Primary neuron cultures were prepared from male and female E18 Arc KO or WT mouse cortex and hippocampus as previously described (). Tissue was dissociated in DNase (0.01%; Sigma-Aldrich, St. Louis, MO) and papain (0.067%; Worthington Biochemicals, Lakewood, NJ), and then triturated with a fire-polished glass pipette to obtain a single-cell suspension. Cells were pelleted at 500xg for 4 min, the supernatant removed, and cells resuspended and counted with a TC-20 cell counter (Bio-Rad, Hercules, CA). Neurons were plated on glass coverslips (Carolina Biological Supply, Burlington, NC) coated with poly--lysine (0.2 mg/mL; Sigma-Aldrich) in 12-well plates (Greiner Bio-One, Monroe, NC) at 90,000 cells/mL, or in 10-cm plastic dishes at 800,000 cells/mL. Neurons were initially plated in Neurobasal media containing 5% horse serum, 2% GlutaMAX, 2% B-27, and 1% penicillin/streptomycin (Thermo Fisher Scientific) in a 37°C incubator with 5% CO. On DIV4, neurons were fed via half media exchange with astrocyte-conditioned Neurobasal media containing 1% horse serum, GlutaMAX, and penicillin/streptomycin, 2% B-27, and 5 μM cytosine β--arabinofuranoside (AraC) (Sigma-Aldrich). Neurons were fed with astrocyte-conditioned media every three days thereafter.

C57BL/6 Arc knock-out (KO) mice (C57BL/6-Arc/J, a kind gift from Dr. Kuan Wang, NIH) have GFP knocked in to the Arc ORF (). Arc KO and wild-type (WT) mice used in these studies were littermates from heterozygous (Arc) crosses. Both male and female mice were used. No differences between sexes in the experiments conducted in this study were noted, and data from both sexes were therefore grouped together. Mice were housed in breeding pairs, or group-housed with littermates of the same sex after weaning (2-5 mice/cage), on a 12:12 h day:night cycle, with food and water provided ad libitum. Hippocampal and cortical primary neuron cultures were prepared from E18 embryos, while brain lysates were taken from P30-50 mice. Mice were test- and procedure-naive before terminal experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Utah.

HEK293T cells were purchased from ATCC (#CRL-11268). Cells were maintained at 37°C with 5% CO 2 in DMEM media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA) and passaged every 3-4 days at 70% confluency. For transfections and transfer experiments, HEK cells were seeded to 10-cm dishes or collagen-coated glass coverslips in 12-well plates.

Method Details

Plasmids The open reading frame (ORF) of full-length rat Arc (NP_062234.1) cDNA was subcloned from pRK5-myc-Arc. The insert was amplified by PCR, digested with BamH1 and Xho1, and ligated into the pGEX-6p1 (GE Healthcare, Little Chalfont, UK) expression vector between the BamH1 and Xho1 restrictions sites. The GST-Arc ORF was similarly amplified and cloned into the pFastBac1 vector (Thermo Fisher Scientific) between the BamH1 and Xho1 restriction sites. prArc-ΔCTD was generated by blunt end cloning after PCR amplification of the Arc ORF from pGEX-6p1-Arc, excluding sequence coding aas 277-374. aas 195-364 of the Arc ORF (CA-prArc) was similarly cloned into the pET11a vector, which contained a His tag. pBluescript-SKII-GFP was generated by restriction digest of mEGFP (BBA16881.1) from pGL4.11-arc7000-mEGFP-ArcUTRs (generously provided by Dr. Haruhiko Bito, University of Tokyo) and subsequent ligation into the KpnI and SacI restriction sites flanking the insert in pBluescript-SKII-ArcUTRs plasmid (generously provided by Dr. Kristen Keefe, University of Utah). The pGEX-4T-1 Drosophila Arc1 (NP_610955.1) construct was provided by Dr. Mark Metzstein, University of Utah. EGFP-C3-Arc and pRK5-myc-Arc were generously provided by Dr. Kimberly Huber (UT Southwestern) and Dr. Paul Worley (Johns Hopkins University), respectively. All protein expression constructs were transformed into DH5α E. coli cells and individual colonies were screened by Sanger Sequencing (GeneWiz, South Plainfield, NJ) sequencing services, using primers synthesized by Integrated DNA Technologies (Coralville, IA). Trace files were analyzed using A Plasmid Editor (APE) freeware available from the University of Utah. Sequenced verified constructs were then transformed into BL21-DE3 bacterial cells for protein expression. See Table S1 for specific oligo primer sequences.

