Supporting InformationGreten-Harrison et al. 10.1073/pnas.1005005107SI TextNumber of Mice Used for Experiments. Body weight of wild-type andsynuclein nullmicewas recordedat 8wkof ageusingmalewild type,n = 27; male synuclein null, n = 16 female wild type, n = 21; andfemale synuclein null, n = 12 (Fig. 1C). Wild type, n = 56; synu-clein null, n = 125 for survival curves (Fig. 1D). Three-month-oldwild type and synuclein null n = 6 mice/genotype for synapsedensity in CA3 (Fig. 2A). For presynaptic terminal area meas-urements of CA3 synapses, n = 18 sections/genotype and n = 3mice/genotype for wild-type and synuclein null mice (Fig. 2B).Presynaptic terminal area of CA3 synapses in rescued mice wasquantified using n=18 sections/genotype and n=3mice/genotypesynuclein null and synuclein transgenic mice (Fig. 2C). For pre-synaptic terminal area measurements of hippocampal synapses inculture, n=10 coverslips/genotype from n=2mice/genotype (Fig.2D). For quantifying synapse density in CA3, n = 50 images/ge-notype from 2 animals/genotype (Fig. 2F). For morphometricanalysis of CA3 synapses, 118 wild-type and 160 synuclein nullsynapses were analyzed (Fig. 2G). For quantifying synapse densityin CA1, wild type, n = 78; synuclein null, n = 76 micrographs, 2mice/genotype (Fig. 2H). For morphometric analysis of CA1 syn-apses, 198 wild-type and 223 synuclein null synapses were analyzed(Fig. 2I). For Schaffer collateral I/O curves in young mice, wildtype, n= 14 slices from 3 animals and synuclein null, n= 10 slicesfrom 3 animals (Fig. 3A) and in old mice, wild type, n = 9 slicesfrom 3 animals; synuclein null, n= 11 slices from 3 animals (Fig. 3E and F). For paired-pulse facilitation in 3-mo-old mice, wild type,n = 14 slices from 3 animals; synuclein null, n = 10 slices from 3animals (Fig. 3C). In 12-mo-oldmice, wild type, n=9 slices from 3animals; synuclein null, n= 11 slices from 3 animals (Fig. 3G). Foraction potential conduction velocity in 3-mo-old mice, wild type,n = 17 slices from 3 animals; synuclein null, n = 13 slices from 3animals (Fig. 3D) and in 12-mo-old mice, wild type, n = 14 slicesfrom 4 animals; synuclein null, n = 13 slices from 4 animals (Fig.3H). For Schaffer collateral I/O curves in young mice, wild type,n = 14 slices from 3 animals; synuclein null, n = 34 slices from 10animals; rescued mouse synuclein transgenics: n = 5 from 1 ani-mal; rescued human synuclein transgenics: n = 12 from 3 animals(Fig. 3 I and J). For electroretinogram (ERG) analysis of retinalfunction at 3 mo, wild type, n = 9 mice and synuclein null, n = 6(Fig. 4A) and at 12 mo, wild type, n = 7 and synuclein null, n = 5(Fig. 4B). For visual placement analysis, 2- to 3-mo wild type, n =21; 2- to 3-mo synuclein null, n = 22; 12- to 18-mo wild type, n =15; 12- to 18-mo synuclein null, n = 7 (Fig. 4C). For inverted gridtest analysis, 2- to 3-mowild type, n=23; 2- to 3-mo synuclein null,n= 28; 12- to 18-mo wild type, n= 8; 12- to 18-mo synuclein null,n = 9 (Fig. 4D). For quantitative immunoblotting at both 3 and12 mo, n = 4 mice/genotype/age (Fig. 5 A and B).

Materials and MethodsAntibodies. Isoform-specific synuclein antibodies and antibodiesto synaptic proteins have been previously published (1, 2). An-tibodies to NeuN and synaptophysin for quantitative immuno-histochemistry experiments were purchased from Chemicon andused at a 1:500 dilution.

