Supporting Information

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Fig. 6. Number of synapses on motor neurons. (A) Example of motor neuron stained with the synaptic marker synaptophysin. (Scale bar, 20 mm.) (B) Quantification of synaptophysin-positive puncta per motor neuron. W/W, Wistar motor neurons on Wistar astrocytes; H/H, Holtzman motor neurons on Holtzman astrocytes. n = 20; P = 0.7.

Fig. 7. Validation of quantification of relative GluR subunit expression by RT-PCR. RT-PCR followed by restriction digest with the four different subunit-specific restriction enzymes was performed on mRNA mixes with known subunit composition [for GluR1 BglI, lane 1 (1); for GluR2 Bsp1286I, lane 2 (2); GluR3 Eco47III, lane 3 (3); GluR4 EcoRI, lane 4 (4); all enzymes, lane 5 (A); no enzyme, lane 6 (U)]. (A-D) Restriction digest products from RT-PCR of only GluR1 (A), GluR2 (B), GluR3 (C), and GluR4 mRNA. (E and F) Example of restriction digest products from RT-PCR of mix of four subunits (1:1:1:1 in E and 1:2:1:1 in F). (G and H) Quantification of relative subunit expression in samples shown in E and F, showing that the measured values approximate the predicted values.

Fig. 8. Expression of GluR2 and other synaptic proteins in the ventral spinal cord of both rat strains. (A-E) GluR2 stainings of ventral spinal cords from both rat strains. Immunostainings of lumbar spinal cord from a Wistar (A, C, and E) and Holtzman (B, D, and E) rat. Sections were stained for GluR2 (A and B; red), SMI32 (C and D; green), and DAPI (blue). Overlays are shown in E and F. (Scale bar, 20 mm.) Up to 75.1 ± 10.1% of SMI32-positive neurons in the ventral horn were intensely stained by the GluR2 antibody in Holtzman rats, whereas this was only the case in 40.3 ± 5.6% of Wistar motor neurons (n = 3; P = 0.039). (F and G) Western blot of ventral part of the spinal cord from Wistar (W) and Holtzman (H) rats stained for synaptophysin, PSD95, and GRIP. Equal loading was demonstrated by b-actin staining. Quantification of Western blots normalized to the b-actin signal (G; n = 3-4 per group; P > 0.5 in all instances).

SI Experimental Procedures

Motor neurons were cultured on a preestablished feeder layer of astrocytes as described previously (1). Astrocytes were also derived from the ventral part of the spinal cord, unless stated otherwise. Almost all cells in the astrocytic feeder layer stained positive with the marker GFAP, and 80.3 ± 5.9, 85.9 ± 1.6, 91.7 ± 1.9, and 92.4 ± 1.9% of neurons grown on top of the astrocytic monolayer stained positive for the motor neuron markers peripherin (2), SMI32, p75NTR, and ChAT, respectively (n = 4).

Motor neuron death induced by a 30-min exposure to 300 mM kainate after 1 week in culture was determined as described previously (3). Similarly, ACM was added (1/4 of the total volume) on days 2 and 6 in culture. ACM was heat-inactivated by heating the medium to >100°C for 5 min and treated with trypsin by using trypsin-bound beads. Membrane fractions were prepared from feeder layers of astrocytes by ultracentrifugation and added to motor neuron cultures as indicated. The purity of membrane fractions was established by measurement of the plasma membrane marker phosphodiesterase (4) (30 units/mg protein in the membrane fraction versus 0 units/mg protein in the supernatant).

Ca²⁺ measurements using Indo-1FF were performed as described previously (5). For perforated patch-clamp recordings, gramicidin was used. Three properties of AMPA receptor currents induced by 100 mM kainate [relative Ca²⁺ permeability ratio, P_Ca/P_Na; rectification index; and sensitivity to 1-naphthyl acetyl spermine (NAS)] were determined, as described previously (6). To estimate the total amount of AMPA receptors expressed at the cell surface, measurements of the AMPA receptor current density were performed. This was done by measuring the AMPA receptor current amplitude in whole-cell recordings at -60 mV induced by 20 mM kainate, divided by membrane capacitance (which reflects the membrane surface).

RT-PCR experiments were performed on single cells aspirated through the patch pipette and on RNA samples derived from ventral spinal cord lysates. Single-cell RT-PCR experiments were carried out as described previously (7, 8), with minor modifications.

RT and first PCR were performed to amplify a 750-bp fragment from the four subunits, a second PCR labeled fragments with [a-³²P]dCTP. Labeled PCR fragments were subjected to a subunit-specific digestion, separated on a 6% polyacrylamide gel, detected by using a PhosphorImager (Storm 840; Molecular Dynamics) and quantified in ImageQuant 5.0. The relative mRNA expression was determined for each subunit and the sum of four different subunits approximated 100% in all instances.

To validate the technique, AMPA receptor subunit RNA mixtures [prepared from cDNA clones, which were a kind gift of Dr. Brorson (University of Chicago, Chicago, IL)] with known composition were subjected to RT-PCR, proving the reliability of this technique (SI Fig. 7).