Protein purification Starter bacteria cultures for protein expression were grown overnight at 37°C in LB supplemented with ampicillin and chloramphenicol. Starter cultures were used to inoculate large-scale 500 mL cultures of ZY auto-induction media. Large-scale cultures were grown to OD 600 of 0.6-0.8 at 37°C at 150 rpm and then shifted to 19°C at 150 rpm for 16-20 h. Cultures were then pelleted at 5000xg for 15 min at 4°C and cell pellets were resuspended in 30 mL lysis buffer (500 mM NaCl, 50 mM Tris, 5% glycerol, 1 mM DTT, pH 8.0 at room temperature (RT) for Arc constructs and GST; 300 mM KCl, 50 mM Tris, 1% Triton X-100, 1 mM DTT, pH 7.4 at RT for Endophilin3A) and flash frozen in liquid nitrogen. Frozen pellets were thawed quickly at 37°C and brought to a final volume of 1 g pellet:10 mL lysis buffer, supplemented with DNase, lysozyme, aprotinin, leupeptin, PMSF, and pepstatin. Lysates were then sonicated for 8-10x45 s pulses at 90% duty cycle and pelleted for 45 min at 21,000xg. For GST-tagged constructs, cleared supernatants were then passed through a 0.45 μm filter and incubated with pre-equilibrated GST Sepharose 4B affinity resin in a gravity flow column overnight at 4°C. Bound protein was then washed twice with two column volumes (20 resin bed volumes each) of lysis buffer, re-equilibrated with 150 mM NaCl, 50 mM Tris, 1 mM EDTA, 1 mM DTT, pH 7.2 at RT, and cleaved on-resin overnight at 4°C with PreScission Protease (GE Healthcare) for the GST-tagged constructs, or thrombin (Sigma-Aldrich) for dArc1. Cleaved proteins were then buffer exchanged to 150 mM NaCl, 50 mM Tris, pH 7.4 at RT to kill protease activity, run on an S200 size exclusion column to separate the cleaved protein, and peak fractions were pooled. GST was affinity-purified as described above using Sepharose 4B resin and eluted directly using 15 mM reduced l -glutathione, 10 mM Tris, pH 7.4 at RT. His-tagged CA-prArc was affinity-purified as described above using Ni+ resin (Roche, Basel, Switzerland) and eluted directly using 250 mM imidazole, 10 mM Tris, pH 7.4 at RT. GST and CA-prArc were then buffer exchanged to 150 mM NaCl, 50 mM Tris, pH 7.4 at RT. To strip Arc protein of nucleic acids for prArc(RNA-) preparations, cell pellets were lysed in 20 mM NaCl, 50 mM Tris, 5% glycerol, 2 mM MgCl 2 , 1 mM DTT, pH 8.0 at RT as described above. Nucleic acids were precipitated from cell supernatants by dropwise addition of 10% PEI, pH 8.0 to a final concentration of 0.1% followed by incubation at 4°C for 20 min and pelleting for 20 min at 27,000xg. The resulting supernatant was then precipitated by addition of saturated ammonium sulfate to a final concentration of 30%. Precipitated protein was pelleted at 10,000xg for 10 min, resuspended in 60 mL lysis buffer, and affinity purified. The cleaved affinity-purified product was then dialyzed to Q-column buffer A (Q-A; 20 mM NaCl, 50 mM Tris, pH 7.4 at RT) overnight. Dialyzed protein was then subjected to anion exchange chromatography (HiTrap Q, GE Healthcare) with a gradient of Q-A buffer to Q-B buffer (1 M NaCl, 50 mM Tris, pH 7.4). Average yields for purified proteins were 10.5 mg (8-13 mg) per liter of cell culture.

