Babu et al. Synaptic activity induces neurogenesis FIGURE 2 | Activation of NMDA receptors induces synchronized increase of intracellular calcium in neurons within hippocampal neuronal network. (A) Primary hippocampal neurons loaded with the Ca2+ indicator dye Fura-2. The neurons were then stimulated with Mg2+-free solution and the intracellular Ca2+ concentration was measured in pairs of neurons. (B–D) Glycine induced increase in intracellular Ca2+ was synchronous in all neurons and showed simultaneous rise in ﬂuorescence in different neurons [blue and red traces (B)]. The ﬁrst derivative of the Ca2+ signals (C) and the cross- correlation coefﬁcient of the ﬁrst derivative calculated from cell pairs (D) indicates highly synchronous network activity. (E) When the stimulus solution was applied there was always an increase in the intracellular Ca2+ concentration all recorded neurons (n = 22). The intracellular Ca2+ concentration increased from a basal level of 87 ± 11 nM to a mean concentration of 456 ± 52 nM. If the intracellular calcium increase within neurons were the cause for the observed synaptic potentiation, sequestering calcium in the cells should decrease LTP. We thus performed patch-clamp recordings similar to those described above while adding the Ca2+ chelator EGTA (0.1 mM) to the patch pipette. The EGTA within the patch pipette would diffuse intracellularly and sequester Ca2+ within the neurons. As expected, addition of EGTA completely blocked LTP (Figure 3C). Taken together, the data indicate that application of glycine added Mg2+-free solution induce change in synaptic network and a robust potentiation of synaptic currents in cultured neurons similar to what was reported previously (Liao et al., 2001; Lu et al., 2001). To investigate how synaptic plasticity induced by glycine in a neu- ronal network would affect neuronal differentiation, the co-cultures were ﬁxed 4–6 days after the stimulus and immunostained for MAP2 and βIII-tubulin expression. This analysis revealed a robust increase (∼79%) of EGFP-labeled cells that had acquired neuronal phenotype (Figure 3D). To rule out a direct activation of the precursor cells by glycine- and Mg2+-free conditions we exposed precursor cells without substrate neurons and scored for the cells that underwent neural differentiation. In the isolated precursor cells we did not ﬁnd an increase in the fraction of neurons generated, consistent with notion that network activity in the mature neurons is required (Figure 3E). This observation indicated that neuronal differentiation was a result of the interaction between the neurons and the precursor cells. It is important to note that glycine was applied for only 5 min but pro- duced an increase in neuronal differentiation from EGFP-labeled neural precursor cells that could be measured several days later. Our ﬁnding suggests that neural precursor cells sense the transient change in synaptic activity surrounding them and translate this stimulus into the initiation of neuronal differentiation. We next addressed, whether these effects might be caused by GABA-dependent mechanisms. However, two observations ruled out a likely role of GABA in our co-culture model. First, the stimu- lation was done in the presence of Bicuculline or SR95531, both potent inhibitors of the GABAA receptor. Therefore, GABAergic transmission to the neuronal precursor cells could not be the acti- vator of neuronal differentiation programs. Second, when we incu- bated the co-cultures in the continued presence of Bicuculline or SR95531 without the glycine induced LTP-like synaptic activity we still found an increase in neuronal differentiation from precur- sor cells (Ctr, 1.0; SR95531, 1.63 ± 0.20; P < 0.03; Figure 3F). The Frontiers in Neuroscience | Neurogenesis September 2009 | Volume 3 | Article 49 | 6