Hippocampal neurons were cultured alone, with ACM or with trypsinized ACM and were immunostained at 4 div with antibodies against GAD and tau to label GABAergic axons and VGAT to label GABAergic presynaptic terminals. (A, B), GABAergic axon length and branching (A) and GABAergic synapse density (B) were significantly increased in neurons cultured with astrocytes (middle) but not with trypsinized ACM compared to neurons cultured alone (left). Scale bar = 25 (top), 10 (bottom) μm. (C), Quantification of GABAergic axon length, branching and synapse density in neuron-only cultures cultured with astrocytes, treated with ACM, trypsinized ACM, ACM + trypsin inhibitor or ACM + trypsinized ACM. All values are shown as mean ± s.d. (n = 76 cells, 4 independent expts.; Kruskal-Wallis nonparametric ANOVA test followed by Dunn’s pairwise multiple comparison test, p < 0.001).