Electron microscopy Negative stain For all negative stain specimens, copper 200-mesh grids coated with Formvar and carbon (Electron Microscopy Sciences or Ted Pella, Redding, CA) were glow discharged for 20-45 s in a vacuum chamber at 30mA. 3.5 μL sample was then applied to the grid for 35-45 s and excess sample was wicked away using filter paper. Grids were then immediately washed 2-4x for 5 s with 30 μL water droplets, then once with 1% uranyl acetate (UA) on parafilm. Excess water/UA was wicked away and then a final droplet of UA was applied for 30 s. Excess UA was wicked away and grids were air-dried for 30-60 s. Imaging was performed using either an FEI T12, FEI Tecnai Spirit microscope operated at 120 kV equipped with a Gatan Orius SC200B CCD camera or JEOL 1400 electron microscope. Cryo-EM Purified Arc protein was dialysed into 300 mM NaCl, 50 mM Tris, pH 7.4 and concentrated twice using Amicon 100 MWCO centrifugal filters (Millipore, Burlington, MA) to yield a final protein concentration of ∼2 mg/mL. 10 nm diameter gold beads were added to the sample. Degassed 2/2-3C C-flat grids (Electron Microscopy Sciences, Hatfield, PA) were glow discharged for 45 s at 30 mA. Sample was applied to the grid 2 times for 30 s, and the grid was plunge frozen in liquid ethane using a FEI Vitrobot Mark IV. Micrographs were acquired using a FEI Tecnai G2 F20 microscope operated at 200 kV, equipped with a FEI Falcon II direct detector. The nominal defocus was 1.3 μm. EM quantification Grids were surveyed visually to check for uniformity of sample application. For each experiment, six images were taken from randomly selected grid squares. Full and partially formed particles between 20-40 nm were then counted manually using ImageJ. Counts were then divided by the image field of view (2.07 μm2) and data presented as oligomer count/μm2. Arc capsid assembly assay GFP mRNA was added to prArc(RNA-) (5 mg/mL in low salt buffer: 20 mM NaCl, 50 mM Tris, pH 7.4 at RT) at a nucleic acid:protein ratio of 7.3% (w/w) (corresponding to 1 molecule of Arc to 10 nucleotides). Reactions were then diluted to 1 mg/mL of prArc(RNA-) by dropwise addition of low salt buffer or capsid assembly buffer (500 mM NaPO 4 , 50 mM Tris, 0.5 mM EDTA, pH 7.5 at RT) and incubated for 2 h at RT. Following incubation, negative stain EM grids were prepared of each reaction at 0.25 mg/mL and capsid formation was quantified by manual counting of 6 images. Fully formed capsids included spherical particles between 20-50 nm with clear double shells. Similar results were seen in three independent protein preparations.

Dynamic light scattering Purified Arc protein was subjected to dynamic light scattering measurements on a Malvern Zetasizer Nano ZSP instrument. The scattering was carried out at 25°C and at a fixed angle of 173° (backward scattering). The scattered intensity is represented as number of particles under the assumption that the scattering intensity from spherical particles is proportional to the size to the sixth power.

Phylogenetic reconstruction NCBI genome sequence databases were queried using the human or Drosophila melanogaster Arc protein sequence using tBLASTn. Repbase was also queried using the CENSOR program to identify known repeat families with high sequence similarity to mammalian or brachyceran Arc genes, respectively. The following sequence IDs were used for analysis: (GenBank locus) Mm ARC—AHBB01089569; Hs ARC—LIQK02016549; Ac ARC—AAWZ02020354; Lc gypsy2—AFYH01030203; CC gypsy—LHQP01046008; Dm ARC1—JSAE01000572; Ds ARC1—CAKG01020471; Sc ARC1—LDNW01019671; Dm ARC2—JXOZ01003752; Ds ARC2—AWUT01001000; Sc ARC2—LDNW01019670; Bm gypsy—BABH01046987; Tc gypsy—AAJJ02003810. Repbase: Lc gypsy—Gypsy2-1-I_Lch; Dr gypsy26—Gypsy-26-I_DR; Lh gypsy11—Gypsy-11_LH-I; Dm gypsy1—Gypsy1-I_DM; ty3—TY3. Protein (Arc and Gag) sequences that were found to have high similarity to Arc proteins and Gags of other related Ty3/gypsy elements were aligned using the MUSCLE program. Trimmed Arc/Gag alignments were uploaded to MEGA7 for subsequent maximum likelihood phylogenetic reconstruction using default parameters, and 500 bootstrap iterations were performed to generate a lineage tree. Drosophila melanogaster dArc1 and dArc2 protein sequences were used to query schizophoran fly protein databases using BLASTp. More hits were observed than expected if darc1 were present in one-to-one orthologs in the species examined. Protein FASTA sequences were aligned using MUSCLE and a maximum likelihood phylogram was generated using MEGA.