Mice. All mice used in this study were maintained under an institu-tional animal care and use committee approved protocol. αβγ-Synuclein tripleKOmiceweremaintained on aC57B6/J backgroundand compared with wild-type C57B6/J mice that were purchasedfrom The Jackson Laboratory. In addition, αβγ-synuclein triple KOmice were crossed to mouse or human wild-type α-synuclein trans-

genic mice (3) to generate synuclein null and rescued littermate an-imals. These rescued mice were used to confirm synuclein depen-dence of synapse size and I/O curves. However, due to the restrictedexpression pattern of the α-synuclein transgene in the retina and theage-dependent phenotypes of α-synuclein transgenics (3), we couldnot perform rescue experiments for the other observed phenotypes.

Quantitative Immunohistochemistry. Immunofluorescence of brainsections was performed as described (4). NeuN was used as a neu-ronal marker and synapsin and synaptophysin as synaptic markers.Blinded confocal images were quantified using ImageJ software.

Electron Microscopy. Hippocampal sections from 3- or 12-mo-oldmale mice were analyzed by electron microscopy (n = 2–3 mice/genotype; see Number of Mice for Experiments for specific num-bers). Electron micrographs were analyzed blind to genotypeusing iTEM software. Only asymmetric synapses (gray type I)were included for these analyses. Synaptic parameters measuredincluded: total number of synaptic vesicles, docked vesicles, ac-tive zone length, pre- and postsynaptic terminal area, number ofmitochondria, and number of synapses. For analysis of Schaffercollaterals, axon diameter and myelin thickness were measured.Data from mice of the same genotype and age were groupedtogether and statistically analyzed using the Student’s t test.

Hippocampal Electrophysiology. Vibratome-cut transverse hippo-campal slices (400-μm thick) from 3-mo and 12-mo-old mice wereused for field recordings as described previously (1). Input/output(I/O) curves were generated using stimulus intensities from 0 to 100μA in increments of 10 μA. Afferent volley responses (fiber volley)were isolated with 50 μM 5-phosphovaleric acid (D-APV), 100 μMpicrotoxin, and 10 μM 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline (NBQX) in the superfusion solution. Fiber volley andfieldEPSPmeasurementswereobtainedwith apatchpipette placedin the stratum radiatum within 200 μm of the stimulating electrodeand 100 μm of the slice surface. During the conduction velocityexperiments, fiber volley responses were recorded within 200 μmand 400 μm of the stimulating electrode. The action potential con-duction velocity was determined by calculating the shift in the timeof the peak of the fiber volley obtained when the responses wererecorded at 400 μm and 200 μm from the stimulating electrode.Frequency-following experiments were performed as previouslydescribed (5, 6). Activity-evoked changes in the excitability of CA1afferents were studied by delivering trains of stimuli to the stratumradiatum. Successive fiber volley responses evoked by trains of100 pulses stimuli at 10, 30, 100, and 300 Hz were recorded. Fre-quency-following capabilities were evaluated by plotting the am-plitude reduction of the afferent volley during repetitive stimulation(A100/A1 ratio) as a function of stimulation frequency.

ERG Recordings.ERG recordings were performed on anesthetizedmice as described (7). All recordings were performed blind togenotype.

Behavior.We performed behavioral tests for vision with cohorts ofage-matched wild-type and synuclein null mice. All behavioralexperiments were done blind to genotype.

Visual Placing Test. This test is used to evaluate visual abilities (8).Mice were suspended by the tail and moved toward an elevatedhorizontal beamwhile ensuring that their whiskers could not touchthe beam.Mice were scored on a scale of 1–5; 5 if they reached forthe beam immediately, 3 if they reached eventually, and 1 if they

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did not. Each mouse was tested in three independent trials andtheir scores were averaged.

Inverted Grid Test.This test was performed as described previously(3). Mice were placed on a 0.6 cm2 wire mesh and inverted ata height of 1 m. The time mice gripped the mesh was measuredfor a maximum of 120 s.

Quantitative Immunoblotting. Brain homogenates from3- or 12-mo-old wild-type and αβγ-synuclein KO mice (n = 4/genotype) wereelectrophoresed on SDS/PAGE gels and immunoblotted using

routine biochemical methods. Protein levels were quantified usingIRDye secondary antibodies on aLi-COROdyssey infrared imagingsystem. Actin and β-tubulin were used as internal loading controls.

Hippocampal Cultures. Dissociated hippocampal cultures wereprepared from P1 pups as previously described (1).

Lipid Analysis. Total brain lipids were extracted by the Bligh andDyer method (n = 3 animals/genotype) (9). The lipid composi-tion of wild-type and synuclein null mice was measured by HPLCat Avanti Polar Lipids.

