Supplemental Experimental Procedures

Cell, Volume 131

Supplemental Data The Classical Complement Cascade Mediates CNS Synapse Elimination Beth Stevens, Nicola J. Allen, Luis E. Vazquez, Gareth R. Howell, Karen S. Christopherson, Navid Nouri, Kristina D. Micheva, Adrienne K. Mehalow, Andrew D. Huberman, Benjamin Stafford, Alexander Sher, Alan M. Litke, John D. Lambris, Stephen J. Smith, Simon W.M. John, and Ben A. Barres

Supplemental Experimental Procedures

Culture of RGCs RGCs were purified by sequential immunopanning to greater than 99.5% purity from P5 SpragueDawley rats, and cultured in serum-free media as described (Barres et al., 1988; Meyer-Franke et al., 1995). RGCs were then cultured in serum -free medium, modified from Bottenstein and Sato (1979), containing Neurobasal (Gibco), bovine serum albumin, selenium, putrescine, triiodothyronine, transferrin, progesterone, pyruvate (1 mM), glutamine (2 mM), B27 (Invitrogen), CNTF (10 ng/ml), BDNF (50 ng/ml), insulin (5 µg/ml), and forskolin (10 µM). Recombinant human BDNF and CNTF were obtained from Peprotech (NJ). All other reagents were obtained from Sigma.

Preparation of astrocytes P1- P2 cortices were papain-digested and plated in tissue culture flasks (Falcon) in a medium that does not allow neurons to survive. After 3-4 days non-adherent cells were shaken off of the monolayer and cells were incubated another 2 days to allow monolayer to refill. Medium was replaced with fresh

medium containing AraC (10 µM) and incubated for 2 days. Astrocytes were trypsinized and plated onto 6- well inserts (Falcon, 1.0 µm).

RGC Gene Expression Analysis Using Affymetrix GeneChip Arrays Total RNA was harvested using RNeasy Mini Kit (Qiagen). cDNA was synthesized from 2 μg total RNA using the Gibco BRL Superscript Choice system and a T7-(dT)24 primer [5’- GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24 - 3’]. Biotinylated cRNA target was prepared by T7 linear amplification using the Bioarray RNA Transcripts Labeling Kit (Enzo) followed by fragmentation. Target was hybridized to Affymetrix Test-2 GeneChip arrays to assess target performance and the Rat U34 genome GeneChip array set following standard Affymetrix protocols. Microarray data was generated in paired triplicates from three independent cultured RGC preparations. Assessment of the quality of sample data and changes in gene expression were analyzed with MicroArray Suite 5.0 software. Genes identified as changing in expression in response to astrocytes increased or decreased at least 2-fold in all three replicates.

Semiquantitative RT-PCR 2x PCR Master Mix kit (Promega) was used as supplied by manufacturer with the exception of the addition of 4 mM MgCl2 (5.5 mM final) to the C1qC reaction mix The following PCR primers were used:

F-C1qB

(5'-CGGAATTCCCTTCTCTGCCCTGAGGACGG-3'),

CCTTTCTGCATGCGGTCTCGGTC-3'), TCACCAACCAG-3'),

R-C1qA

F-C1qA

R-C1qB

(5'-CGGGAT-

(5’-CGGAATTCGACAAGGTCC-

(5'-CGGGATCCGGGGTCCTTCTCGATCC-3',

F-C1qC

(5'-

CCGGGGGAGCCAGGTGTGGAG-3'), R-C1qC (5’-GCACAGGTTGGC-CGTATGCG-3'). GAPDH primers

were

added

to

all

reactions

as

an

internal

control

(gapdh-s:

5’-

GGTCTTACTCCTTGGAGGCCATGT-3’; gapdh-as: 5’-GACCCCTTCA-T-TGACCTCAACTACA3’). PCR program was as follows: initial denaturation for 4 min at 94°C; cycle denaturation for 1 min at 94°C, annealing for 30s at 55°C, and extension for 30 sec at 72°C (30 cycles total); final extension for 10 min at 72°C. The PCR products were fractionated on 1.5% agarose gel and visualized by ethidium bromide staining.

