Primary neuron culture and immunofluorescence labeling
Spinal neuron cultures from stage 22 Xenopus laevis (Xenopus 1, Dexter, MI, USA) embryos of either sex were prepared by methods previously described [76, 77]. All experiments and animal housing were conducted according to National Institutes of Health (NIH, Bethesda, MD, USA) guidelines for animal care and safety, with the approval and under the auspices of the Mayo Clinic Institutional Animal Care and Use Committee. Unless indicated, spinal neuron cultures were grown on non-coated coverglass at room temperature (20 to 22°C) and experiments were performed 12 to 20 h after plating. Coating with poly-D-lysine (PDL, 0.5 mg/ml; Sigma, St. Louis, MO, USA) and fibronectin (FN, 20 μg/ml; Sigma) was performed in Dulbecco's phosphate-buffered saline (D-PBS) for one hour (37°C) followed by repeated washes in calcium- and magnesium-free PBS. Culture medium consisted of Leibovitz medium (87.1% vol/vol, GIBCO, Grand Island, NY, USA), fetal bovine serum (0.4% vol/vol, HyClone, Logan, UT, USA), and saline solution (12.5% vol/vol; 10 mM D-glucose, 5 mM sodium pyruvate, 1.26 mM calcium chloride (CaCl2), and 32 mM HEPES, pH 7.5). Cultured spinal neurons were chemically fixed (20 minutes; 2.5% formaldehyde, 0.01% glutaraldehyde), permeabilized with Triton X-100 (0.1%) and processed for immunofluorescence labeling as described [12, 78]. Microtubules were labeled with polyclonal anti-β-tubulin (0.4 μg/ml; Abcam, Cambridge, England, UK) and an Alexa488 conjugated secondary antibody (2 μg/ml; Invitrogen, Carlsbad, CA, USA). Filamentous actin was labeled with Alexa555-conjugated phalloidin (260 nM; Invitrogen).
Image acquisition and processing
We acquired digital time-lapse DIC images using a Zeiss (Jena, Germany) Axiocam CCD camera mounted on a Zeiss Axiovert 200 M inverted microscope (100 × oil immersion objective, 1.4 numerical aperture, 1.6 × optical zoom). For rapid time-lapse imaging of endocytic membrane retrieval, we used a Zeiss LSM 5LIVE confocal microscope equipped with a 63 × water immersion objective (1.2 numerical aperture, 2 × optical zoom). Individual frames were acquired at a rate of 100 ms per capture. We generated all representative movies using Image J software (NIH, LSM toolbox plugin) by exporting time-lapse stacks to a QuickTime format (MOV, MPEG4 compression, three frames per second) . Images of immunolabeled growth cones were captured on a Zeiss LSM 5LIVE confocal microscope using a 63 × oil immersion objective (1.4 numerical aperture, 1.6 × optical zoom).
Focal endocytic assays
All focal endocytic assays were performed in a serum-free modified Ringers (MR) solution (120 mM sodium chloride (NaCl), 2.2 mM potassium chloride (KCl), 2 mM CaCl2, 1 mM magnesium chloride (MgCl2), 5 mM HEPES, 2 mM sodium pyruvate; pH 7.6, 20 to 22°C). Spinal neuron cultures on glass-bottomed uncoated dishes were positioned over an inverted confocal microscope. Using a micromanipulator stabilized by a floatation table, we positioned a micropipette 100 μm in front of the leading edge of the growth cone in the direction of neurite extension. We fabricated micropipettes to an approximate 1-μm opening by heat-pulling capillary glass (1 mm OD, 0.58 mm ID, Warner Instruments, Hamden, CT, USA) with a micropipette puller (Flaming/Brown, Sutter Instruments, Novato, CA, USA; and PC-10, Narishige, East Meadow, NY, USA). A stock solution of FM 5-95 or FM 2-10 (10 mM in H20, Invitrogen) was diluted to 1 mM or 2 mM, respectively, in MR and 2 to 4 μL were loaded into each micropipette. A picospritzer (Picospritzer III, Parker Instrumentation, Huntsville, AL, USA) controlled focal dye application by applying four repetitive pulses (2 Hz, 400 ms pulse duration, 2.5 p.s.i.) immediately after the onset of confocal imaging. Cytochalasin D (30 nM; Sigma) was added 30 minutes before dye application and confocal imaging. For focal application of fluorescent dextran, the micropipette was loaded with fluorophore-conjugated dextran (Alexa488 or tetramethylrhodamine-labeled, 10,000 MW, neutral charge, Invitrogen; 1 mM in MR) and positioned 80 μm in front of the growth cone in the direction of neurite extension. A second micropipette containing MR was used to focally wash away uninternalized dextran. A picospritzer controlled both micropipettes by delivering 10 to 20 repetitive pulses of fluorescent dextran (2 Hz, 120 ms duration, 2.5 p.s.i.) and subsequently washing away uninternalized dextran with the second micropipette (2 Hz, 120 ms duration, 2.5 p.s.i.) until the background fluorescence intensity subsided and internal vesicles could be visualized (approximately 5 to 10 s). For co-internalization of FM 5-95 (100 μM in the micropipette) and fluorescent dextran (Alexa-488 conjugated; 500 μM in the micropipette), we simultaneously applied both dyes for 10 s from the same micropipette (2 Hz, 120 ms duration, 2.5 p.s.i.). A second micropipette was used to wash away uninternalized fluorescent dextran as previously described.
