Figure S1. Assessment of the viability of cultures. Representative bright-field images, and raster plots of spontaneous electrophysiological spiking activity recorded over 300 s using MEAs at DIV21. (a) Hi-Spots that had a rough surface and dark patches showed very little or no spontaneous electrophysiological activity as shown in (b). (b) Voltage recordings are routinely made simultaneously from eight separate electrodes. (c) Hi-Spots with a smooth surface, light appearance and no dark patches were spontaneously active (d), and hence classed as viable.

Figure S2. eGFP and Parvalbumin positive neurones in Hi-Spot cultures. Cultures were transfected with adenovirus containing the sequence for eGFP driven by the neurone-specific synapsin promoter. The cortices were pre-incubated with adenovirus (50 moi, 30 min, 37°C) then dissociated by trituration before the Hi-Spot production protocol. Hi-Spots expressing neuronal eGFP were imaged ‘live’. The teflon disk carrying the Hi-Spot was placed on a glass slide and a silicon gasket placed around it. A glass cover slip was then placed on top to create a sealed chamber in which the Hi-Spot could be imaged for at least 30 min without any signs of deterioration. (a) eGFP fluorescence was observed in many cells with extensive arborisations forming a network of connections. (b) The high level of fluorescence allowed for the imaging of morphological details such as synaptic spines shown at higher magnification and marked with *, calibration bar 7.5 μm. Immunohistochemical staining of Hi-Spots at DIV21 identifies parvalbumin containing neurones (c and d). Results are representative images from two different experiments showing neurones that are immunopositive for parvalbumin, which is a marker for a subset of high-frequency firing neurones in the cortex.

Figure S3. Western blots showing specificity of neuronal and synaptic markers. Individual western blots of mature rat cortical tissue (positive control) for the neuronal and synaptic marker antibodies used in analysis of Hi-Spot development. These blots show selective labelling of proteins at the appropriate molecular weight band as indicated by the asterisks for each antibody. The bands at the corresponding positions were taken from western blots of Hi-Spot samples at different DIV and used to create the composite illustrating Hi-Spot maturation in Fig.  3(b).

Figure S4. Western blot analysis of the relative ratios of b III Tubulin to GFAP expression in Hi-Spots compared to in-vivo. Western blots of tissue homogenates from P14 rat cortex and different batches of Hi-Spots at DIV14 (n = 4). The quantities of b III Tubulin in each sample was estimated by western blotting (data not shown) and samples resolved by SDS–PAGE based on equivalent b III Tubulin content. Quantification of b III Tubulin and GFAP was by Licor fluorescence imaging (see Methods). Immunoblotting for GFAP in these samples identified a differential expression in Hi-Spots at DIV14 compared to in-vivo cortex at P14. The GFAP: b III Tubulin ratio is ~ 8-fold greater in Hi-Spots (8.0 ± 1.2, n = 4) than at the equivalent time point in-vivo.

Figure S5. Insensitivity of cultures to glutamate in media containing serum. Quantification of the levels of propidium iodide fluorescence in Hi-Spots subjected to 120-min exposure to the indicated glutamate concentrations in serum-containing media. No evidence of cell damage was observed under these conditions. Bars indicate full range of data (n > 18 for each dose).

Table S1. Western blot parameters. Proteins were separated by 10% SDS-PAGE (12.5% for synaptobrevin) and wet transferred overnight onto nitrocellulose membrane at 4°C. Primary antibodies were incubated at the dilution and time shown in Table 1. All secondary antibodies (Alexa 700 and 800 nm) were incubated for 1 h at 1 : 10 000 RT. Specific immunoreactivity was determined using fluorescent secondary antibodies and visualized with Odyssey Licore. NR1 was visualized using a Pearce ECL detection kit.

Table S2. Immunohistochemistry parameters. Details of tissue fixation conditions and primary antibody dilutions used for the different antibodies employed in the immunohistochemistry experiments.

Table S3. Confocal microscope settings. Hi-Spots were imaged with a Leica TCS SP2 TCS MP microscope. The gain, offset, expansion, pinhole and laser power were setup for each wavelength and kept constant for all samples imaged.

Table S4. MEA data analysis.

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