Plasmids encoding PSD95 or Arc fused to TimeSTAMP cassettes11
were modified by standard molecular biology techniques including polymerase chain reaction, restriction enzyme digestion, and ligation to create new TimeSTAMP variants. New linker sequences were introduced with synthetic oligonucleotides. All subcloned fragments were sequenced in their entirety to confirm successful construction. Full sequences of all plasmids in this study are available upon request.
The following compounds were obtained from Sigma: bicuculline, CHPG, DHPG, actinomycin D, and cycloheximide. Alexa Fluor 647 carboxylic acid succinimidyl ester was obtained from Invitrogen. BILN-2061 and ITMN-191 were synthesized by a contract synthesis company (Acme). BDNF was obtained from Chemicon, Primary antibodies used were mouse monoclonal anti-PSD95 (Neuromab), mouse monoclonal anti-AU1 (Covance MMS-130R), rat monoclonal anti-HA (Roche), and rabbit anti-synapsin (Chemicon). For immunoblotting, primary antibodies were used at 0.1–0.4 μg/mL and HRP-conjugated goat secondary antibodies (Zymed) at 0.1 μg/mL. For immunofluorescence, primary antibodies were used at 0.5–1 μg/mL and Alexa Fluor 568- and 647-conjugated goat secondary antibodies (Invitrogen) at 0.5 μg/mL.
All cell culture reagents were obtained from Invitrogen unless otherwise indicated. HEK293A cells (Invitrogen) were cultured in DMEM medium with 10% v/v FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin and transfected with Lipofectamine 2000. Hippocampal neurons were dissociated by papain from embryonic day 18 (E18) or postnatal day 0 (P0) Sprague Dawley rats, transfected by Amaxa electroporation (Lonza AG), cultured in Neurobasal with B27 supplement, 2 mM GlutaMAX, 50 U/mL penicillin, and 50 μg/mL streptomycin as previously described11
. All animal procedures were approved by Institutional Animal Care and Use Committee of the University of California, San Diego or of Stanford University.
Compartmentalized neuronal culture chambers
Silicon wafer masters were made by soft photolithography on two layers of photoresist with patterning provided by two transparency masks created in CAD software and printed on a 20,000 dots-per-inch printer. Polydimethylsiloxane (PDMS) prepolymer and catalyst (Dow Corning) were mixed at a 10:1 ratio and allowed to polymerize on the masters at 70°C overnight. Blocks were cut out, sterilized, and adhered to washed coverglasses, then the channels were coated with poly-D-lysine. More detailed procedures have been previously published27
For analysis of TimeSTAMP cleavage by immunoblotting, HEK293A cells were transfected with Lipofectamine for 3 h, then transferred to fresh medium without or with BILN-2061. 1–2 days later, cells were rinsed quickly in Hank’s Buffered Saline Solution (HBSS) and immediately lysed in 0.1% boiling sodium dodecyl sulfate (SDS) loading buffer. Lysates were sonicated to shear DNA and then run on NuPage 4–12% Novex Bis-Tris SDS polyacrylamide gels (Invitrogen). Proteins were transferred onto polyvinylidene fluoride membrane (Millipore) by electroblotting, which were then blocked with 10% nonfat dried milk in tris-buffered saline with 0.1% Tween-20 (TBST), incubated in primary antibody overnight in 5% bovine serum albumin in TBST at 4 °C, washed in TBST to remove excess primary antibody, incubated in HRP-conjugated secondary antibody in 10% nonfat dried milk in TBST for 45 min at room temperature, and finally rinsed 3 times for 10 min each in TBST. Proteins were visualized by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Thermo) and film (Amersham Hyperfilm Blue). For analysis of stimulus-dependent PSD95 synthesis in neurons by immunoblotting, transfected dissociated P0 rat hippocampal neurons were plated and maintained in 6-well dishes. Following treatment by the desired pharmacological agents, cells were rinsed quickly with HBSS and immediately lysed in boiling SDS, then immunoblotting was performed as above.
To quantify effects of TimeSTAMP expression on synaptic density, hippocampal neurons were dissociated, electroporated, and plated on washed coverslips coated with poly-D-lysine. Neurons were cultured in the absence of BILN-2061 or in its presence from 4 to 14 DIV. Neurons were fixed by the addition of one culture volume of 8% paraformaldehyde for 15 minutes at room temperature, then washed in phosphate-buffered saline (PBS), blocked in PBS with 5% non-immune goat serum, and probed for synapsin and HA according to standard procedures. Specificity of secondary antibodies was confirmed in control samples without primary antibody. Coverslips were mounted in Vectashield (Chemicon) and sealed with nail polish. Conditions were randomized and blinded, then images of 7–10 neurons per condition were obtained on a Zeiss Axiovert 200M with a LSM 5 Live confocal scanner, using a 40× water-immersion lens with numerical aperture (NA) 1.2 and one of the following filter sets: 495/10 nm excitation, 515 nm dichroic, 535/25nm emission (YFP); 460/20 nm excitation, 515 nm dichroic, 535/25 nm emission (miniSOG); or 540/25 nm excitation, 560 nm dichroic, 595/50 nm emission (mOrange, mKO2, or mCherry). A stack of optical sections with 1024 × 1024 resolution at 1-μm intervals through each neuron was obtained and then flattened in a maximum intensity projection. Blinded quantitation of synaptic density was performed as previously described11
. Analysis was performed on an Apple Macintosh notebook computer using the public domain NIH ImageJ program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image
To follow fluorescence development of TS:YFP tags in transfected HEK293A cells, cells were cultured and transfected on glass-bottom dishes in DMEM with 10% fetal bovine serum. One day after transfection, DMEM was exchanged for HBSS with B27. Cells were imaged on a Zeiss Axiovert 200M equipped with a xenon arc lamp, a 20×0.7 NA lens, a Photometrics Cascade II 1024 camera, and a stage-top environmental chamber with temperature set to 35°C and humidity to 100%. Images were acquired before and at various times after BILN-2061 addition with exposure times selected to avoid sensor saturation. To plot normalized fluorescence versus time, total background-subtracted fluorescence from individual cells at each time point was measured using ImageJ software and normalized to the maximum value for each cell.
