R1, D3 and C57BL/6 ESC lines were obtained from ATCC [39–41]. Pure botulinum holotoxin serotypes /A (2.5 × 108 LD50/mg), /B (1.1 × 108 LD50/mg), /C (3.5 × 107 LD50/mg), /D (0.9 × 108 LD50/mg), /F (0.2 × 108 LD50/mg) and /G (1.2 × 107 LD50/mg) were obtained from Metabiologics (Madison, WI) at 1 mg/mL in Ca2+/Mg2+-free phosphate buffered saline, pH 7.4 (PBS), and stored at −30°C. In the case of BoNT/G, toxin was first activated by a 60 min incubation at 37°C in 0.05 M sodium phosphate buffer (pH 6.5), 0.3 mg/mL TPCK-treated trypsin (Sigma-Aldrich, St Louis, MO) and 10% glycerol. Activated toxin was diluted 1:1 with soybean trypsin inhibitor and stored at −30°C until use. α-Latrotoxin (LTX; Sigma-Aldrich) was resuspended to 300 nM in H2O and stored at −20°C. Mono-sodium glutamate (Sigma-Aldrich) and γ-aminobutyric acid (GABA; Sigma-Aldrich) were resuspended to 20 mM in PBS and stored at 4°C. Solutions were diluted to the indicated concentrations in basal electrophysiologic buffer (BEB; 10 mM glucose, 1 mM MgCl2, 10 mM HEPES, 2 mM CaCl2, 3 mM KCl, 136 mM NaCl and 0.1% BSA, pH 7.4, 310 ± 10 mOsm; Sigma-Aldrich). R-2-amino-5-phosphonopentanoate (AVP, 50 μM, Sigma) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM, Sigma) were prepared in BEB and added 1 h prior to and concurrent with the addition of glutamate. Fluo-4 (Life Technologies, Carlsbad, CA) was prepared per the manufacturer’s instructions. Neurons were maintained in BEB during time-lapse imaging. High potassium electrophysiologic buffer (KEB) was prepared similarly to BEB, except with substitutions of 60 mM KCl and 79 mM NaCl. Electrical field stimulation (1 msec, 100 mV pulses at 10 Hz) of neurons on 18-mm cover slips was applied via a field stimulation perfusion chamber (PC-49FS; Warner Instruments, Hamden, CT) with a Grass S88 stimulator (Grass Medical Instruments, Quincy, MA).
Suspension adaptation and continuous mESC culture
R1, D3 and C57BL/6 cell lines that had been previously maintained in adherent culture with mouse embryonic fibroblasts (MEFs) were thawed and maintained at 37°C at 5% CO2 in 90% relative humidity in 10-cm bacterial plates in 10 mL ESM (Knockout DMEM supplemented with 100 uM β-mercaptoethanol, 15% ES qualified fetal calf serum [ATCC], 0.1mM nonessential amino acids, 2.0 mM L-glutamine and 5000 units/mL penicillin/streptomycin [Life Technologies] and 1000 units/mL recombinant mouse leukemia inhibitory factor [LIF; Chemicon International, Temecula, CA]). Alternatively, ESCs were maintained in commercially prepared complete ESC medium (Millipore, Billerica, MA). Cells were observed daily and passaged once aggregates became clearly visible (typically 4–8 days). Cells that were adherent or failed to divide during adaptation were discarded. Surviving aggregates were trypsinized, and 1.5 × 106 cells were inoculated into a fresh 10-cm bacterial dish with 10 mL ESM. ESCs were subcultured every 48 h. For passaging, aggregates were allowed to settle by gravity, washed once with 0.5 mL PBS and dissociated for 3 min at 37°C with 0.5 ml of TrypLE Express (Life Technologies). Dissociation was terminated with 0.5 mL of ESM, cells were gently triturated and 1.5 × 106 mESCs were transferred to a fresh 10-cm dish.
Generation of GCaMP3-expressing ESCs
Suspension-adapted R1 ESCs were stably transfected with a genetically encoded Ca2+ construct (GCaMP3) driven by the synthetic CAG promoter (Addgene plasmid 22692, Cambridge, MA) . Five μL Lipofectamine 2000, 5 μg plasmid and 5 μL PLUS reagent were prepared per manufacturer’s instructions (Invitrogen) in a total volume of 100 μL DMEM and added to 100 μL of 1 × 106 ESCs/mL in DMEM for 10 min at 37°C. Suspensions were transferred to 10 mL ESM in a bacterial dish and returned to an incubator. Media was changed at 1 d and G418 selection (250 μg/mL; Sigma-Aldrich) started at 2 d. Transient transfection rates exceeded 40%, as estimated from basal fluorescence levels at 2d. Media was subsequently changed every 4 d until G418-resistant ESC aggregates developed. Stably transfected ES cell aggregates (20–50 per transfected population) were isolated and cultured as above until differentiation.
