We purchased all reagents at ACS grade from Sigma unless noted otherwise. We purchased brain–derived neurotrophic factor (BDNF) from Promega, and used it at a concentration of 50 µg/ml. We purchased HPG from Chiralix (The Netherlands).
We prepared AHA and the triazole ligand as described previously47
. We prepared the TexasRed–PEO2
–Alkyne by dissolving TexasRed–PEO2
–propionic acid succinimidyl ester (Biotium Inc.) in excess neat propargylamine (Sigma–Aldrich). After 30 min, we added the solution dropwise to anhydrous diethyl ether. We collected the resulting precipitate by centrifugation (5 min, 10°C, 10.000 × g). Then, we washed the precipitate three times with anhydrous diethyl ether, dried and characterized it by ion spray MS to confirm the formation of the product with a molecular weight of 802.90 g/mol (Supplementary Fig. 1a
). We synthesized the azide–bearing fluorescein tag in a similar way using the amine–reactive 5–carboxyfluorescein–PEO8
–propionic acid succinimidyl ester (Biotium Inc.) and 3–azidopropan–1–amine20
to yield a product with a molecular weight of 881.20 g/mol (Supplementary Fig. 1b
Cultured Hippocampal Neuron and Organotypic Hippocampal Cultures
We prepared and maintained dissociated hippocampal neurons as previously described12
. Brieﬂy, we dissected out and dissociated hippocampi from postnatal day 0 to 2 rat pups (strain Sprague–Dawley) by either trypsin or papain and plated at a density of 40,000 cells/cm2
onto poly–D–lysine–coated glass–bottom Petri dishes (Mattek). We maintained these cultures in Neurobasal A medium containing B–27 and Glutamax supplements (Invitrogen) at 37°C for 18–24 days before use. We prepared organotypic hippocampal cultures according to Gogolla et al.48
, and maintained in culture for three weeks before use.
Copper–Catalyzed [3+2]–Azide–Alkyne–Cycloaddition Chemistry (CuAAC) and Detection of Tagged Proteins
In all culture experiments, we removed the growth medium from neuronal cultures and replaced it with either HEPES–buffered solution (HBS)12
or methionine–free Hibernate A (HibA) medium (BrainBits LLC.) for 30 min to deplete endogenous methionine. We observed no difference in protein synthesis between neuronal cultures incubated in HBS or HibA for time points tested (up to 2 hours). However, we opted to use HibA after it became available as a defined and customizable minimal neuronal growth media to optimize and standardize our labeling procedure. For AHA labeling, we supplemented HBS and HibA with 2 mM AHA, 2 mM AHA plus 40 µM anisomycin, or 2 mM methionine. After incubation at 37°C, 5% CO2
, we washed cells with chilled PBS–MC (1mM MgCl2
, 0.1 mM CaCl2
in PBS) on ice to remove excess amounts of AHA and methionine followed by immediate fixation with chilled 4% paraformaldehyde, 4% sucrose in PBS–MC for 20 min at room temperature (RT).
For CuAAC, in order to avoid copper bromide–derived precipitates, we used TCEP in combination with copper sulfate to generate the Cu(I) catalyst during the CuAAC reaction. Briefly, a CuAAC reaction mix composed of 200 µM triazole ligand (stock solution dissolved at 200 mM in DMSO), 2 µM fluorescent alkyne or azide tag, 400 µM TCEP and 200 µM CuSO4 was mixed in PBS (pH 7.6 ) with vigorous vortexing after each addition of a reagent. We incubated hippocampal primary cultures or organotypic hippocampal slices overnight at 20°C with the CuAAc reaction mix in a humid box under gentle agitation. Following incubation, we washed cells or slices three times for 10 min each at RT with 1% Tween–20, 0.5 mM EDTA in 1x PBS pH 7.4 followed by three rinses with 1x PBS pH 7.4 prior to immunostaining using standard conditions. We tested AHA and HPG concentrations ranging from 0.1 mM to 4 mM and observed saturated labeling at a 2 mM concentration without any apparent toxicity or any change in gross cellular morphology (data not shown). Therefore, we used 2 mM AHA or HPG in all subsequent experiments.
For immunolabeling after AHA incorporation and cycloaddition, we treated primary cells sequentially with PBS, blocking solution (0.1% Triton X–100, 2 mg/ml BSA, 5% sucrose, 10% normal horse serum in PBS), primary Ab in blocking solution at 4°C overnight or at RT for 2 h, PBS–Tx (0.1% Triton X–100 in PBS), Alexa488– or Alexa568–conjugated secondary Ab (Invitrogen) in blocking solution, PBS–Tx and PBS, and mounted in Gold Prolonged Antifade reagent (Invitrogen) prior to imaging. For immunolabeling and cycloaddition of AHA–tagged proteins in organotypic cultures, we fixed slices overnight at 4°C, rinsed extensively several times, blocked and permeabilized overnight in blocking solution before performing CuAAC for at least 12 h at RT. We performed immunostaining for MAP2 as described above with extensive washes and prolonged incubation periods with secondary antibodies. We used the following primary antibodies: rabbit anti–microtubule–associated protein 2 (anti–MAP2) polyclonal (1:1000, Chemicon), mouse anti–MAP2 monoclonal antibody (Sigma, 1:500), mouse anti–bassoon monoclonal (1:1000, Stressgen Bioreagents Corp.), mouse anti–Tau (1:400, Chemicon), goat anti–LAT1 (L13) polyclonal antibody (Santa Cruz, 1:100), rabbit anti–methionyl tRNA synthetase polyclonal antibody (abcam, 1:500). For secondary antibodies, we used anti–rabbit or anti–mouse conjugated Alexa Fluor 488, 568, or 647 (1:500; Invitrogen).