HEK cell experiments Transfections HEK cells were transfected using polyethyleneimine (PEI) at a ratio of 3 μg PEI:1 μg DNA diluted in Opti-MEM (Thermo Fisher Scientific). Cells were transfected at approximately 60%–70% confluency. For EV isolation and media transfer experiments, culture media was exchanged 4-6 h post-transfection to remove PEI and DNA, and media was harvested 24 h later. Transfection and transfer Media from transfected HEK cells was harvested 24 h post-transfection and centrifuged at 500xg for 4 min to remove dead cells and debris. Media from untransfected, naive cells was removed and replaced with the cleared transfected media and incubated for an additional 24 h. Following incubation, cells were fixed and combined immunocytochemistry/fluorescence in situ hybridization (ICC/FISH) for Arc or GFP protein and RNA was performed as described below. Endocytosis blockade To block endocytosis, a group of naive HEK cells plated on coverslips in 12-well plates that were receiving media from GFP-Arc-transfected HEK cells were treated at the same time with 80 μM Dynasore (Abcam, Cambridge, MA) for the first 6 h, then the media was removed and replaced with fresh HEK media. 18 h later, Dynasore-treated and untreated HEK cells were fixed. The entire 18-mm coverslip was viewed with a 20x objective and the number of clusters of GFP-Arc-transferred cells was manually counted. Representative images were obtained using a 20X objective on an Olympus FV1000 confocal microscope (Tokyo, Japan).

Neuron transfer experiments DIV15 cultured neurons were used for all neuronal experiments. For purified Arc protein incubation experiments, neurons were treated with 4 μg of purified prArc, prArc-ΔCTD, CA-prArc, or prArc(RNA-) protein in normal neuronal feeding media and incubated for 1 or 4 h. For extracellular vesicle (EV) incubation experiments, neurons were treated with 10 μg protein from the purified EV fraction obtained from eight 10-cm dishes of DIV15 cultured cortical neurons in which E18 WT cortical neurons had been plated at 800,000 cells/mL (see “Cell Culture” methods), and incubated for 1 or 4 h. A subset of neurons in the purified protein- and EV-treated experiments was treated with 100 μM of the group 1 mGluR agonist dihydroxyphenylglycine ((S)-3,5-DHPG; Tocris Bioscience, Bristol, UK) for 5 min, which was then washed out and replaced with previously conditioned neuronal media, and neurons were allowed to rest for 25 min before fixation. To block protein translation during DHPG treatment, a subset of neurons was pretreated with 180 μM cycloheximide (CHX, Sigma-Aldrich) 30 min before DHPG. CHX was left in the media for 1 h total. To block endocytosis, neurons were pretreated with 80 μM Dynasore (Abcam, Cambridge, MA) for 30 min before adding purified protein. For RNase treatments, a sample of either prArc or WT EV was incubated with RNase A (1:1000; Omega Bio-tek, Norcross, GA) for 15 min, then SUPERase-In RNase Inhibitor (1 U/μL; Thermo Fisher Scientific) immediately before being added to neurons. The treated samples were then added to neurons and incubated for 4 h.

Immunocytochemistry After treatments, neurons were washed twice with 37°C 4% sucrose/1X phosphate-buffered-saline (PBS; 10X: 1.4 M NaCl, 26.8 mM KCl, 62 mM Na 2 HPO 4 , 35.3 mM KH 2 PO 4 , pH 7.4), then fixed for 15 min with 4% sucrose/4% formaldehyde (Thermo Fisher Scientific) in 1X PBS. Neurons were washed 3 × 5 min with 1X PBS, permeabilized for 10 min with 0.2% Triton X-100 (Amresco, Solon, OH) in 1X PBS, and blocked for 30 min in 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) in 1X PBS. Neurons were then incubated in primary antibody diluted in block for 1 h at RT, washed 3 × 5 min in 1X PBS, and incubated in secondary antibody diluted in block for 1 h at RT. Neurons on coverslips were mounted on glass slides in Fluoromount (Thermo Fisher Scientific) and dried overnight at RT. Primary antibodies used were: rabbit anti-Arc (1:1000; custom-made; ProteinTech, Rosemont, IL); rabbit anti-Arc (1:1000; Synaptic Systems, Goettingen, Germany); chicken anti-MAP2 (1:5000; ab5392; Abcam); mouse anti-Rab5 (1:1000; BD Biosciences, San Jose, CA); DAPI nuclear stain (Molecular Probes, Thermo Fisher Scientific). Secondary antibodies used were: Alexa Fluor 405, 488, 555, or 647 for the appropriate animal host (1:750; Thermo Fisher Scientific or Jackson ImmunoResearch).

Combined FISH/ICC in neurons and HEK cells Daberkow et al., 2007 Daberkow D.P.

Riedy M.D.

Kesner R.P.