A modified 4/4 protocol was employed to differentiate ESCs into neural progenitor cells [11, 14]. Following routine sub-passaging, 3.5 × 106 dissociated ESCs were transferred to 25 mL of differentiation medium (ESM modified with 10% ES qualified fetal calf serum and without LIF) in a 10-cm ultra-low attachment culture dish (Corning, Lowell, MA). Differentiating aggregates were maintained on a rotary shaker at 45 rpm at 37°C, 5% CO2 and 90% relative humidity. Complete media changes were conducted at 48 h intervals, with the addition of 6 μM retinoic acid (Sigma-Aldrich) at 4 and 6 days after starting differentiation.
On DIV 0, aggregates were dissociated with TrypLE Express for 5 min at 37°C. Trypsinization was halted by adding 5 mL of 1% soybean trypsin inhibitor (Life Technologies), the aggregates were gently dissociated by triturating with a 10 mL pipet, and the cell suspension was filtered through a 40-μm cell strainer (Thermo Scientific). Cells were pelleted for 5 min at 300 × g, washed in N2 medium (Neurobasal-A medium with 1x N2 vitamins, 2 mM glutamine and antibiotics [Life Technologies]) and counted. Cells were plated at 125,000 (coverslips) or 150,000 cells/cm2 (dishes) in N2 medium. Complete washes were conducted at 4 h and 24 h to remove residual serum, gliotrophic factors secreted by glial cells and non-adherent cells, and at 48 h after plating (DIV 2), N2 was replaced with B27 medium (Neurobasal-A supplemented with antibiotics, 2 mM glutamine and 1x B27 vitamins [Life Technologies]). Subsequently, cells underwent full medium changes with B27 on DIV 4 and 8, and then 50% media changes with B27 every fifth day. Glial cell elimination by serum-starvation starting at DIV 0 resulted in the loss of roughly 30% of plated cells between DIV 2–4 .
Tissue culture treated dishes ranging in size from 10 cm to 24-well plates were prepared by coating with 0.5 μg/mL poly-D-lysine (PDL, Sigma-Aldrich) for at least 3 h, followed by two quick washes with sterile ddH20 and storage in N2 at 37°C until plating. Coverslips (18 mm, Thermo Fisher Scientific, Waltham, MA) were coated with 200–300 μL of 0.5 μg/mL PDL for 24 h at 37°C followed by 5 μg/mL laminin (Sigma-Aldrich) in Knockout DMEM for 3 h and transferred coated-side up to 12-well dishes. All characterization of differentiated neurons was conducted exclusively with neurons derived from R1 ESCs between 5–30 passages.
ESN cultures were lysed with 250 μL denaturing cell extraction buffer (Life Technologies) and clarified by centrifugation through a Qiashredder (Qiagen, Valencia, CA); total protein concentration was determined by bicinchoninic acid (BCA) analysis (Thermo Scientific, Rockford, IL). Fifteen micrograms of total protein was separated on a 10% (BoNT/B, BoNT/D, BoNT/F or BoNT/G) or 12% (BoNT/A or /C) Nupage gel (Life Technologies) with MOPS running buffer. Gels were transferred to PVDF and probed for SNARE proteins with a mouse anti-SNAP-25 antibody (SMI81; Covance, Gaithersburg, MD), a mouse anti-VAMP2 antibody (Synaptic Systems, Gottingen, Germany), and a mouse anti-Syntaxin-1 (Synaptic Systems) diluted 1:1000 in TBS Superblock with 0.05% Tween-20 (TBST; Life Technologies). Proteins were visualized with goat anti-mouse Alexa-488 labeled antibodies diluted 1:2500 in TBST and imaged with a Versadoc MP4000 (Bio-Rad, Hercules, CA).