To visualize new synaptic protein synthesis in neurons, dissociated P0 hippocampal neurons were transfected with TimeSTAMP reporters and a mCherry marker by electroporation. They were then cultured in Neurobasal with B27 in glass-bottom dishes or PDMS chambers. Cultures were maintained at 37°C in 5% carbon dioxide and 100% humidity. Half of the medium was replaced every 2 days. On the day of imaging, medium was exchanged for HBSS with B27. Cells were imaged by epifluorescence on the Zeiss Axiovert 200M as above. They were also imaged on an Olympus IX81 using a 20× 0.7 NA lens and a FV1000 confocal scanning system. For confocal imaging, the following settings were used: excitation with a 488-nm argon-ion laser line at 10% power and a 559-nm laser diode at 10% power, pinhole 200 μm, scan resolution 1024 × 1024 pixels, scanning speed 1 μs per pixel, photomultiplier voltage 700 V, digital gain 1. In either imaging system, fields including transfected neurons identified by mCherry fluorescence were imaged before and at various times after BILN-2061 addition. A stack of optical sections at 1-μm intervals through each neuron was obtained at each position and time point and then flattened in a maximum intensity projection.
Immunocytochemistry and photo-oxidation for EM
For conventional immuno-EM labeling, cells were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in PBS (0.1 M, pH 7.4) for 30 min, rinsed several times in chilled buffer, and incubated in permeabilizing buffer (0.1% Triton X-100 in PBS) for 5 min. Then cells were incubated in primary antibody (monoclonal antibody to HA 1:1000, Roche clone 12CA5) in 0.01% Triton X-100 and 5% normal goat serum (NGS) in PBS at 4°C for 3 h to overnight, secondary conjugated to biotin (Goat anti mouse, 1:1000) in 5% NGS in PBS at 4°C for 1–2 h, and streptavidin-HRP (1:100) in 5% NGS in PBS at 4 °C for 1 hour, allowing for sufficient washing in 5% NGS in PBS on ice after each incubation. Fluorescent cells were identified and registered by the grid on the cover glass of these dishes. Confocal images of these cells were taken with minimum exposure using a MRC-1024 inverted confocal microscope (BioRad) to identify transfected cells and for correlative light microscopic imaging. Then the buffer was replaced with a solution of 1 mg/mL DAB and 0.001% H2
in PBS. Cells in the registered grids were monitored under a transmission microscope until a light brown reaction product became visible. The dish was then removed from the microscope and washed in chilled buffer (5 times for 2 min) and post-fixed in 2% glutaraldehyde in 0.1 M PBS on ice for 20 min and then in 1% osmium tetroxide (Electron Microscopy Sciences) in 0.1 M PBS on ice for 30 min. Cells were then washed in chilled buffer twice and rinsed in distilled water and stained with 2% aqueous uranyl acetate (Ted Pella Inc.) for 1 h to overnight at 4°C. The samples were then dehydrated in a cold graded ethanol series (20%, 50%, 70%, 90%, 100%, 100%, 100%) 2 min each, rinsed once in room temperature anhydrous ethanol, and infiltrated in Durcupan ACM resin (Electron Microscopy Sciences) using 1:1 anhydrous ethanol and resin for 30 min, then 100% resin twice for 1 h each, then into fresh resin and polymerized in a vacuum oven at 60°C for 48 h. Sample preparation for photo-oxidation was performed as described previously.24
Incubation with mersalyl acid (5 mM in cacodylate buffer for 30 min on ice) followed by several rinse steps in chilled buffer before photo-oxidation was added in some experiments to reduce DAB staining of mitochondria.
Labeled and imaged areas of embedded cultured cells were identified by transmitted light microscopy. Areas of interest were sawed out using a jeweler’s saw and mounted on dummy acrylic blocks with cyanoacrylic adhesive. The coverslip was carefully removed, ultrathin sections were cut using an ultramicrotome, and electron micrographs were imaged using a 1200 TEM (JEOL) operating at 80 keV.
Statistical comparisons between two groups for a measure of interest were performed with two-tailed t-tests assuming unpaired samples with significance level set at α = 0.05. Comparisons between more than two groups for a measure of interest were performed by one-factor ANOVA followed by pairwise Tukey’s tests if ANOVA revealed unequal means at a significance level of α = 0.05. Prior to t-tests and Tukey’s tests, F-tests were first used to reject a null hypothesis of equal variances for a two-tailed distribution with significance level set at α = 0.05. If the null hypothesis of equal variances was accepted then Tukey’s test (for ANOVA post-hoc analysis) or a t-test for samples with equal variances was performed; otherwise Dunnett’s T3 test (for ANOVA post-hoc analysis) or a t-test for samples with unequal variances was performed. For analysis of dendritic segment TS:YFP fluorescence or synaptic density, sample numbers were determined by the number of healthy transfected neurons in each condition. For analysis of synaptic TS:YFP fluorescence in chambers, sample numbers were determined by the number of puncta in each segment. Statistical tests were performed in Excel (Microsoft) and Prism (Graphpad).