Keefe K.A. Arc mRNA induction in striatal efferent neurons associated with response learning. 2 O, denatured at 90°C for 5 min, put on wet ice for 2 min, then mixed with RNA hybridization buffer (23.75 mM Tris-HCl, 1.19 mM EDTA, 357 mM NaCl, 11.9% dextran sulfate, 1.19X Denhardt’s solution (Thermo Fisher Scientific), 2.5% nuclease-free water, 60% formamide (Fisher Scientific, Hampton, NH)). The Arc probe (1:500) or GFP probe (1:750) was hybridized to the cultured cells at 56°C for 16 h. The following day, cells underwent a series of washes to decrease background signal: 3 × 5 min 2X SSC, 15 min in RNase A (1:1000; Omega Bio-tek) at 37°C, 10 min 2X SSC at RT, 10 min 0.2X SSC at RT, 15 min 0.2X SSC at 56°C, 10 min 0.2X SSC at RT, 5 min TNT (0.1 M Tris-HCl, 0.15 M NaCl, 0.05% Tween-20, pH 7.5). Cells were then blocked in TNB (0.1 M Tris-HCl, 0.15 M NaCl, 0.5% w/v blocking reagent (Sigma-Aldrich), pH 7.5) with 2.5% sheep serum (Jackson ImmunoResearch) and 2.5% donkey serum for 30 min. In the primary antibody step, a DIG-HRP (1:1000; Sigma-Aldrich) and either MAP2 (1:2500; Abcam), Arc (1:500; custom-made), or Rab5 (1:500; BD Biosciences) antibody were diluted together in TNB with 2.5% sheep serum and 2.5% donkey serum and incubated on the cells for 1 h. After 3 × 5 min washes in TNT, the DIG-HRP signal was developed using a TSA Plus Cyanine 3 kit (1:50; PerkinElmer, Waltham, MA) for 30 min. Cells were washed for 5 min in TNT and 5 min in 1X PBS, then secondary antibody was diluted 1:750 in 5% donkey serum and 1X PBS and incubated on the cells for 1 h to detect MAP2, Arc, or Rab5. Nuclei were stained with DAPI (Thermo Fisher Scientific), then coverslips were mounted on glass slides with Fluoromount and dried overnight at RT. The fluorescent in situ hybridization (FISH) procedure for Arc and GFP was based on a previously published protocol (). We used a full-length rat Arc ribonucleotide probe (rat and mouse Arc are 99% identical at the aa level) or EGFP (see cloning strategy above in “Plasmids”) as in the published protocol, but modified the protocol for use in cultured neurons and HEK cells instead of brain sections. Arc and GFP plasmids were linearized with Not1 and purified via standard phenol/chloroform extraction. The linearized antisense Arc or GFP were used to make a ribonucleotide probe that had DIG-UTP incorporated using a T7 DIG RNA labeling kit (Sigma-Aldrich), then purified with a G-50 spin column (GE Healthcare). Cells were washed once with 37°C 4% sucrose/1X PBS, then fixed for 15 min with 4% sucrose/4% formaldehyde in 1X PBS. Cells were washed 3 × 5 min with 1X PBS, permeabilized in 0.2% Triton X-100 for 10 min, washed 2 × 5 min in 1X PBS, then 5 min with 2X saline-sodium citrate (SSC; 20X: 3 M NaCl, 300 mM citric acid trisodium salt dihydrate, pH 7). Cells were prehybridized in 1X prehybridization solution (Sigma-Aldrich) for 30 min. The DIG-labeled Arc or GFP ribonucleotide probe was diluted 1:3 with ddHO, denatured at 90°C for 5 min, put on wet ice for 2 min, then mixed with RNA hybridization buffer (23.75 mM Tris-HCl, 1.19 mM EDTA, 357 mM NaCl, 11.9% dextran sulfate, 1.19X Denhardt’s solution (Thermo Fisher Scientific), 2.5% nuclease-free water, 60% formamide (Fisher Scientific, Hampton, NH)). The Arc probe (1:500) or GFP probe (1:750) was hybridized to the cultured cells at 56°C for 16 h. The following day, cells underwent a series of washes to decrease background signal: 3 × 5 min 2X SSC, 15 min in RNase A (1:1000; Omega Bio-tek) at 37°C, 10 min 2X SSC at RT, 10 min 0.2X SSC at RT, 15 min 0.2X SSC at 56°C, 10 min 0.2X SSC at RT, 5 min TNT (0.1 M Tris-HCl, 0.15 M NaCl, 0.05% Tween-20, pH 7.5). Cells were then blocked in TNB (0.1 M Tris-HCl, 0.15 M NaCl, 0.5% w/v blocking reagent (Sigma-Aldrich), pH 7.5) with 2.5% sheep serum (Jackson ImmunoResearch) and 2.5% donkey serum for 30 min. In the primary antibody step, a DIG-HRP (1:1000; Sigma-Aldrich) and either MAP2 (1:2500; Abcam), Arc (1:500; custom-made), or Rab5 (1:500; BD Biosciences) antibody were diluted together in TNB with 2.5% sheep serum and 2.5% donkey serum and incubated on the cells for 1 h. After 3 × 5 min washes in TNT, the DIG-HRP signal was developed using a TSA Plus Cyanine 3 kit (1:50; PerkinElmer, Waltham, MA) for 30 min. Cells were washed for 5 min in TNT and 5 min in 1X PBS, then secondary antibody was diluted 1:750 in 5% donkey serum and 1X PBS and incubated on the cells for 1 h to detect MAP2, Arc, or Rab5. Nuclei were stained with DAPI (Thermo Fisher Scientific), then coverslips were mounted on glass slides with Fluoromount and dried overnight at RT.

