For cell proliferation, we used an EdU (5-ethynyl-29-deoxyur- idine) assay kit (Invitrogen), following the manufacturer’s instruc- tions. Primary antibodies The following primary antibodies were used: rabbit monoclonal anti-Tuj1 (Covance, Princeton, NJ), mouse monoclonal anti- MyoD (BD Pharmingen, San Diego, CA), mouse monoclonal anti- desmin (Sigma), rabbit polyclonal anti-S100b (GeneTex, Irvine, CA), rabbit polyclonal anti-neurofilament L (anti-NF-L; Chemi- con-Millipore, Temecula, CA), rabbit polyclonal anti-neurofila- ment-H (anti-NF-H; Chemicon-Millipore), mouse monoclonal anti-GFP (Invitrogen), rat monoclonal anti-mouse CD31 (BD Pharmingen), mouse monoclonal anti- a smooth-muscle actin (Sigma), and mouse monoclonal anti-Pax7 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) Microscopy, Cell Imaging, and Counting An inverted motorized fluorescent microscope (Olympus, IX81, Tokyo, Japan) with an Orca-R2 Hamamatsu CCD camera (Hamamatsu, Japan) was used to acquire images. Camera drive and acquisition were controlled by a MetaMorph Imaging System (Olympus). Ten arbitrary microscopic fields were counted in each immunostained plate, and values pooled from parallel duplicates per time point and individual experiment. Time-lapse recording FDB muscles were digested by gentle agitation in 0.2% (w/v) type-2 Sigma collagenase diluted in Krebs solution at 37uC for 2 hours. They were then dissociated into single myofibers by trituration with a Pasteur pipette, pretreated with serum to prevent fibers from attaching to the walls. After brief centrifugation, the supernatant was removed, and the pellet resuspended in plating medium (see above). The cell suspension was cultured in flasks coated with laminin, using plating medium in 5% CO2 at 37uC, and followed for 4–5 days in a time-lapse system until the fluorescence disappeared. The FDB myofibers were cultured on the stage of a time-lapse microscopy system (37uC, 5% CO2) using an Olympus inverted phase-contrast microscope fitted with a video camera. Cells were observed for 4–5 days, and their morphology monitored with phase-contrast. Cell fluorescence was monitored at 2-hour intervals. Pictures were recorded on standard VHS tape and digitized for analysis. Fluorescence-activated cell sorting FACS was carried out on a BD FACS (Aria Sorter, San Jose, CA) at 4uC and a pressure of 20 psi, using a laser at the 488 nm line, a 530/30 band pass filter, a 100 mm sorting tip, and a 34.2 kHz drive frequency, sterilized with 10% bleach. This instrument allowed us to characterize cells by size as well as fluorescence. Low flow rate improved the purity of cell sorting. Data acquisition and analyses were performed using BD FACS Diva 5.0.3 software, gated for a high level of GFP expression. The clear separation of GFP+ from GFP- cells explains the ease of sorting. Sorted cells were re-analyzed to confirm that all were GFP+. They were then plated on laminin-coated dishes. Patch-clamp recording Cells were voltage-clamped using an Axopatch-200B amplifier (Molecular Devices, Silicon Valley, CA) in the whole-cell configuration of the patch-clamp technique [20]. Nestin-GFP+ cells were cultured for 12–16 days on glass coverslips and Skeletal Muscle Neural Precursor continuously perfused with an external solution (see below), using a push-pull syringe pump (WPI). Only those exhibiting 3 or more multipolar processes were transferred to a small, flow-through Lucite chamber positioned on an Axiovert 100 (Zeiss) microscope stage. Patch pipettes were pulled from borosilicate glass (Boralex, WPI, Sarasota, FL) using a Flaming Brown micropipette puller (P97, Sutter Instrument Co., Novato, CA), then fire-polished to obtain electrode resistances ranging from 1.5 to 3.0 GV. The pipette was filled with the following solution (mM): 140 CsCl, 4 NaCl, 0.5 CaCl2, 5 K-EGTA (ethylene glycol tetraacetic acid), and 10 K-HEPES (N-[2-hydroxyethyl]piperazine-N’-[2-ethane- sulfonic acid]). The composition of the bath solution was (mM): 140 NaCl, 2.8 KCl, 1.0 CaCl2, and 10 HEPES [21]. To test Ca2+ permeation through glutamate receptors, 0.5 mM MgCl2 was added in some experiments. Solution pH was adjusted to 7.2 with NaOH. All experiments were conducted at room temperature (21–22uC). Glutamate was flushed a few microns away from the patch-clamped cells using a Picospritzer II (General Valve Co., Pine Brook, NJ) to control the pressure and duration of the perfusion pulse. Statistical Analysis Results are expressed as the mean 6 SEM. Statistical significance was assessed using analysis of variance (ANOVA) followed by t-test using Prism GraphPad. P,0.05 was considered significant. Results Skeletal muscle-derived neural stem cells are nestin-GFP+ and Tuj1+ Figure 1A shows the change in appearance over time of neural phenotype cells expressing nestin-GFP and Tuj1 in FDB fiber culture. Tuj1 is commonly used as an axonal marker in young neurons [22,23,24]. Nestin-GFP+/Tuj1+ cells increased from day 1 to 14, representing a fraction of the total number of cells in culture, based on Hoechst 33342 staining. Merged fluorescence and brightfield images indicate that at day 1, the nestin-GFP+ cells were loose in the chamber or associated with myofibers, but all were Tuj1-, while at day 4, nestin-GFP+/Tuj1+ cells with neural phenotype were apparent. At days 7 and 14, neural cells became the dominant population among nestin-GFP+ cells. To determine how early nestin-GFP+/Tuj1+ cells appear in our cultures, we examined their phenotype and immunoreaction profile daily during the first week and at two weeks after plating. Figure 1B shows nestin-GFP, MyoD, and Tuj1 expression in cell cultures derived from FDB muscles. Nestin-GFP allowed us to identify either muscle or neural precursor cells [15,25], while MyoD and Tuj1 revealed myogenic and neurogenic cells, respectively [26,27]. At day 1, myofiber nuclei and activated muscle satellite cells are MyoD+ and Tuj1-, and nestin-GFP fluorescence was obvious; 10.261.6% of the nestin GFP+ cells were also MyoD+. The use of collagenase type-2, an enzyme that seems to destroy myofibers’ basal lamina, consequently releasing most satellite cells, may account for the large number of myofibers with no satellite cells attached [28]. Merged Pax7 and nestin-GFP images confirm the presence of quiescent muscle precursors in satellite position with respect to the myofiber (see below). Brightfield images support the presence of cells expressing nestin-GFP, either attached to satellite cells or detached from the myofiber. At day 2, the population of nestin-GFP+/MyoD+ increased to 13.362.4% due to proliferation of released and attached satellite cells. At the same time, a population of Tuj1+ cells appeared, representing 24.563.3% of the nestin-GFP+ cells. Notably, all Tuj1+ cells were also nestin-GFP+, PLoS ONE | www.plosone.org 3 February 2011 | Volume 6 | Issue 2 | e16816