). (B) Nigral cell bodies are present mainly at the contralateral
hemisphere (arrowheads), while ipsilateral cell bodies could hardly be found (silver staining,
at the top). Cell bodies and myelinated bundles are identified bilaterally in striatal slices
adjacent to autoradiographic slices (cresyl violet staining at the middle and silver staining at
the bottom, respectively). (C) Both groups showed asymmetric turning behavior in response
to apomorphine (on the left) and forelimb use (on the right) 2 weeks before (b) treatment (n =
7 - 10 per group, with error bars denoting SD; filled and open symbols indicate BoNT-A and
sham treatment, respectively). There was no difference between groups, but GLM analysis
Wedekind et al. - page 34 of 35
detected reduced asymmetry 2 weeks after (a) BoNT-A treatment (only turning behavior).
Significance indications: *** p < .001.
Figure 3 Effect of BoNT-A on D2R in vivo availability. Longitudinal [11C]raclopride PET in
6-OHDA rats showed that 5 months after treatment, BoNT-A diminishes the interstriatal
differences compared with sham treatment. BoNT-A treated rats display reduced interstriatal
BPND difference compared with sham-treated rats 5 months after treatment. (A) Coronal
(upper row) and horizontal (lower row) parametric images of sham and BoNT-A-treated rats
(n = 4 - 5 per group) show increased and normalized [11C]raclopride binding in the ipsilateral
(ipsi) CPu compared with contralateral (contra), respectively. At the top, a rat brain atlas
(Schiffer) is depicted to illustrate high striatal (red) and low extrastriatal (dark blue) SRTM2modeled BPNDs. (B) Individual regional TACs of [11C]raclopride concentrations (kBq/cc) and
the corresponding SRTM-derived modeling curves (solid lines) show that ipsilateral
concentrations in the CPu of BoNT-A-treated rats (filled symbols) appear to be normalized
compared with contralateral (contra). Both BoNT-A and sham-treated rats (open symbols)
were simultaneously investigated 3 months after treatment. (C) BoNT-A diminished the
interhemispheric differences in the CPu 5 months after treatment. Filled and open symbols
indicate BoNT-A and sham treatment, respectively (n = 5 per group). Top: Individual SRTMderived BPNDs illustrate the interhemispheric difference with stacked lines for increased and
solid lines for reduced ipsilateral BPND. Bottom: The mean percent change (with error bars
denoting SD) was calculated as (ipsilateral BPND–contralateral BPND)/contralateral BPND*100.
Significance indications: * p = .045 (unpaired Student t test; n = 5).
Figure 4 Correlations of behavioral outcome and in vivo D2R availibility. Percent changes in
BPND, determined in PET 1.5 months after treatment (first scan), were modestly correlated to
net contraversive rotational behavior (A) and forelimb usage (B) of 6-OHDA rats (n = 5 per
Wedekind et al. - page 35 of 35
group), tested 2 weeks after treatment. Percent changes in SRTM-derived BPND were
calculated with (ipsilateral BPND – contralateral BPND)/contralateral BPND *100.
Figure 5 Effect of BoNT-A on striatal in vitro dopamine receptor concentrations. BoNT-A
reduced striatal D1R and D2R in the CPu of 6-OHDA rats (5 months after treatment). (A)
Striatal ipsilateral (ipsi) D1R concentration is reduced compared with contralateral (contra).
Representative, color-coded coronal images show the total binding (upper row) and the nonspecific binding (NS, lower row) with the scale assigning colors to D1R concentrations in
picomoles per milligram protein. D1R concentrations decreased ipsilaterally only after BoNTA treatment (p = .004; error bars denoting SD). Filled and open symbols indicate BoNT-A
and sham treatment, respectively. Individual changes are diplayed in the insert on the right
side as solid lines for a decrease and as stacked lines for an increase. Significance indications:
**** p < .0001 (Bonferroni-corrected paired Student t test). (B) BoNT-A treatment
normalized striatal ipsilateral (ipsi) D2R concentration compared with contralateral (contra).
Representative, color-coded coronal images show the total binding (upper row) and the nonspecific binding (NS, lower row) with the scale assigning colors to D2R concentrations in
picomoles per milligram protein. D2R concentrations increased ipsilaterally only in shamtreated animals (p = .004; error bars denoting SD). Filled and open symbols indicate BoNT-A
and sham treatment, respectively. Individual changes are displayed in the insert on the right
side as solid lines for a decrease and as stacked lines for an increase. In vitro changes were
highly correlated to in vivo changes in SRTM-derived BPND determined 5 months after
treatment (n = 5 per group) and prove a consistent definition of striatal ROIs in both binding
approaches. Percentage changes in SRTM-derived BPND were calculated with (ipsilateral
binding – contralateral binding)/contralateral binding *100. Significance indications: * p =
.039 (Bonferroni-corrected paired Student t test).