Local perfusion experiments were performed with an Olympus IX–70 confocal laser–scanning microscope using Plan–Apochromat 40x/0.95 air or 40x/1.0 oil objectives. We excited Alexa 488 with the 488 nm line of an argon ion laser, and collected the emitted light between 510 and 550 nm. For restricted treatment of isolated somata or dendritic segments, we used a dual micropipette local delivery system. The delivery micropipette was pulled as a typical whole–cell recording pipette with an aperture of ~ 0.5 µm. We controlled the area of local perfusion by a suction pipette, using it to draw the treatment solution across one or more dendrites and to remove the perfusion solution from the bath. In all microperfusion experiments we monitored the dimension of the perfusion spot with the fluorescent dye Alexa Fluor 488 hydrazide (1 µg/ml, Invitrogen) throughout the duration of the experiment; perfusion spot diameters ranged from 30 to 50 µm). We used only the experiments in which the affected area changed by less than 20% for analysis. In all local perfusion experiments, we maintained the set–up at 32°C with a closed box–incubator around the microscope and used multiple small water pans to keep the system humid. We started somatic perfusions with anisomycin (40 µM ) 20 min before bath application of 2 mM AHA or 2 mM AHA + BDNF (50ng/ml; 30 min incubation) to decrease somatic translation to minimal levels. After fixation, we performed FUNCAT using 1 mM TRA tag and immunostained for MAP2. We determined the size of the treated area for each soma or dendrite based on Alexa 488 fluorescence integrated across all images (typically 6–10) taken during local perfusion
Microscopy and image analysis
Unless otherwise specified, we acquired images with a Zeiss 510 Meta confocal laser scanning microscope. We excited Alexa 488 and 5–carboxyfluorescein with the 488 nm line of an argon ion laser, and collected the emitted light between 510 and 550 nm. We excited TexasRed with the 568 nm line of a krypton ion laser, and collected the emitted light above 600 nm. In experiments where two channels were acquired simultaneously, we chose settings to ensure no signal bleed–through between channels. For between–dish comparisons on a given day, we acquired all images using the same settings, without knowledge of the experimental condition during image acquisition. We performed all postacquisition processing and analysis with ImageJ (NIH) and Imaris (Bitplane Scientific Software). To facilitate the analysis of fluorescence signal as a function of distance from the soma, we linearized dendrites and extracted their unprocessed full–frame images using the Straighten plugin for ImageJ. For the analyses of local perfusion experiments, we calculated the TRA signal intensity per volume using a 3D–mask that was generated from the corresponding MAP2 stacks of straightened dendrites in 10 µm volume segments using Imaris.
Copper–free click chemistry and single particle imaging
We performed QD experiments on 8–12 DIV hippocampal neurons. First, we deprived cells of methionine for 30 min in HBS, and then incubated with HBS supplemented with 2 mM AHA, 2 mM AHA + 40 µM anysomycin, or 2 mM methionine for 2–4 hours at 37°C, 5% CO2
. For the click chemistry reaction, we washed and incubated cells in 1µM DIFO–biotin29
for 5 minutes at room temperature (20–25°C). Following 5–10 washes, we then incubated coverslips for 1 min at 37°C with streptavidin–coated quantum–dots emitting at 605 nm (1 nM, Invitrogen) in borate buffer (50 mM) supplemented with sucrose (200 mM). We extensively (~5 times) rinsed cells in HBS and exposed them to KCl (40 mM) and FM4–64 (2 µM, Invitrogen) for 30 s to stimulate vesicle recycling at presynaptic sites. We then washed and imaged cells in an open chamber mounted on an inverted microscope (IX71, 60X objective, NA=1.45, Olympus). We detected QDs and FM4–64 using a Hg+ lamp and appropriate excitation and emission filters (QD: D455/70x, HQ605/20m, FM4–64: D535/50x, E590lpv2; Chroma Technology). We recorded QDs for 38.5 s at 13Hz (500 consecutive frames) with a CCD Camera (Cascade 512BFT, Roper Scientific) and Metaview (Meta Imaging). Average number of QD per movie in AHA, AHA+Anisomycin and Methionine conditions: 25, 3.5 and 6.2s; average number of mobile QDs: 8.3, 3.6, 0.8, respectively. For the analysis we did not take into account QDs with D<10−4
/s. We performed GABAA
receptor experiments on 15 DIV neurons immunolabeled with QDs as previously described 49
Results are presented as mean s.d./SEM for the indicated number of experiments. Statistical analyses were performed using one–way ANOVA and Student's t–test.