57, p < 0.05), and P7 (fold change = 2.86, p < 0.01) IP-astrocyte inserts (Figures 5G and 5H). Thus, IP-astrocytes are as capable of inducing structural synapses in RGC cultures as MD astrocytes are. Structural synapses are not indicative of functional synapses, thus we analyzed synaptic activity of the RGCs in the presence of a feeder layer of astrocytes. Previous studies have shown that the number of functional synapses increases significantly with an MD-astrocyte feeder layer (Ullian et al., 2001). Bortezomib in vivo We found that

both the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) increased significantly and to a comparable degree with feeder layers of IP-astrocytes P1 or P7, to that observed with an MD-astrocyte feeder layer (Figures 5I–5L). Taken together, these results show that IP-astrocytes MDV3100 cell line retain functional properties characteristic of astrocytes. Intracellular calcium oscillations have been observed in astrocytes in vivo and are considered an important functional property of astrocytes and may aid in regulation of

blood flow or neural activity (Nimmerjahn et al., 2009). Several stimuli have been implicated in initiating calcium waves in MD-astrocytes. We used calcium imaging with Fluo-4 to investigate if IP-astrocytes exhibit calcium rises in response to glutamate, adenosine, potassium chloride (KCl), and ATP and if the nature of their response was similar to MD astrocytes (Cornell-Bell et al., 1990, Jensen and Chiu, 1991, Kimelberg et al., 1997 and Pilitsis and Kimelberg, 1998). Few calcium oscillations were observed at rest in IP-astrocytes, contrary to MD-astrocytes. A single cell in confluent cultures of P7 IP-astrocytes would respond independently of its neighbors. Such isolated and spontaneous firing of astrocytes has previously been observed in brain slices (Nett et al., 2002 and Parri and Crunelli, 2003).

In contrast, rhythmic calcium activity and regular spontaneous activity were observed in MD-astrocytes grown in the same media as cultured IP-astrocytes P7 (Figures 6A and 6C). Both MD-astrocytes and IP-astrocytes responded to 10 μM of adenosine (100% of MD-astrocytes, 89.6% ± 5.5% of IP-astrocytes; Figures S2C and S2D), 50 μM of glutamate (100% of MD-astrocytes, 88.1% ± 7.9% of IP-astrocytes; Figures S2E and Ketanserin S2F), and 100 μM of ATP (94.4% ± 5.5% of MD-astrocytes, 92.5% ± 1.5% of IP-astrocytes; Figures 6A and 6B) with increased frequency of calcium oscillations and/or amplitude of calcium oscillations. Both have several P2X and P2Y receptors and adora1 and adora2b receptors and thus can respond to these stimuli. Both MD and IP-astrocytes express mRNA for ionotropic glutamate receptors, but only the latter have metabotropic receptors (accession record number, GSE26066). Thus, the second phase calcium response observed with glutamate in IP-astrocytes after a period of quiescence, could be a metabotropic response. This was not observed in MD-astrocytes.