Mice C3 KO mice were generated by using the approach of homologous recombination as described (Wessels et al., 1995) and obtained from The Jackson Laboratory (stock number 003641). C3KOs had a C57BL/6J background. They were compared to control C57BL/6J mice (stock number 000664). DBA/2J (D2) mice were from our colony (SJ) maintained at The Jackson Laboratory. This colony is derived from and periodically intermixed with the Jackson Laboratory D2 stock (000671) and is essentially identical to that stock. Glaucoma in our colony has been studied extensively (Libby et al. 2005a). D2 Gpnmb+ is a control DBA/2J substrain (full name DBA/2J-Gpnmb+/Sj) that has a wild type version of the glaucoma-inducing gene Gpnmb. Other than the absence of a point mutation in Gpnmb, the Gpnmb+ substrain is genetically matched to the standard DBA/2J strain with no other known genetic differences. This substrain was recently characterized for glaucoma phenotypes and does not develop high intraocular pressure or glaucoma (Howell et al., 2007).

In situ hybridization In situ hybridization was performed on 12 μm fresh frozen PBS perfused retinal sections as previously described (Schaeren-Wiemers and Gerfin-Moser, 1993). The C1q specific probes used were designed against the coding regions of mouse C1qA, C1qB, and C1qC in pCMV-SPORT6 (Open Biosystems

clone Id: 5337411, 5101106, and 6442329 respectively). The plasmids were digested with EcoRI, and in vitro transcription was performed with T7 polymerase. The detection of hybridized mRNA in sections was performed using the NBT/BCIP developing system (Roche Applied Science).

Array Tomography The tissue was processed for array tomography as described in Micheva and Smith, 2007. In brief, the LGN were dissected out, further fixed using microwave irradiation (PELCO 3451 laboratory microwave system; Ted Pella), then dehydrated in ethanol and embedded in LRWhite resin. Serial ultrathin sections (70 nm) were cut on an ultramicrotome (Leica), mounted on subbed glass slides and immunostained using C1q antibodies (C1q goat, Quidel, C1q goat, Sigma and a monoclonal C1q rat from Hycult) and antibodies against synaptic proteins: synapsin I (rabbit, Chemicon), SV2 (mouse, DSHB, Iowa), PSD-95 (mouse, NeuroMabs), PSD-95 (rabbit, Zymed). For secondary antibodies, Alexa 488 and Alexa 594 from the appropriate species were used. In some occasions, applied antibodies were eluted and the sections were restained with different antibodies. Sections were mounted using SlowFade Gold antifade reagent with DAPI (Invitrogen). Imaging was done on a Zeiss Axiovert 200M fluorescence microscope with AxioCam HRm CCD camera, using a Zeiss 63x/1.4 NA Plan Apochromat objective.

Labeling of retinogeniculate afferents Cholera Toxin β subunit (CTβ) conjugated to Alexa 488 (green label) was injected into the left eye, and CTβ conjugated to Alexa 594 (red label) into the right eye (2 -3µl; 0.5% in sterile saline; Invitrogen (Molecular Probes); CTβ has no biological activity) as described in Bjartmar et al., (2006) with slight modifications. 24 hours later brain tissue was harvested and postfixed overnight in 4% PFA,

cryoprotected in 30% sucrose and then sectioned coronally at 40µm, mounted onto slides and coverslipped with Vectashield (Vector Laboratories; Burlingame, CA).

Quantification of LGN images and preparation of photomicrographs Images were digitally acquired with a color CCD camera (SPOT). All images were collected and quantified “blind”, and age matched littermate controls were used in addition to age matched standard C57BL/6J mice. Universal gains and exposures were established for each label. Raw images of the dLGN were imported to Photoshop (Adobe) and cropped to exclude the vLGN and IGL, then the degree of left and right eye axon overlap was quantified using the multi-threshold protocol described in Torborg et al.(2005). This technique is designed to compare overlap across a range of signal:noise values in WT versus transgenic mice. This approach best allows for direct statistical comparison of overlap between various strains of mice at different ages.