1. Chandra S, et al. (2004) Double-knockout mice for alpha- and beta-synucleins: Effect onsynaptic functions. Proc Natl Acad Sci USA 101:14966–14971.

2. Rosahl TW, et al. (1995) Essential functions of synapsins I and II in synaptic vesicleregulation. Nature 375:488–493.

3. Chandra S, Gallardo G, Fernández-Chacón R, Schlüter OM, Südhof TC (2005) α-Synucleincooperates with CSPalpha in preventing neurodegeneration. Cell 123:383–396.

4. Ho A, et al. (2006) Genetic analysis of Mint/X11 proteins: Essential presynapticfunctions of a neuronal adaptor protein family. J Neurosci 26:13089–13101.

5. Malenka RC, Kocsis JD, Ransom BR, Waxman SG (1981) Modulation of parallel fiberexcitability by postsynaptically mediated changes in extracellular potassium. Science214:339–341.

6. Poolos NP, Mauk MD, Kocsis JD (1987) Activity-evoked increases in extracellularpotassium modulate presynaptic excitability in the CA1 region of the hippocampus.J Neurophysiol 58:404–416.

7. Vistamehr S, Tian N (2004) Light deprivation suppresses the light response of innerretina in both young and adult mouse. Vis Neurosci 21:23–37.

8. Crawley JN (2007) What’s Wrong with My Mouse? (Wiley, New York), 2nd Ed, pp86–110.

9. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. CanJ Biochem Physiol 37:911–917.

Fig. S1. Characterization of synuclein nulls rescued with a human α-synuclein transgene. (A) Western blotting of brain homogenates from αβγ-Syn−/− andαβγ-Syn−/− mice crossed to human α-synuclein transgenics (αβγ-Syn−/−; htg). Vasolin-containing protein (VCP) is used as a loading control. (B) Immunohisto-chemistry on αβγ-Syn−/− and αβγ-Syn−/−; htg hippocampal neurons. Like endogenous synuclein, human transgenic α-synuclein is present at synapses of culturedneurons (red, α-synuclein; green, dendrite marker MAP2). (C) Colocalization of human transgenic α-synuclein with presynaptic marker synapsin (red, α-syn-uclein; green, synapsin). (D) Synuclein expression in 3-mo-old wild-type αβγ-Syn+/+ and synuclein null αβγ-Syn−/− hippocampus as determined by immunohis-tochemistry. (E) Hippocampal sections of synuclein null αβγ-Syn−/− mice were stained with the presynaptic marker, synapsin (Scale bar, 100 μm.) (Right) Highmagnification view of CA3 stratum lucidum region (Scale bar, 5 μm.) (F) Presynaptic terminal area of CA3 synapses of synuclein nulls αβγ-Syn−/− and littermatesexpressing human wild-type α-synuclein (αβγ-Syn−/−; htg, red bar) as quantitated by synaptophysin staining. (G) Paired-pulse facilitation in Schaffer collateralsynapses in the hippocampus of 3-mo-old wild-type αβγ+/+, synuclein null αβγ−/−, synuclein nulls rescued by a mouse α-synuclein transgene αβγ−/−; mtg ora human transgene αβγ−/−; htg. (H) Action potential conduction velocity of Schaffer collaterals in 3-mo-old wild-type, synuclein null, synuclein nulls rescued bya mouse α-synuclein transgene, or a human transgene.

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Fig. S2. Normal brain architecture in aged synuclein null mice. Nissl stained saggital brain sections from 3-, 12-, and 24-mo-old wild-type (αβγ+/+) and synucleinnull (αβγ−/−) mice. (Scale bar, 200 μm.)