RNA isolation was performed by using TriPure (Roche, Indianapolis, IN). After DNase treatment, the RNA concentration was measured by using Picogreen (Invitrogen, Carlsbad, CA). Reverse transcription was performed by using mMuLV (Invitrogen). For real-time quantitative PCR of GluR2, the forward primer was 5′-GATCGAAGAGAAACACAAAGTAG-3′, and the reverse primer was 5′-GGAGGAGATTATGATCAGGGTA-3′. Real-time PCR was carried out by using SYBR Green I (Eurogentec, San Diego, CA) and thermal cycling was performed on a 7000 Sequence Detection System (Applied Biosystems, Bedford, MA). Each reaction was performed in triplicate and a standard curve with GluR2 cDNA was run along each experiment. All samples were normalized to the level of 18S RNA (TaqMan ribosomal RNA control reagents; Applied Biosystems), which was detected by using TaqMan universal PCR master mix (Applied Biosystems).

Wistar and Holtzman female rats 4-5 months of age, weighing 200-300 g, were subjected to spinal cord ischemia, as described previously (9). Rats were intubated and ventilated with 3% isoflurane during the procedure. During the thoracotomy, spinal cord ischemia was induced by clamping the aortic arch and the left subclavian artery for 14 min. A clinical score (0, no deficit; 6, maximal deficit) was used to estimated the deficit (9). After 24 h, rats were killed with CO₂ and transcardially perfused with PBS containing 4% paraformaldehyde. Spinal cords were dissected and embedded in paraffin. Sections of the lumbar spinal cord were stained with hematoxylin and eosin. Normal appearing neurons with nucleole were counted in the ventral horn by using Lucia Image (version 4.60; Laboratory Imaging Ltd.) at 20´ magnification.

Genomic DNA was isolated and purified from the tails of three different Wistar and Holtzman rats by using standard techniques. Intronic primer pairs used for PCR amplification of exon 1, intron 1, and 3,285-bp genomic sequence upstream of the GluR2 start codon were designed by using the GenBank sequences (GenBank accession no. AF250875.1). cDNA of three different Wistar and Holtzman rats was isolated. cDNA primer pairs for PCR amplification of GluR2 were designed by using the GenBank reference sequence NM_017261.1. All PCR products were sequenced by using the BigDye term V3.1 Sequencing kit (Applied Biosystems). The sequencing reactions were loaded on the ABI3730 DNA Analyzer (Applied Biosystems). The data were collected and analyzed with the ABI FOUNDATION DATA COLLECTION, version 1.0, and SEQUENCING Analysis, version 5.0, respectively.

GluR2 (1/50; Chemicon, Temecula, CA) and SMI32 (1/500; Sternberger Monoclonals, Lutherville, MD) stainings were performed on frozen sections of the lumbar spinal cord of 4- to 5-month-old Wistar and Holtzman rats. Motor neuron cultures were stained with GFAP (1/5,000; DakoCytomation, Glostrup, Denmark), synaptophysin (0.5 mg/ml; Chemicon), p75NGF receptor (1/100), ChaT (1/200), and SMI32 (1/500). As secondary antibodies, Alexa 555-labeled antibody (1/500; Molecular Probes, Eugene, OR) was used mostly, GFAP was detected by using FITC and SMI32 was detected by using Alexa 488.

Spinal cords from Wistar and Holtzman rats were isolated, separated into ventral and dorsal parts, homogenized in RIPA buffer, as described previously (10). When mutant SOD1^G93A animals were studied, mutant SOD1^G93A and nontransgenic littermates of 40 days old were used (before motor neuron loss occurred in the ventral spinal cord). The blots were incubated with anti-GluR2 antibody (1/250; Chemicon), anti-GRIP (1/1,000; Synaptic Systems), anti-PSD95 (1/1,000; Synaptic Systems), anti-synaptophysin (1/1,000; Chemicon). After incubation with anti-IgG alkaline phosphatase (Roche Diagnostics), blots were scanned on a STORM 840 scanner (blue fluorescence; Molecular Dynamics) and the intensity of the bands on the Western blot was quantified by using ImageQuant version 5.0. b-Actin (Sigma, St. Louis, MO) was blotted to demonstrate equal loading of the gel.

A 1,357-bp fragment of the GluR2 promoter was amplified from DNA of SH-SY5Y neuroblastoma cells (CRL-2266) using 5′-ACG CGA TGG GGA AAC AGA TTT CTA AGT AAG TC-3′ as sense and 5′-TTC CAA GAA AAG TAG AGC ATC CAC-3′ as antisense primer. This fragment was cloned upstream of the luciferase gene in a pGL3-basic vector (Promega, Madison, WI). Primary cortical neurons were transfected with this vector together with a CMV-b-galactosidase vector using Nucleofector (AMAXA, Gaithersburg, MD), and 48 h later, cells were lysed by using "cell culture lysis buffer" (Luciferase assay system; Promega). In the cell lysate, the luciferase activity was measured after adding "bright light" substrate (PerkinElmer, Wellesley, MA) in a VIKTOR 2 (PerkinElmer). "Tropix Galacto-Light Plus Systems" (Applied Biosystems) was used to measure b-galactosidase activity.

Media and additives were obtained from Invitrogen, TTX was from Calbiochem (San Diego, CA), MK-801 was from Tocris Cookson (Bristol, UK), pluronic acid was from Molecular Probes, Indo 1FF AM was from Tef Labs (Austin, TX), and gramicidin was from Fluka (Seelse, Germany). RNase inhibitor and all restriction enzymes were from Promega. MMLV reverse transcriptase was from Invitrogen, and random hexamers and dNTPs were from Applied Biosystems. Ready-to-Go PCR beads were from Amersham Biosciences (Piscataway, NJ). All other chemicals were from Sigma.

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