Cell imaging and analysis Imaging Coverslips were imaged using a 60X oil objective on an Olympus FV1000 confocal microscope (Tokyo, Japan) and images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD). Neurons included for analysis were selected in an unbiased manner by looking at MAP2 dendritic morphology for cell health. Coverslips were viewed blind to find the brightest immunofluorescence in each independent experiment, and this value was then used to set the image acquisition settings for that experiment. Images from all coverslips in that experiment were then acquired using the exact same settings. Analysis of dendritic Arc protein and mRNA expression During analysis, images were blindly thresholded (to remove background fluorescence and to ensure images were analyzed in the linear range) to the brightest immunofluorescence in an individual experiment, and the same threshold was applied to all other images in that experiment. Integrated density (average pixel intensity x area) of two 30-μm dendritic segments/neuron was measured from each coverslip. In general, thick proximal dendritic branches were avoided in our analysis to control for potential differences in dendritic volume. The KO control group in each experiment, whether ICC or FISH, was set as “1,” and the integrated density values in the other groups were normalized to this and are displayed in the graphs as fold-change ± SEM. For representative images in the figures, the Smart look-up table (LUT) in ImageJ was applied to highlight differences in Arc expression between groups. Analysis of Arc/Rab5 colocalization Two 30-μm dendritic segments/neuron were selected for analysis of Arc protein or mRNA colocalization with Rab5 protein. The Arc channel and Rab5 channel were thresholded to the same value across all images. Using ImageJ, a mask was made of the thresholded section of dendrite for both Rab5 and Arc. The Arc mask was applied to the Rab5 mask and the number of overlapping puncta was quantified. The number of Arc particles overlapping Rab5 was divided by the total number of Arc particles in the stretch of dendrite to determine the Arc/Rab5 colocalization.

Western blots Immunoblotting and analysis Western blot samples were mixed with 4X Laemlli buffer (40% glycerol, 250 mM Tris, 4% SDS, 50 mM DTT, pH 6.8) and heated at 70°C for 5 min. SDS-PAGE gel electrophoresis was used to separate protein samples. Separated samples were transferred to a nitrocellulose membrane (GE Healthcare). Following transfer, membranes were briefly stained with 0.1% Ponceau stain, then destained with 1% acetic acid to remove background, for imaging of total protein. Membranes were blocked in 5% milk + 1X tris-buffered saline (TBS; 10X: 152.3 mM Tris-HCl, 46.2 mM Tris base, 1.5 M NaCl, pH 7.6) for 30 min at RT, then incubated in primary antibody in 1X TBS for either 1 h at RT or overnight at 4°C. Membranes were washed 3 × 10 min in 1X TBS, then incubated in an HRP-conjugated secondary antibody (Jackson ImmunoResearch) in block for 1 h at RT. After 3 × 10 min in 1X TBS, a chemiluminescent kit (Bio-Rad, Hercules, CA) was used to detect the protein bands, and the membranes were imaged on an Azure c300 gel dock (Azure Biosystems, Dublin, CA). Blots were analyzed and quantified using the Gel Analysis plugin in ImageJ. Antibodies Antibodies were used at the following concentrations: Arc (1:000; mouse monoclonal, Santa Cruz), Arc (1:000; rabbit polyclonal, custom, Protein Tech), ALIX (1:500; rabbit polyclonal, custom, provided by Dr. Wesley Sundquist), actin (1:1000; HRP-conjugated, Abcam), GFP (1:1000; chicken polyclonal, Aves). All secondary antibodies were used at a dilution of 1:10,000 (HRP-conjugated goat anti-rabbit, goat anti-mouse, goat anti-chicken, Jackson ImmunoResearch).