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Fig. S3. Analysis of neurodegeneration and gliosis in CA3 and CA1 subfields of hippocampus. (A) Representative images of 3-mo-old wild-type (αβγ-Syn+/+) andsynuclein null (αβγ-Syn−/−) hippocampi stained with NeuN. (B) Quantification of NeuN positive nuclei in the CA3 subfield of hippocampus at 3, 12, and 24 mo ofage (wild-type αβγ+/+ blue bar; synuclein null αβγ-Syn−/− green bar; n = 3 mice/genotype/age). (C) Quantification of NeuN positive nuclei in the CA1 subfield ofhippocampus at 24 mo of age (n = 3 mice/genotype). (D) Representative images of 3-, 12-, and 24-mo-old wild-type (αβγ-Syn+/+) and synuclein null (αβγ-Syn−/−)hippocampi stained with glial fibrillary acidic protein (GFAP), a marker for astrogliosis, and developed using diaminobenzidine precipitation. (Scale bar, 200 μm.)(E) Striatum sections from young (3 mo) and old (∼12 mo) wild-type and synuclein null mice stained with antibodies to tyrosine hydroxylase to reveal dopa-minergic neurons and termini. (Upper) Low magnification; (Lower) high magnification images of the striatum. (Scale bar, 100 μm.) (F) Quantification of tyrosinehydroxlyase staining shown in E. *P < 0.05.

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Fig. S4. Synapse density and size quantification in aged synuclein null mice. (A) Quantification of synapse density in the CA3 region of the hippocampus of 3-and 12-mo-old wild-type (αβγ+/+) and synuclein null (αβγ−/−) mice using synapsin staining (n = 6 mice/genotype/age). (B) Quantification of synapse density in theCA3 region of the hippocampus of 3- and 24-mo-old wild-type (αβγ+/+) and synuclein null (αβγ−/−) mice using synapsin staining (n = 3 mice/genotype/age). (C)Quantification of synapse density in CA1 region using blinded electron micrographs derived from 3- and 12-mo-old wild-type (αβγ+/+) and synuclein null (αβγ−/−)mice. (3 mo, αβγ+/+, n = 78 micrographs; αβγ−/−, n = 76 micrographs; 12 mo, αβγ+/+, n = 52 micrographs; αβγ−/−, n = 54 micrographs). (D) Presynaptic puncta sizeof 3-, 12-, and 24-mo-old wild-type (αβγ+/+) and synuclein null (αβγ−/−) mice using synapsin staining (n = 3–6 mice/genotype/age). Data for 3-mo-old mice aretaken from Fig. 2C. Statistical analysis by Student’s t test **P < 0.001, ***P < 0.0001.

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Fig. S5. Changes in excitability of Schaffer collaterals. Excitability changes during repetitive stimulation (100 pulses) after abolition of postsynaptic activity inslices of 12-mo-old wild-type mice (αβγ-Syn+/+, n = 15 slices from 4 animals; blue triangle) and synuclein null mice (αβγ-Syn−/−, n = 13 slices from 4 animals; greensquare). (A) Fiber volley frequency-following capabilities: plots of the amplitude reduction of the fiber volley (A100/A1 ratio) as a function of stimulationfrequency (10, 30, 100, and 300 Hz). (B) Representative traces show the comparison of afferent volley responses at pulse 1 (thin line) and pulse 100 (thick line)obtained from wild-type mice and synuclein null mice. Change in amplitude of the presynaptic volley as a percentage of baseline value (first response in train)for responses recorded during 1 s of stimulation (100 pulses) at 100 Hz. Statistical difference indicated by *P < 0.05, **P < 0.01, and ***P = 0.001.

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Fig. S6. Ultrastructural analysis of Schaffer collaterals. (A) The diameter of Schaffer collaterals and (B) thickness of myelin sheaths were quantified fromblinded electron micrographs of wild-type (αβγ+/+) and synuclein null (αβγ−/−) hippocampi. (Left) Age = 3 mo; (Right) age = 12 mo. (C) g, the ratio of axondiameter to total fiber diameter (including myelin) for wild-type (αβγ+/+) and synuclein null (αβγ−/−) mice at 3 mo (Left) and 12 mo (Right) of age. Statisticalanalysis by Student’s t test *P < 0.05, **P < 0.001.

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Fig. S7. Retinal immunohistochemistry of wild-type and synuclein null mice. Following the ERG recordings, wild-type (αβγ-Syn+/+) and synuclein null(αβγ-Syn−/−) mice were killed and the retina analyzed by immunohistochemistry. Representative images from 3-mo (Left) and 12-mo (Right)-old mice of thedenoted genotypes. (Top) Immunostaining of retina for rhodopsin to reveal outer segments (OS) of photoreceptors and the presynaptic marker, CSPα for theouter plexiform layer (OPL). (Middle) immunostaining of CSPα to demarcate outer- and inner plexiform layers (IPL). (Scale bar, 100 μm.) (Bottom) Low mag-nification Nissl staining of retinal cell layers. Data show no cellular degeneration or synaptic disorganization in either wild-type or synuclein null mice at either3 or 12 mo.

