Chen et al.
Apamin rescues parkinsonian symptoms
Supplement materials
SK channel blockade reverses cognitive and motor deficits induced by nigrostriatal
dopamine lesions in rats.
Chen et al.
Supplement 1
Detailed protocol of [125I] apamin binding and autoradiography
Brain sections were incubated with 25 pM [125I]-apamin at 48°C in 100 mM Tris–Cl buffer,
containing 0.5% bovine serum albumin, pH 7.4. Nonspecific binding was assessed by adding
a large excess of native apamin (Sigma, 0.1 M), before adding [125I]-apamin. Sections were
incubated for 60 minutes and rinsed three times, each for 20 seconds, in the same buffer.
The sections were rinsed a fourth time, for 20 seconds, in water. Dried sections were placed
on Kodak BioMax MR films. Sections cut at the same level of the brain, from sham and 6OHDA-lesioned rats were analyzed together in the same binding experiment and exposed
side-by-side on the same autoradiographic film to prevent experimental artifacts during
analysis of autoradiograms. Serial sections of one naive rat were added to the experimental
sections, serving as internal standards for labeling to exclude possible experimental artifacts
in the comparison of labeling. Autoradiograms were exposed for 12 days to obtain
unsaturated labeling and thus to allow the detection of increases or decreases in labeling.
Films were then processed in a Kodak Industrex developer. Azur II-stained sections were
used for reference. Autoradiograms were analyzed and radioactivity quantified with Image J
software. [14C] plastic standards (Amersham) were used to calibrate the [125I] concentration.
Mean receptor density was calculated in kbq/g tissue equivalent for each nucleus, using six
to eight bilateral measurements in each animal. The group-value presented for each
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Chen et al.
Apamin rescues parkinsonian symptoms
structure studied is the mean of values obtained for each animal ± SEM. Nonspecific binding
corresponded to around 15% of total binding. Specific binding was calculated as the
difference between total and nonspecific binding for a given area. Rat brain regions were
identified and named using the Paxinos and Watson’s rat brain atlas (2007).
Supplement 2
Technical details of lesion verification
-
[3H]-Mazindol binding and autoradiography
Brain sections were air-dried and were preincubated at 4°C for five minutes in a Tris 50 mM
buffer with NaCl 120 mM, KCl 50 mM (pH 7.9). Then, they were incubated for 40 minutes at
4°C with 7 nM [3H]mazindol (specific activity 27 Ci/mmol; PerkinElmer Life and Analytical
Sciences, Zaventem, Belgium) in 50 mM Tris buffer containing 300 mM NaCl and 5 mM KCl
(pH 7.9), with 3mM desipramine (hydrochloride, Sigma-Aldrich) added to block the
norepinephrine reuptake. Sections were rinsed twice for three minutes in the same
incubation buffer and then for ten seconds in distilled water before air-drying.
Autoradiographs were realized by applying the slides to a Kodak Biomax MR film (SigmaAldrich) for at least six weeks. After exposure, the films were processed in a developer
(Kodak GBX developer, Sigma-Aldrich), at room temperature for 30 seconds, then fixed,
washed in water and air dried. All autoradiograms were scanned. The surface of striatum
was delimitated in pixels with reference to Paxinos and Watson (2007). The extent of
bilateral striatal 6-OHDA lesions was estimated by delimitating the extent of the lesioned
areas in each hemisphere as the sum of pixels with low gray levels. As no difference was
found in either the surface measured on each side of the brain, the values obtained were
averaged. The extent of the lesion was determined as the ratio between the lesioned and
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Chen et al.
Apamin rescues parkinsonian symptoms
total striatum area, expressed as the percentage of the total striatal surface. Then, a mean ±
S.E.M. was calculated for each experimental group.
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Tyrosine hydroxylase immunohistochemistry
After five minute postfixation in 3% paraformaldehyde and washing [5 x 5 minutes in Tris
buffer (50mM)], brain sections were pretreated for quenching of endogeneous peroxidase
activity [2 x 10 minutes in Tris buffer (50mM) containing Lysine (18.3 mg/ml) and hydrogen
peroxide (0.3%) followed by 6 x 5 minutes in Tris buffer (50mM) washing] and for blocking of
non-specific antibody binding [1 x 20 minutes in Tris buffer (50mM) containing bovine serum
albumin (5%)(BSA)]. Then they were incubated overnight at 4°C with monoclonal mouse
antibody [1:500; Chemicon, Temecula, CA; in Tris buffer containing BSA (1%)], for the
detection of TH. After washing [6 x 5 minutes in Tris buffer (50mM)], sections were exposed
for 1 hour at room temperature, to biotinylated goat anti-mouse [1:200; Vector
Laboratories, Burlingame, CA; in Tris buffer (50mM) containing BSA (1%)] followed by
washing [5 x 5 minutes in Tris buffer (50mM)] and by a streptavidin–biotin–horseradish
peroxidase solution incubation [Elite ABC Kit, Vector, in Tris buffer (50mM) containing BSA
(1%) for 1 hour at room temperature]. Immunolabeling was revealed after washing [3 x 5
minutes in Tris buffer (50mM)] by incubating the sections in a 0.5% solution of
diaminobenzidine tetrahydrochloride in Tris buffer containing 3% hydrogen peroxide for 3
minutes at room temperature. Following washing [5 x 5 minutes in Tris buffer (50mM)],
sections were dehydrated in an ascending series of alcohols, cleared in xylene, and
coverslipped using DPX mounting medium (Sigma-Aldrich). Control staining was performed
without the primary or secondary antibodies. Four coronal photomicrographs of the
ipsilateral and contralateral SNc and VTA were captured using a Leitz Aristoplan light
microscope equipped with a Nikon high-resolution digital camera (756 x 581 pixels; Nikon,
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Chen et al.
