We also observed this inside out layering again when we

We also observed this inside-out layering again when we stained for nestin and MAP2, with strong nestin staining on the cells and processes growing at the outer 75μM of the SFEB (Fig. 2C), an observation we have made with Tuj1 staining as well (Fig. 1E). Within our SFEBs, nestin expression decreased from 36% at day 15 to 12% at day 30. At the outer edge of the SFEB, nestin expression decreased slightly from 21% at day 15 to 18% at day 30. In the center of the SFEB nestin expression decreased from 11% at day 15 to 7% at day 30. In addition, the cell processes at the edge of the SFEBs grew in a circular morphology around the edge of the SFEB in the majority of our samples. Additionally, we observed numerous Nkx2.1 and MAP2 positive cells within the outer 200μM of the SFEB. This organizational structure does not exist in the center of the SFEBs (Fig. 1E). This pattern of staining suggests that SFEBs grown using our method are producing early cortical layers at the outer edges of the structure, possibly of lateral moving cells. To further characterize this layer, we asked whether the cells we observed were akin to reelin positive Cajal–Retzius cells observed in the outer layers of the developing cortex (Gaspard et al., 2008). We observed reelin-positive staining of the outer fibers of the cells around the edge of the SFEB in a circular pattern that mimicked the nestin staining we observed (Fig. 2B). At 15days, reelin-positive cells increased from 2% of the total neuronal population to 6% at 30days. Additionally, we asked whether the cells in these structures were post-mitotic by staining for Sox-1 and Tuj. Although we did observe small pockets of Sox-1 staining, the majority of the cells were not Sox-1-positive but Tuj1-positive, suggesting that they were primarily post-mitotic neurons. (Fig. 3C). We also observed GFAP staining at the outer edge our SFEBs (Fig. 3E). This staining was sparsely scattered throughout the SFEBs we imaged and was variable between SFEBs. Further studies will be needed to completely characterize the cell-types differentiated using our method within our SFEBs. Large-scale synchronous network activity is an integral part of early tgf beta receptor 1 structure development and aids in the development of mature, functional synapses (Ben-Ari et al., 1997; Garaschuk et al., 1998). Spontaneous synchronous network activity is composed of barrages of action potentials, which induce network-wide calcium transients (Ben-Ari, 2001). This spontaneous rise in intracellular calcium has been implicated in the developmental expression of AMPA and NMDA receptors and a decrease in the number of silent synapses during development (Voigt et al., 2005). Thus, spontaneous network activity plays an important role in the maturation of individual neurons. Therefore we tested the hypothesis that SFEBs grown using our method could demonstrate both spontaneous and evoked Ca activity that was synaptic in nature. SFEBs were incubated in Fluo-4 NW (Invitrogen) at 37°C for 45min and allowed to rest at room temperature for 15min before time-lapse images were taken. Spontaneous Ca activity was measured during a 2.5-minute time window. We observed spontaneous calcium oscillations at a frequency of 0.05Hz, much slower then what has been observed in other systems. In most studies of developing cortical neurons spontaneous Ca oscillation frequency has ranged from 0.1Hz to 2Hz with some studies citing faster frequencies combined with oscillatory patterns (Mao et al., 2001; Tang et al., 2003). There was great variability in the intrinsic Ca signaling in our SFEBs with frequencies ranging from 0.1Hz to 0.02Hz. This variability may be a reflection of the heterogeneous mix of neurons in these preparations and may be improved in the future by using a cell-sorting strategy to isolate more specific populations of neurons. After application of TTX (1μM), almost all calcium activity was blocked within minutes, supporting our hypothesis that evoked calcium was due to synaptic activity within the SFEB (Fig. 4A). We were also able to induce Ca activity using a standard Tyrode\'s solution and then block most of the evoked activity after 5min with a cocktail of blockers including TTX (1μM), APV (50μM), and CNQX (30μM) (Fig. 4B). While a few calcium signals were observed during incubation with TTX in a subset of SFEBs, we attribute those signals to the spontaneous differentiation of TTX-insensitive cells such as neuroglia. Although the expression of these cells is low in our SFEBs and as such may not fully account for these signals, TTX-insensitive Ca-transients have been previously observed in cultured cortical neurons (Murphy et al., 1992). We next asked whether we could evoke calcium transients after local electrical stimulation using a tungsten microelectrode inserted into the SFEB (Figs. 4C, D). SFEBs were incubated in Fluo-4 NW as indicated before the application of a stimulus of 200mV delivered once every 30s, using a standard stimulator. After stimulation, calcium transients were evoked in the local region adjacent to the microelectrode, and these transients were blocked after the application of TTX, suggesting that they are synaptic in nature. However, it is difficult to interpret this data further because SFEBs do not contain the type of circuit cytoarchitecture commonly observed in areas of the brain such as the hippocampus or cerebellum. Nonetheless, this does demonstrate that SFEBs grown using our method can give rise to small networks of mature neurons that are electrically excitable by methods commonly used in slice electrophysiology.