Determination of endocytic density
We determined the distribution of endocytic vesicles in the peripheral and central regions of the growth cone by counting individual vesicles within the defined regions of interest. All analyses were performed within ImageJ software (Bio-Formats ZVI plug-in, Madison, WI, USA). Individual vesicles were identified 15 s after the initial focal FM 5-95 application. Vesicles that had originated within 1 μm of the outline of the growth cone were considered peripheral. In order to determine the area of individual growth cones, we set fluorescence thresholds slightly above the background fluorescence levels and generated binary images (background fluorescence = 0, membrane fluorescence = 1). We then selected the outline of the growth cone as a region of interest in order to measure the total area. In order to determine the area of the central domain, we eroded the peripheral region of the binary growth cone image (1-μm diameter), redefined the region of interest outlining the new growth cone (central region), and measured the area within. The area of the peripheral region was determined by subtracting the central area from the total growth cone area. Endocytic density values were determined by dividing the number of endocytic events by the area of the respective region. Data from multiple growth cones was then averaged to determine the mean endocytic density (the number of vesicles per μm2).
Quantitative fluid-phase endocytic assay
For comparisons of the rate of membrane retrieval, we incubated spinal neuron cultures with fluorescent dextran (150 μM; Texas Red conjugated, 3000 MW, lysine fixable, Invitrogen) for 10 minutes at room temperature followed by consecutive rinses (10 minutes) at reduced temperature (10°C). Neurons were then chemically fixed with 5% formaldehyde in a cytoskeleton-stabilizing buffer for 20 minutes and mounted with Prolong Gold (Invitrogen) . Clostridium difficile Toxin B (20 ng/ml, Calbiochem, Gibbstown, NJ, USA) was applied at the time of plating. Culture medium was used for all dye incubations and washes. We acquired digital fluorescence images using a Zeiss Axiocam mounted on a Zeiss Axiovert 200 M inverted microscope (20 ×, 0.8 numerical aperture, 1.6 × optical zoom). Identical acquisition parameters were used for all experimental groups and the original 14-bit images were analyzed using ImageJ software. A region of interest encompassing the entire growth cone (defined as the distal 40 μm of the axon) was used to determine the mean fluorescence intensity of dextran-labeled endocytic vesicles in the growth cone. A threshold was set above the background intensity, identical for all conditions, and the fluorescence intensity of the region of interest was measured. Data were background subtracted and the final corrected intensity value for each growth cone was normalized to the appropriate mean control.
Axonal growth assays
To determine the effect of increasing doses of cytochalasin D on axon outgrowth, we measured the rate of axon extension during a 1-h assay performed 12 to 14 h after plating. Cytochalasin D (10 to 100 nM) or dimethyl sulfoxide (DMSO) was added 30 minutes prior to the growth assay. For measurements of neurite length on different substrates, spinal neurons were plated on PDL or PDL + FN substrates. After 14 h in vitro, cultures were chemically fixed and phase-contrast digital images were captured using a cooled CCD camera (ProgRes C10 plus, Jenoptik, Jupiter, FL, USA) mounted on a Zeiss (Axiovert 40CFL) inverted microscope (10 × objective). Axon lengths were determined using the ImageJ plug-in NeuronJ . We measured only the longest neurite, or branch of each neurite, and only axons > 50 μm in length were included in the analysis. To determine the effect of Toxin B on axon outgrowth, we measured the rate of axon extension during a 1-h assay performed 12 to 20 h after plating . Toxin B (20 ng/ml) was added at the time of plating.
Statistical analyses were performed using Graphpad Prism software (v5, La Jolla, CA, USA). The D'Agostino and Pearson omnibus test was used to assess the data for normality. Statistical comparisons with normal distributions used either a two-tailed t-test or one-way analysis of variance (ANOVA; Tukey post-test), as indicated in the figure legends. All other comparisons utilized the non-parametric Mann-Whitney U-test.