Electrophysiological recordings Mice aged P26-P34 were euthanized and the brain was rapidly removed and placed in a 4°C cholinebased cutting solution containing (in mM): NaCl 78.3, NaHCO3 23, glucose 23, choline chloride 33.8, KCl 2.3, NaH2PO4 1.1, MgCl2 6.4, and CaCl2 0.45, pH 7.4. Parasagittal brain slices containing the LGN (250 μm) were cut on a vibratome following the method of Turner and Salt (1998) with adaptations (Hooks and Chen, 2006). Generally only one slice containing the LGN and optic tract was obtained per animal. Slices were allowed to recover at 31°C for 25 min in the choline cutting solution and for 30 min in isotonic saline solution (in mM: NaCl 125, NaHCO3 25, glucose 25, KCl 2.5, NaH2PO4 1.25, MgCl2 1, and CaCl2 2, pH 7.4). Oxygenation (95% O2/5% CO2) was continuously supplied during cutting, recovery and recording. Whole-cell voltage-clamp recordings of thalamic

relay neurons from the contralateral monocular region of the dorsal LGN were performed following the method of Chen and Regehr, 2000. Recordings were carried out at room temperature in flowing isotonic saline containing the GABAA receptor antagonist 40 μM bicuculline to inactivate local inhibitory circuits, and the corticothalamic tract was cut to prevent recurrent excitation of the LGN neuron via cortical excitation. Patch pipettes with resistance of 2-4MOhm were pulled from thickwalled borosilicate glass capillaries and filled with an internal solution containing (mM) CsCl 130 (Cs+ was used as the main cation rather than K+ to improve voltage uniformity), NaCl 4, HEPES 10, EGTA 5, CaCl2 0.5, MgATP 4, Na2GTP 0.5, QX-314 5 (to suppress voltage-gated sodium currents), pH adjusted to 7.2 with CsOH. Access resistance was monitored throughout the recording and was between 4-8MOhm after 70% compensation. A stimulating electrode (either bipolar concentric or a pair of wires, results were identical with each) was buried just below the surface of the optic tract next to the ventral LGN. Stimuli were delivered every 45 seconds to allow recovery between trials. Stimulus duration was 100-200 μs, and stimulus intensity ranged between 0-40 μA. In general, once an input was found the stimulus intensity was reduced to a level where no response was seen and then increased in 0.5μA increments in order to recruit individual axonal inputs. Recordings were carried out at two different holding potentials: at -70mV in order to measure responses mediated predominantly by activation of AMPA receptors, and at +40mV in order to measure responses mediated by NMDA receptors.

Dye Fills and cell morphology Mice were anesthetized with inhalant isofluorane, and received intravitreal injections of CTβ conjugated to Alexa 488 into the left eye, and CTβ conjugated to Alexa 647 into the right eye (2-3µl; 0.5% in sterile saline). Mice were euthanized 24 – 48 hours later and LGN slices were prepared for

electrophysiological recordings as described above. The internal solution used was as described above except it also contained Alexa 594 hydrazide at 0.2mg/ml, which filled the cell during the recording to allow visualization of the entire cell. Immediately after recording slices were fixed in 4% paraformaldehyde for 1 hour at room temperature on a shaker, followed by 3 washes in phosphate buffered saline. Slices were mounted in Vectashield to prevent fading of the fluorophores, and slides were stored at 4°C until they were imaged. Confocal images were collected on a Leica confocal. Cells were imaged using a 40x objective so that the entire dendritic tree was visible in the image. Optical sections were taken every 0.2um, and the entire z-stack was reconstructed as a 3D projection using Volocity software.

Retinal cell counts Retinal flat mounts were prepared by dissecting out retinas whole from the eyecup and placing four relieving cuts along the major axis, radial to the optic nerve. Each retina was stained with DAPI (Vector Laboratories, Burlingame, CA) to reveal cell nuclei. Measurements of total cell density in the ganglion cell layer (which includes both ganglion cells and displaced amacrine cells) were carried out blind to genotype from matched locations in the central and peripheral retina for all four retinal quadrants of each retina. Quantification was limited to P30 retinas, which is an age subsequent to ganglion cell genesis and apoptosis in the mouse.