Fig. S8. Electron microscopy of synuclein null retina. Ultrastructure of wild-type (αβγ-Syn+/+) and synuclein null (αβγ-Syn−/−) retina showing the outer segment,OPL, inner nuclear layer, IPL, and retinal ganglion layer. (Left) Age = 3 mo; (Right) age = 12 mo.

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Fig. S9. Lipid analysis of synuclein null mice. Total brain lipids were extracted from wild-type (αβγ-Syn+/+, blue bar) and synuclein null (αβγ-Syn−/−, green bar)brains by the Bligh and Dyer method and analyzed by HPLC. PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; LPS, lyso-phosphatidylserine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; SM, sphingomylein; PA, phosphatidic acid; and PS, phosphatidylserine (n = 3mice/genotype).

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Table S1. Levels of neuronal proteins in wild-type and synuclein null brains

Wild-type αβγ+/+

averageWild-type αβγ+/+

SEMKO αβγ−/−

averageKO αβγ−/−

SEMt testP value

α-Synuclein 1 0.061 0.002 0.002 3.240E-06β-Synuclein 1 0.093 0.024 0.024 5.34E-05γ-Synuclein 1 0.157 0.008 0.005 7.46E-4Parkin 1 0.121 1.275 0.142 0.118LRRK2 1 0.099 0.790 0.037 0.062UCHL-1 1 0.058 1.068 0.057 0.435PINK 1 0.036 0.923 0.080 0.412DJ-1 1 0.060 0.961 0.102 0.750Synphilin 1 0.108 1.011 0.166 0.957Tyrosine hydroxylase 1 0.012 0.952 0.056 0.435Nurr1 1 0.091 1.259 0.112 0.123VMAT2 1 0.120 0.897 0.124 0.573COMT 1 0.038 1.199 0.048 0.017AADC 1 0.050 0.967 0.129 0.816Amphiphysin 1 0.056 1.193 0.153 0.257Dynamin 1 0.049 1.104 0.125 0.468Dynamin I 1 0.059 1.002 0.025 0.981Dynamin III 1 0.077 1.309 0.153 0.625Auxilin 1 0.031 1.073 0.086 0.473Epsin 1 0.046 1.187 0.102 0.107Clathrin light chain 1 0.070 1.207 0.066 0.075AP180 1 0.123 0.813 0.127 0.332Synaptojanin 1 0.103 1.195 0.090 0.203CSPα 1 0.066 0.991 0.104 0.941HSC-70 1 0.056 1.108 0.051 0.174Syntaxin I 1 0.039 0.933 0.041 0.255Syntaxin Ia (U6250) 1 0.118 0.963 0.056 0.785SNAP-25 (P913) 1 0.095 1.100 0.042 0.373Synaptobrevin 1 0.040 1.033 0.054 0.328Complexin II 1 0.043 1.484 0.110 0.006Complexin I + II 1 0.046 1.297 0.099 0.015NSF 1 0.090 0.870 0.035 0.230Synaptotagmin I 1 0.013 0.882 0.057 0.091Synapsin Ia + Ib 1 0.047 1.177 0.080 0.078Synapsin IIa 1 0.026 0.952 0.040 0.357Synapsin IIb 1 0.056 1.314 0.066 0.003Rab 3A 1 0.041 1.045 0.070 0.592Rabphillin 1 0.143 1.082 0.031 0.597Tomosyn 1 0.045 1.005 0.071 0.958APPL1 1 0.066 0.986 0.015 0.842SynCAM 1 0.085 1.209 0.070 0.107N-cadherin 1 0.049 0.876 0.066 0.183Munc 18 1 0.034 1.057 0.046 0.35514-3-3 β 1 0.051 1.251 0.067 0.01614-3-3 ε 1 0.061 1.292 0.106 0.041

Levels of neuronal proteins in wild-type (αβγ+/+) and synuclein null (αβγ−/−) mice (3 mo old; n = 4 brains/genotype) were determined by quantitative immunoblotting. All antibodies have been described (1) or arecommercially available.

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