Coomassie gels Samples for analysis via SDS-PAGE were mixed with 4X Laemlli buffer and heated at 70°C for 5 min. Protein samples were separated on 10% SDS gels. Gels were then stained with 0.1% Coomassie blue stain (0.1% w/v Coomassie blue, 50% methanol, 10% acetic acid, 40% water) for 30 min and destained overnight in destain solution (50% methanol, 10% acetic acid, 40% water). Gels were visualized using an Azure c300 gel dock under the auto-exposure setting on the visible channel. Gel exposures were analyzed and quantified using the Gel Analysis plugin in ImageJ.

Immunoprecipitation WT and Arc KO cortices were dissected out and homogenized in 150 mM NaCl, 50 mM Tris, 1% Triton X-100, 0.5% sodium deoxycholate, 0.05% SDS, pH 7.4 (IP lysis buffer), with protease inhibitor added fresh (Roche). Homogenates were pelleted at 200xg for 5 min at 4°C to remove tissue debris. Supernatants were removed, diluted from 2 mL to 4 mL, and rocked at 4°C for 10 min before being pelleted at 17,000xg for 10 min at 4°C to remove insoluble material. Cleared supernatants were removed, a small aliquot was taken as the input, and the remainder used for immunoprecipitation. Supernatants were immunoprecipitated with either Arc antibody (rabbit polyclonal, custom-made; Protein Tech) or normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) at 1 μg/500 μL lysate for 2 h at 4°C with gentle rocking. Following antibody incubation, a 10% volume of washed 50/50 Protein A bead slurry (Thermo Fisher Scientific) was added to the antibody/lysate mixture and incubated for an additional hour at 4°C with rocking. Bead-antibody complexes were then pelleted briefly at low speed, supernatants were removed, and beads were washed three times with IP buffer. Washed beads were then resuspended in 200 μL IP buffer. With half of the bead slurry, protein was eluted from the beads with 17 μL 4X Laemlli buffer for 5 min at RT, then 50 μL IP buffer was added and the solution was removed from the beads into a new tube and heated at 70°C for 5 min. The input (10% lysate volume) and 30 μL each of the IgG and antibody elutions were separated by SDS-PAGE on a 10% acrylamide gel and immunoblotted as described above. The bands for the input and IgG and Arc elutions were analyzed using the Gel Analysis plugin in ImageJ, and the data were represented graphically as a ratio of the signal from each elution over the input signal from each individual mouse. With the other half of the bead slurry, the IP buffer was adjusted to 1% SDS and 0.8 mg Proteinase K (New England Biolabs, Ipswich, MA) was added. Samples were then incubated at RT for 30 min with rocking and total RNA was extracted as described below.

Chemical crosslinking of Arc proteins in situ Transfected HEK cells expressing myc-Arc-WT or a GFP control were briefly trypsinized, quenched with DMEM (Thermo Fisher Scientific), and pelleted. Media was removed and pelleted cells were then crosslinked with 0.4% formaldehyde in PBS for 10 min with rocking at RT. Cell suspensions were immediately quenched with Tris to a final concentration of 50 mM and repelleted. Supernatants were removed and cell pellets were then lysed with 150 mM NaCl, 50 mM Tris, 1% Triton X-100, pH 7.4 (lysis buffer) for 20 min at 4°C with rocking. Lysates were cleared by centrifugation at 21,000xg for 10 min at 4°C and cleared supernatants were then run on a 4%–8% gradient gel and analyzed via western blot with antibodies for Arc (mouse monoclonal, Santa Cruz) and GFP (chicken polyclonal, Aves).

RNA extraction For all samples, total RNA was extracted using TRIzol (Thermo Fisher Scientific). TRIzol-extracted samples were mixed 5:1 with chloroform, incubated at RT for 3 min, and pelleted at 12,000xg at 4°C for 10 min. The resulting aqueous phase was taken and mixed 1:1 with isopropanol, incubated at RT, and pelleted at 12,000xg at 4°C for 10 min. The resulting supernatant was removed and pellet washed with cold 75% ethanol. Washed pellets were then repelleted at 7500xg for 5 min at 4°C. The supernatant was removed and dried pellets were resuspended in ddH 2 O.

