Time lapse images were taken on

Time-lapse images were taken on an inverted Zeiss LSM 510 microscope using Plan-Neofluar 20× (0.8 N.A.) objective. For Ca analysis, images were taken every 1.5s for 3min using 512×512 pixel resolution and cells were excited using the 488nm laser line. On each cell soma a 5μM×5μM ROI was drawn and change in fluorescence intensity over time was calculated within the imaging software. When drugs were added to the bath solution, a 5-minute wash-in period was maintained before resuming imaging. To stimulate Ca activity in some experiments an excitatory Tyrode\'s solution was perfused into the dish containing the SFEB. This solution contained (in mM): NaCl 32, KCl 10, CaCl2 2, MgCl2 2, Glucose 30, and Hepes 25. After background subtraction, Ca signals were then analyzed by hand and with a custom written routine in Matlab (Mathworks, Natick, MA) based on the “PeakFinder” algorithm. For every maximum in the signal, our algorithm calculates the area of the signal with vertices being the maximum itself and the furthest monotonically decreasing minima on either side. This is used to model the relevance of every peak in the signal. Maxima that have an associated area greater than one standard deviation from the mean of the signal are chosen as relevant events. Relevant peaks are then used in calculation of inter-event intervals and event frequencies. To perform electrophysiological experiments, SFEBs were left on the mesh and cut from the MIs and were placed into a recording chamber. Cells were viewed using an Olympus BX51WI microscope with a 40× water immersion lens and differential interference contrast (DIC) optics and imaged using a Hammmatsu Orca R2 CCD camera. SFEBs were constantly perfused with a recording solution containing (in mM): 119 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 40 Sucrose, 30 Glucose, and 20 HEPES titrated to a pH of 7.3 and osmolarity of 330. For whole-cell current-clamp recordings, low resistance recording pipettes (9–12mΩ) were pulled from borosilicate capillary glass. Recording pipettes were backfilled with a solution containing (in mM): 130 K-gluconate, 10 KCl, 2 Mg-ATP, 0.2 Li-GTP, 0.6 CaCl2, 5 MgCl2, 0.6 ethylene glycol-bis (b-aminoethyl ether)-N,N,N′,N′-tetraacetic Lomustine (EGTA), and 5 HEPES titrated to a pH of 7.1 and an osmolarity of 310. Action potentials were evoked with current clamp steps (0pA for 100ms, steps from −60pA to +120pA, 20pA each, for 1s). Na+ and K+-currents were obtained using voltage clamp mode (holding potential: −70mV for 100ms, steps from −90mV to +20mV, 10mV each, for 1s). Data were acquired at 22°C using an Axon Multiclamp 700B amplifier and a Digidata 1440a acquisition system, with pClamp 10 software (Molecular Devices). Data analysis was carried out using Clampfit 10.2 software (Axon Instruments). Data is presented as mean±standard error.
Results In order to better understand the kinetics of neuronal differentiation in our SFEBs, a time course study, looking at three different time points during differentiation: 15, 30, and 45 days was performed. These experiments were performed on free-floating SFEBs that were flash-frozen and cryosectioned. We quantified the neuronal marker NeuN, as well as the glutamate reuptake transporter VGLUT1 co-stained with the nuclear marker DRAQ5 or the neural marker MAP2. Both NeuN and VGLUT expressing cells statistically increase from day 15 to day 30 (p<0.003 for Student\'s t-Test, n=4 images per condition) (Fig. 2A). We repeated this experiment using both vGLUT and Tuj1 in SFEBs grown on MIs and observed similar results, namely that 47% of our total cells co-expressed Tuj1 and vGLUT at 30 days as compared to 11% at 15days. Thus, we performed all subsequent experiments at 30days of differentiation. SFEBs can be kept longer than 30days on the mesh and do thin after that time, but the most significant thinning occurs during the first 14days on the mesh insert (days 21–30 from the start of the experiment, data not shown). We then questioned whether plating onto MIs would alter the normal differentiation of the SFEBs into cortical layers. We found extensive Tuj1-positive neurons throughout the SFEBs, and Tuj1- and Pax6-positive progenitors localized on the external surface of the SFEBs, resembling an “inside-out” ventricular zone and indicating developing forebrain neural progenitors of the cortical subventricular zone in the developing CNS (Figs. 3A, B). We observed that at day 15, Pax6-positive progenitors comprised 44% of the total number of cells in our SFEBs. This slightly decreased to 31% at 30 days. At the outer-edge of the SFEB, Pax6-positive cells decreased from 50% of the total cells at day 15 to 20% at day 30. In the center of the SFEB, Pax6-positive cells increased from 40% at day 15 to 57% at day 30. This differs from what has been previously reported for SFEBs5, where progenitors appear on the inside of the SFEBs rather than the superficial surface. There are some differences in our protocols, including the use of LDN-193189 instead of recombinant BMPR1A and different cell types; however it is not currently clear what underlies this discrepancy. The morphological organization of our SFEBs may be due in part to our differentiation protocol and not their placement on MIs. Free floating SFEBs created using our same differentiation protocol showed a similar inside-out organizational structure when cryosectioned and stained (Fig. 3D).