Images were collected on a Zeiss LSM-700 confocal microscope with a constant-temperature environmental chamber. For Fluo-4 staining, ESNs on 18-mm coverslips were stained and loaded in a perfusion chamber as previously described . Zen 2009 (Carl Zeiss, Inc, Oberkochen, Germany) was used to determine the mean fluorescence intensity over a fixed area using Fluo-4 and GCaMP3 as indicators for the presence of cytosolic Ca2+. Ca2+ uptake was induced with the application of KEB or LTX at indicated quantities or by field stimulation. To elicit action potentials by field stimulation, electrical current was applied through platinum wires located in the microscope field of view, using 1 msec, 100 mV pulses. Neurons were stimulated with 3 cycles of approximately 300 action potentials at 10 Hz, with a 10 sec rest. The data were normalized to ΔF/F0 via the following equation: y = (F
Δ min − F
Plate reader based quantification of Ca2+ uptake
ESNs were plated in either 24- or 48-well dishes, stained and mounted as previously described and maintained at 25°C . Ca2+ uptake was induced with the application of chemical stimulation at indicated concentrations. Changes in fluorescence were monitored with a Synergy MX plate reader (BioTek Instruments, Inc., Winooski, VT) with excitation of 490/10 nm and emission of 520/10 nm. The data were normalized via the following equation: y = (F
Coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature and blocked and permeabilized for 10 min in PBS with 0.1% saponin and 3% bovine serum albumin (BSA) (PBSS). Coverslips were incubated for 1 h with anti-MAP-Tau and anti-MAP2 primary antibodies (Synaptic Systems, Gottingen, Germany) diluted 1:1000 and 1:500, respectively, in PBSS, washed three times with PBSS and incubated for 1 h with goat anti-mouse or anti-rabbit Alexa-labeled secondaries (Invitrogen) diluted 1:500 in PBSS. Coverslips were washed three times in PBSS and incubated for 1 h with anti-synapsin 1-Oyster 650 primary antibody (Synaptic Systems) diluted 1:500 in PBSS. Coverslips were washed three times in PBS and mounted with Prolong Gold DAPI mounting media (Life Technologies). Images were collected with a Zeiss LSM 700 confocal microscope or Zeiss LSM 510 confocal microscope and analyzed with Zen 2009 or 2008, respectively.
Differential interference contrast (DIC)/bright field microscopy
For evaluation of neuronal morphology, images were collected on a Zeiss LSM 700 confocal microscope with a constant temperature chamber and analyzed with AxioVision LE release 4.8.1. For evaluation of aggregate morphology, images were collected on a Zeiss AxioObserver.A1 epi-fluorescence microscope and analyzed with AxioVision release 4.7.1.
mESCs were collected during routine subculture and washed twice with PBS. Cells were fixed with paraformaldehyde, permeabilized and stained for Oct3/4 expression or with appropriate isotype controls using the Human and Mouse Pluripotent Stem Cell Analysis Kit (BD Biosciences, San Jose, CA). Stained cells were analyzed on a BD FACSAria II flow cytometer using manufacturer-specified laser excitation wavelength and emission filter sets. At least 10,000 gated events were recorded and analyzed for each sample. Density plots and fluorescence intensity histograms were generated using FCS Express, and experimental samples were normalizing to isotype controls.
Quantitation of ESN viability
ESNs were plated in either 24- or 48-well dishes and maintained under indicated conditions. PrestoBlue (Invitrogen) was added per manufacturer’s protocols in fresh NBA-B27, and ESNs were incubated for 45 min at 37°C. Metabolic conversion of PrestoBlue was measured using an excitation of 535 nm and emission of 595 nm with a Synergy MX plate reader.
RNA was harvested from 10-cm dishes of DIV 14 ESNs (n=5) using RNeasy mini kit (Qiagen) and submitted to Expression Analysis (Durham, NC) for RNA sequencing (2 × 25 paired-end, on an Illumina HiSeq 2000 [San Diego, CA]). Transcriptome data were aligned using UCSC’s mouse knowngene transcriptome with Tophat. Unaligned reads were then aligned to the genome using BWA and merged with the Tophat alignments . Transcript abundances were determined using RSEM v1.1.13 and normalized by calculating the fragments of exon per million mapped reads (FPKM) [44, 45]. Normalized data were analyzed for enrichment of canonical pathways, gene function and tissue expression by Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, California). The most abundant 3000 transcripts were also analyzed for gene functional classification and tissue enrichment using Database for Annotation, Visualization, and Integrated Discovery (DAVID) [16, 17].
BoNT and LTX EC50 values were calculated using a four -parameter sigma model from average dose response values determined from densitometry of western blot images and presented as median values with 95% confidence intervals (C.I.). Multiple comparisons were performed using one-way analysis of variance (ANOVA) to determine significance. Differences among means were determined and calculated with the Student's t-test. * indicates a P < 0.05. ** indicates a P < 0.01. *** indicates a P < 0.001. IPA and DAVID generated the p-values adjusted for multiple hypotheses.