In Vitro Multielectrode Array Recording P5-6 animals were sacrificed by rapid decapitation. The eyes were enucleated and transferred to room temperature Ame's Medium (Sigma A1420). The retina was isolated by gently peeling the sclera and pigment epithelium away from the retina. The lens and vitreous humor were then removed, the retina

hemisected, and mounted ganglion cell side up on filter paper discs (Millipore). The retina was transferred, ganglion cell side down, to a multielectrode array (Litke, et. al. 2004) and held in place using dialysis membrane (Spectrum 132678) and a platinum ring (Warner Instruments). The array contained 512 electrodes spaced at 60 µm and arranged in a planar array that covered 1890 x 900 µm. Retinas were superfused with oxygenated Ame's Medium at a rate of 3 ml/min at room temperature for 10 minutes, then 32 C for the remainder of each recording session. Retinas were allowed to equilibrate for 30 minutes prior to collecting data. Spontaneous activity was then recorded for 1.25-2 hours in semi-darkness. Analog waveforms from the 512 electrodes were digitized and sampled at 20 kHz before being written to hard disk for off-line analysis. Signals from individual neurons were isolated as described previously using a threshold equal to 3.5 times the typical noise level on each electrode (Litke et. al. 2004, Shlens et. al. 2006). Analysis and visualization of multielectrode data were performed using custom-written software. The population firing rate was calculated as the sum of all spikes recorded from all neurons divided by the duration of the recording. Wave numbers were counted by manually setting a threshold for each recording. Time bins above this threshold the retina was considered to be participating in a wave. This threshold was determined by visually inspecting the time-varying spike rate of all neurons binned at 1 second intervals. The correlation index between neurons was calculated as described previously (Wong 2000). Briefly, this was calculated by counting the number of spikes from cell A that occurred within +/- 100 ms of a spike in cell B and then dividing this by the number of spikes that would have occurred by chance.

Labeling of Neuromuscular junctions Postnatal mice (between P5 and P15) were anesthetized with a standard juvenile ketamine cocktail and perfused with 2% paraformaldehyde. Sternomastoid muscles were then dissected and placed in petri dishes in physiological saline. Junctional AChRs were labeled for 15 min with tetramethyl rhodamineconjugated -bungarotoxin (10 µg/ml) (Molecular Probes, Eugene, OR). The muscle was then washed three times with PBS and fixed for 5 min with cold methanol. Teased neuromuscular preparations were then incubated overnight at 4 degrees with antibody against neurofilament, followed by incubation with Secondary Alexa (488) conjugated antibodies (Invitrogen). All labeled junctions were mounted with Vectashield and imaged with a color CCD camera (SPOT).

Determining stage of Glaucoma The damage level of each nerve (no or early, moderate and severe) was determined using the consensus of a series of masked investigators. This system of determining damage levels has been previously validated (Anderson 2005, Libby 2005b, Howell et al., 2007). In ‘no or early nerves,’ there is no or very mild damage (< 5% of axons) with healthy axons having a clear axoplasm and intact myelin sheath [average number of axons, 50504 ± 1988 (±S.E.M)]. These nerves have a level of damage that is indistinguishable from those of age-matched mice that do not develop glaucoma. Due to the very minor degree of damage, we previously classified these nerves as mild. Although these nerves are indistinguishable from those of normal mice, some of the aged mice with these nerves must have early, disease related molecular changes that are not detectable morphologically in the optic nerve or retina (even by counting retinal ganglion cells or their axons in the nerve). Thus, we have renamed this category ‘no or early’ glaucoma. In moderate nerves, darkly stained, degenerating axons are readily detectable throughout much of the nerve but the majority of axons appear normal (average

number of axons, 31410 ± 2199). In severe nerves, there is extensive axon damage throughout the optic nerve. There is extensive RGC and axon loss with the vast majority of remaining axons being visibly damaged (average number of axons, 7970 ± 2150). In eyes classified as severe, there is prominent optic nerve excavation. The RGC axon number is significantly different between optic nerves of each damage level (P