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SUPPLEMENTARY MATERIALS AND METHODS
nAChR subunit in situ hybridization
In situ probes were transcribed in vitro using -[35S]-UTP as the sole source of UTP. 4
subunit probes were derived from EcoRI linearized pHYA23-1E(2) using Sp6 RNA polymerase,
5 subunit probes were derived from EcoRI linearized PC989 in pBluescript using T3 RNA
polymerase and 2 subunit probes were derived from HindIII linearized pSP65-49 using Sp6
RNA polymerase. Full length antisense transcript production was verified using denaturing
agarose gel electrophoresis and probes were stored at -20°C as precipitates in 70% ethanol.
500bp average length probe fragments were prepared by alkaline hydrolysis prior to
hybridization.
For hybridization, 14m tissue sections mounted on gelatin/chrome alum/poly-l-lysine
treated slides were thawed to room temperature under vacuum. The tissue was then fixed by
immersion in 4% paraformaldehyde in phosphate buffered saline (PBS: 137mM NaCl, 2.5mM
KCl, 16mM Na2HPO4 and 4mM NaH2PO4, pH 7.4) for 15 min. Three 5 min washes in PBS were
then followed by air drying. Immersion for 10 min in 15mM acetic anhydride in 0.1M
triethanolamine, pH 8.0 acetylated the tissue. After a 2 min wash in 2X SSC (1X SSC: 150mM
NaCl, 15mM trisodium citrate, pH 7.0 with HCl), the tissue was dehydrated sequentially (3 min
washes) in 50, 70, 95 and 100% ethanol, with a final immersion in 100% ethanol before air
drying and vacuum storage.
nAChR subunit probes were added with a final concentration of 5x106 cpm/mL to
hybridization solution (50% formamide, 10% dextran sulphate, 300mM NaCl, 10mM Tris, 1mM
EDTA, 500g/mL yeast tRNA, 10mM DTT, 1X Denhardt’s solution, pH 8.0). Each slide
containing tissue sections was hybridized with 100L of hybridization solution with probe
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applied to a glass coverslip and sealed with DPX. Hybridization was performed at 58°C for 18
hrs.
The DPX was removed after hybridization and a wash in 4X SSC for 15 min with
agitation was used to remove the coverslip. After four further washes (5 min) in 4X SSC, the
slides were immersed in an RNase A solution (20g/mL RNase A, 500mM NaCl, 10mM Tris
(pH 8.0), 1mM EDTA) for 30 min at 37°C. 5 min sequential washes in 2X SSC/1mM DTT
(three times), 1X SSC/1mM DTT and 0.5X SSC/1mM DTT were used to desalt the samples.
Slides were then immersed in 0.1X SSC/1mM DTT at 60°C for 30 mins, allowed to cool in fresh
0.1X SSC/1mM DTT and then dehydrated sequentially (3 min washes) in 50, 70, 95, 100, 100
and 100% ethanol. When dry, samples were exposed for 3-10d to Amersham Hyperfilm-3H.
To quantify hybridized signal intensity, eight [35S] tissue standards generated by mixing
known amounts of isotope (0-25 nCi/mg) with whole brain homogenate were applied to each
film. This allowed radioactivity, optical density and gray level to be correlated by generating
standard curves and therefore relative radioactivity levels (cpm/mg) could be produced for the
tissue samples. Between 5 and 40 measurements were recorded for each brain region analyzed
for each animal and the mean of all measures for each region was used for further analysis.
Nonspecific hybridization was assessed by analysis of at least two regions which did not exhibit
any detectable signal.
Crude synaptosomal preparation
Cortical and thalamic tissue was manually homogenized in 10 volumes of 0.32 M sucrose
buffered to pH 7.5 with 5 mM HEPES hemisodium (Roche Diagnostics, Indianapolis, IN) using
a glass/Teflon tissue grinder. Homogenates were then centrifuged at 12000g for 20 min. The
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crude synaptosomal pellets produced were suspended in isotonic uptake buffer (140 mM NaCl,
1.5 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 20 mM glucose and 25 mM HEPES hemisodium salt,
pH 7.5). 800 μL was used for cortical and 350 μL for thalamic samples.