Apamin rescues parkinsonian symptoms
Tokyo, Japan) interfaced to a PC computer and Image software (Lucia, Nikon), x20). An
experimenter, blind to the lesion, quantified TH immunoreactivity (cell body) by counting the
total number of TH-positive neurons per section containing the ipsilateral and contralateral
SNc and VTA using Image J software. The average of all counts represented the total number
of neurons per section. In addition, cell counts were performed on Nissl-stained neurons in
the SNc and VTA for detecting neuronal cell bodies in accordance with the Paxinos and
Watson’s atlas (2007).
Paxinos G and Watson C (2007) The Rat Brain in Stereotaxic Coordinates, Elsevier
Academic Press. Amsterdam.
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Chen et al.
Apamin rescues parkinsonian symptoms
Supplemental Results: Figures S1 and S2
6-OHDA lesion extent Bilateral intrastriatal 6-OHDA infusions produced lesions restricted to
the dorsal striatum sparing the nucleus accumbens (Fig. S1). The loss of [3H]-mazindol
labeling extended across the whole rostro-caudal levels of the striatum in 6-OHDA-lesioned
subjects. There was no difference in [3H]-mazindol labeling between the three groups
injected with apamin 0.0, 0.1 or 0.3 mg/kg in the dorsal striatum (mean decrease: 59% ±
0.02, 54% ± 0.02, 57% ± 0.02, respectively; F2,27=0.803, NS). Unilateral SNc 6-OHDA infusions
produced a near-complete depletion (> 90%) of DA in the ipsilateral striatum assessed by the
loss of [3H]-mazindol binding in the striatum and to a lesser extent in the nucleus accumbens
area (Fig. S1).
Supplemental Figures Captions
Figure S1. 6-OHDA lesion extent. [3H]-mazindol binding to dopamine uptake sites at the
striatum level (here, at + 1.20 mm related to bregma). Examples of [3H]-mazindol binding
autoradiography (Top) in a sham (left) and a bilaterally 6-OHDA-lesioned rat (right), (Bottom)
in a sham (left) and an unilaterally 6-OHDA-lesioned rat (right). Scale bar: 1 mm.
Figure S2. Illustration of the bilateral 6-OHDA lesion and microdialysis probes in the striatum
of a representative animal. A: [3H]-mazindol binding autoradiography. The numbers indicate
the rostro-caudal coordinates (mm) from bregma (Paxinos and Watson, 2007). B: Cresylviolet stained section showing placements of guide cannula (arrows) in the striatum of the
same animal. Bar = 2.5mm.
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Chen et al.
Apamin rescues parkinsonian symptoms
Table S1: Locomotor behavior in the different tests
The total covered distance (mean ± S.E.M) was measured in cm in each maze.
ANOVA, 1 F5,52=0.88, NS; 2 F5,54=0.17, NS; 3 F5,43=0.28, NS.
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Chen et al.
Apamin rescues parkinsonian symptoms
Table S2: Variations of apamin binding in DA mesolimbic and nigrostriatal pathways
Brain structures
Prefrontal cortex
Cingulate cortex
Accumbens nucleus, core
Accumbens nucleus, shell
Striatum, dorso-lateral part
Striatum, dorso-medial part
Septohippocampal nucleus
Lateral septal nucleus
Medial septal nucleus
Basolateral amygdaloid nucleus
Central amygdaloid nucleus
Medial amygdaloid nucleus
CA1 hippocampal field
CA3 hippocampal field
Dentate Gyrus
Entorhinal cortex, ventral part
Ventral tegmental area
Substantia nigra, pars compacta
Apamin binding
(%)
100.00 ± 1.43
100.06 ± 0.82
99.98 ± 0.75
101.63 ± 1.12
98.45 ± 1.90
100.92 ± 1.66
99.69 ± 1.41
100.22 ± 1.26
102.14 ± 2.47
99.60 ± 2.58
98.77 ± 3.53
100.16 ± 3.09
100.82 ± 4.48
100.57 ± 1.48
99.10 ± 1.48
102.33 ± 2.70
100.34 ± 3.97
80.47 ± 1.73*
Values are the percentage of the apamin binding (relative unit) in 6-OHDA-lesioned rats
relative to sham rats. Values are expressed in mean ± S.E.M. *: P<0.05 (6-OHDA vs sham,
Student’s test).
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Chen et al.
Apamin rescues parkinsonian symptoms
Figure S1
Figure S2
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