Wedekind et al. - page 1 of 2
Table 1: Parameters of PET scans.
PET 1
PET 2
PET 3
1.5 months after treatment
3 months after treatment
5 months after treatment
Sham
BoNT-A
Sham
BoNT-A
Sham
BoNT-A
Weight (g)
471 (44)
453 (32)
527 (56)
490 (30)
573 (70)
525 (46)
Anesthesia (min)
99 (21)
99 (18)
89 (14)
84 (15)
88 (15)
83 (9)
Activity (MBq)
10.8 (8)
8.9 (5.9)
22.5 (6.7)
20.7 (4.7)
11.9 (4.5)
10.1 (2)
Dose (MBq/kg)
23.3 (16.3)
20.2 (14.6)
42.4 (11.1)
42.1 (7.9)
20.6 (6.6)
19.2 (3.7)
Mass (nmol/kg)
0.78 (0.1)
0.71 (0.09)
0.71 (0.18)
0.72 (0.16)
0.75 (0.03)
0.73 (0.05)
Calculated receptor Occupancy (%)
4.4 (0.6)
4 (0.5)
4 (1.0)
4.0 (0.9)
4.2 (0.2)
4.1 (0.3)
Mixed 1-way RM-ANOVA showed no significant differences for each parameter between sham- and BoNT-A-treated animals at any of the 3 PET
scans. Parameters are given as mean (SD), n = 5 per treatment group. Activity, dose and mass are adjusted to time of injection (Ti); duration of
anesthesia is displayed only as time elapsed until start of emission, and receptor occupancy is calculated based on mass at Ti and ED50 = 17.1
nmol/kg with receptor occupancy = activity/(weight*specific activity*ED50) + 1 (Hume et al., 1998).
Wedekind et al. - page 1 of 1
Table 2: Striatal in vivo binding potentials (BPNDs).
PET 1
PET 2
PET 3
1.5 months after treatment
3 months after treatment
5 months after treatment
Contralateral
Ipsilateral
SRTM-derived
Contralateral
Ipsilateral
Contralateral
SRTM-derived
Ipsilateral
SRTM-derived
Sham
2.02 (0.33)
2.45 (0.43)
1.91 (0.18)
2.4 (0.47)
1.94 (0.20)
2.27 (0.44)
BoNT-A
2.03 (0.19)
2.03 (0.22)
1.97 (0.19)
2 (0.4)
1.83 (0.1)
1.68 (0.29)
Logan-derived
Logan-derived
Logan-derived
Sham
2.08 (0.31)
2.58 (0.46)
2.00 (0.21)
2.60 (0.54)
2.03 (0.25)
2.44 (0.52)
BoNT-A
2.12 (0.22)
2.13 (0.23)
2.1 (0.2)
2.16 (0.44)
1.80 (0.27)
1.72 (0.34)
Mixed 2-way RM-ANOVA showed no significant differences between sham- and BoNT-A-treated animals at any of the 3 PET scans. BPNDs are
displayed as mean (SD) with n = 5 per treatment group. SRTM and Logan reference modeling were performed on striatal (referenced to cerebellar)
TACs. Modeling was performed from minute 20 onwards and with fixed k2’ = 0.24/min in case of Logan-derived BPND.
Wedekind et al. - page 1 of 1
Table 3: Striatal in vitro receptor concentrations.
D1R
Contralateral
Ipsilateral
(fmol / mg protein)
D2R
DAT
Change
Contralateral Ipsilateral
Change
(%)
(fmol / mg protein)
(%)
Contralateral
Ipsilateral
(fmol / mg protein)
Change
(%)
Sham
3542 (204)
3418 (294)
-3.5 (5.4)
600 (62)
671 (48) $
12.5 (9.6)
2518 (192)
349 (296) $$$$
-86.6 (10.2)
BoNT-A
3714 (213)
3330 (261) $$$$
-10.4 (3.5) **
637 (28)
610 (64)
-4.2 (9.8) **
2429 (323)
189 (186) $$$$
-92.4 (7.5)
Mixed 1-way RM-ANOVA with post hoc testing detected a decrease in ipsilateral D1R (p = .004) and no significant difference in ipsilateral D2R
concentrations 5 months after BoNT-A treatment. Unpaired Student t test identified that these findings are different when compared with sham
treatment (p = .006 and p = .003, respectively). The decrease in DAT concentration was detected in both groups without intergroup differences (p <
.0001). Values are displayed as mean (SD) with n = 7 - 10 per group. The interhemispheric change is calculated for each animal separately by
(ipsilateral binding – contralateral binding)/contralateral binding*100 and shown as average mean percent change per group. Absolute values: $ p <
.05 and
$$$$
p < .0001 versus contralateral binding (Bonferroni-corrected post hoc paired Student t test); percent change: ** p < .01 versus sham
treatment (unpaired Student t test).