[86Rb+] uptake
25 μL of the synaptosomal preparation was added to 10 μL of uptake buffer which
contained approximately 4 μCi of [86Rb+]Cl. Samples were then incubated at 22°C for 30 min.
Acetylcholinesterase activity was inhibited by subsequent addition of 5 μL of 80 μM
diisopropylfluorophosphate during the last 5 min of incubation. Uptake was terminated by
collection of the synaptosomes on A/E glass fiber filters (Gelman Instrument Co., Ann Arbor,
MI) under 0.8 atmosphere vacuum. Filters were then washed with 0.5 mL of uptake buffer.
Sample preparation for [125I]-epibatidine binding
Excess synaptosomal samples from the [86Rb+] efflux assays stored frozen at -70°C were
thawed, diluted with hypotonic buffer (14 mM NaCl, 0.15 mM KCl, 0.2 mM CaCl2, 0.1 mM
MgSO4, 2.5 mM HEPES, pH 7.5) and centrifuged at 20000g for 20 min. Pellets were
resuspended in fresh hypotonic buffer and centrifuged again. This cycle was repeated a total of
four times. Following the final centrifugation, fresh hypotonic buffer was added to the pellets,
which were then frozen until [125I]-epibatidine binding was assessed.
Membrane and 2% Triton X-100 extract preparation
Cortex and thalamus were dissected, frozen on dry ice and stored at -80°C until ready for
use. Tissue was homogenized for 2 min in an UltraTurrax homogenizer (Janke and Kunkle,
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Staufen, Breisgau, Germany) in an excess (10 mL) of buffer (50 mM Na-phosphate, pH 7.4; 1 M
NaCl, 2 mM EDTA, 2 mM EGTA and 2 mM PMSF). Homogenates were diluted and then
centrifuged at 60000g for 90 min. 2% Triton X-100 extracts were prepared as described (Gotti et
al, 2005a; Gotti et al, 2005b). Protein content was measured using the BCA assay (Pierce
Chemical, Rockland, IL) with bovine serum albumin standards.
Western Blotting
Protein samples were diluted 1:1 (v/v) with Laemmli buffer and then electrophoresed
through 9% acrylamide SDS gels. After SDS-PAGE, the proteins were transferred to 0.45 μm
pore nitrocellulose membranes (Whatman Schleicher and Schuell, Dassel, Germany).
Membranes were blocked overnight in 5% non-fat milk and 0.3% Tween-20 in tris-buffered
saline, incubated for 2 hr with primary antibody (1-2.5 μg/mL) and then incubated with the
appropriate peroxidase-conjugated secondary antibody. Membranes were then washed and
peroxidase activity was detected using a chemiluminescent substrate.
Quantification of Western Blot bands
Films of developed blots were scanned using an Epson 4500 gel scanner, as eight-bit gray
scale TIFF images at 300 dpi. All film images were acquired in the same way and scanned in
parallel with a calibrated optical density tablet (Stouffer, Mishawaka, IN).
Images were analyzed using ImageJ software from the National Institutes of Health
(http://rsb.info.nih.gov/ij/). Pixel values were transformed to optical density values by the
software using the calibration curve derived from imaging the calibrated tablet with the same
parameters as used for the images.
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SUPPLEMENTARY REFERENCES
Gotti C, Moretti M, Clementi F, Riganti L, McIntosh J, Collins A, et al (2005a). Expression of
nigrostriatal alpha 6-containing nicotinic acetylcholine receptors is selectively reduced, but not
eliminated, by beta 3 subunit gene deletion. Mol Pharmacol 67(6): 2007-2015.
Gotti C, Moretti M, Zanardi A, Gaimarri A, Champtiaux N, Changeux J, et al (2005b).
Heterogeneity and selective targeting of neuronal nicotinic acetylcholine receptor (nAChR)
subtypes expressed on retinal afferents of the superior colliculus and lateral geniculate nucleus:
identification of a new native nAChR subtype alpha3beta2(alpha5 or beta3) enriched in
retinocollicular afferents. Mol Pharmacol 68(4): 1162-1171.
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