RT-PCR Total RNA concentrations were measured by A 260/280 on a Nanodrop (Thermo Scientific). Reverse transcription reactions were carried out using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) with 100-200 ng of RNA as template. Resulting cDNAs were amplified using rat Arc, GAPDH primer sets for 35 cycles with a 60°C annealing temperature. Resulting PCR products were analyzed on 1.5% agarose gels stained with SYBR Safe (Thermo Fisher Scientific). Rat Arc primers: Fwd, ACCATATGACCACCGGCGGC; Rev, TCCAGCATCTCAGCTCGGCAC. GAPDH primers: Fwd, CATGGCCTTCCGTGTTCCTA; Rev, GCCTGCTTCACCACCTTCTT. RT-PCR gels were quantified using the ImageJ gel analyzer tool.

qRT-PCR To determine the amount of RNA associated with Arc protein, quantitative RT-PCR was performed on mRNA prepared from 1: whole mouse cortices immunoprecipitated with Arc and IgG protein, 2: EV fractions prepared from HEK cells (see below, “Extracellular vesicle purification”), and 3: lysate and purified protein from bacteria (BL21, Thermo Fisher Scientific) transfected with rat Arc plasmid (pGEX-GST-ArcFL). Some samples were treated with RNase (25 μg, RNase A, Thermo Fisher Scientific) to determine if the mRNA associated with Arc protein was protected from degradation relative to exogenously added GFP antisense RNA (generating using T7 RNA polymerase from linearized pBluescript-SKII-GFP). Preparation 1: Mice were sacrificed after 24 h of dark-housing and 2 h of enriched environment. Whole cortices were dissected and homogenized in IP lysis buffer as described above. After immunoprecipitation, bead slurry was incubated in guanidine thiocyanate containing RLT lysis buffer and column purification of RNA was performed using QIAGEN RNeasy Micro Kit (QIAGEN, Hilden, Germany). Total eluate was used for reverse transcription using High Capacity cDNA Reverse Transcription Kit with 50 U of Multiscribe Reverse Transcriptase and random oligo primers (Thermo Fisher Scientific). Preparations 2 and 3: total RNA was extracted using TRIzol (Thermo Fisher Scientific) as described above (“RNA extraction”). Reverse transcription reactions (25°C for 10 min, 37°C for 2 h, 85°C for 5 min) were carried out using a High Capacity cDNA Reverse Transcription Kit. Resulting cDNA was prepared for qPCR using PowerUp SYBRgreen Master Mix (Thermo Fisher Scientific) in a 96-well plate with primers against rat Arc, GAPDH and asnA (see above, “RT-PCR”; asnA primers: Fwd, GCGTGGATGCCGACACGTTG; Rev, ATACCGCCGCCGATGGTCTG). qPCR was performed on a QuantStudio 3 Real Time PCR System (Thermo Fisher Scientific) using the following protocol: Pre-incubation: 50°C for 2 min, 95°C for 2 min. Amplification: 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 1 min. Melt curve: 95°C for 1 s, 60°C for 20 s, continuous ramp at 0.15°C/s up to 95°C. Ct values of greater than 30 were considered undetectable. Differences in expression were determined using the standard curve method, where a standard DNA sample was serially diluted (10-fold), analyzed for the gene of interest, and the linear equation calculated. The resulting linear equation was used to determine where the Ct values of test samples fell within the standard curve and the result was transformed (log 10 ) to reflect the dilution of the standard sample. Differences were calculated measuring the fold-change from the average of the control values for any given group (test/average control).

Extracellular vesicle purification Lachenal et al., 2011 Lachenal G.

Pernet-Gallay K.

Chivet M.

Hemming F.J.

Belly A.

Bodon G.

Blot B.

Haase G.

Goldberg Y.

Sadoul R. Release of exosomes from differentiated neurons and its regulation by synaptic glutamatergic activity. Extracellular vesicles (EVs) were purified from HEK cell and primary neuronal cultures as previously described (). Media was spun successively at 2,000 and 20,000xg to remove dead cells and debris, and then at 100,000xg to pellet EVs. The crude EV pellet following the initial high-speed spin was resuspended in cold PBS and repelleted at 100,000xg for 1 h at 4°C in an SW41 rotor. The washed EV pellet was further purified by centrifugation over a 10%–20% sucrose-PBS gradient at 100,000xg overnight at 4°C. The resulting pellet was washed in cold PBS to remove excess sucrose and then repelleted at 100,000xg for 1 h at 4°C. The final, washed pellet was resuspended in PBS and used for downstream analysis with EM, western blotting, and neuron treatments.

Trypsin digestion and RNase assays Trypsin was added to prArc and EVs at 0.05 mg/mL for 30 min at RT followed by addition of 1 mM PMSF for 10 min to inactivate trypsin. Untreated and trypsin-treated samples were then analyzed by western blot. RNase A was added to WT neuron lysates and EVs at 50 μg/mL for 15 min at 37°C. Untreated and RNase-treated samples for RT-PCR were then directly extracted with TRIzol.