Babu et al. Synaptic activity induces neurogenesis descriptions of this spatial relationship see for example (Filippov et al., 2003; Kronenberg et al., 2003; Plumpe et al., 2006; Steiner et al., 2006). To mimic the intimate in vivo-relationship between neurons and precursor cells we co-cultured precursor cells on a layer of neurons and astrocytes derived from the hippocampus. This co- culture allowed us to subject the precursor cells to deﬁned external changes in the neuronal layer. Precursor cells from the adult dentate gyrus grow under adher- ent conditions and show stem cell-like properties (Palmer et al., 1997; Babu et al., 2007). We infected precursor cells with a retroviral vector constitutively expressing EGFP and puriﬁed them by FACS. The labeled precursor cells were then layered onto primary neuro- nal cultures from postnatal mice cultured for 10–14 days. This time period was chosen because after this interval most neurons have matured, synapse formation is almost complete, and the neurons elicit synaptic currents. The co-culture of the non-labeled neurons with GFP-labeled precursor cells enabled us to clearly distinguish the precursor cells and their progeny from the substrate culture. After 6 (or in some instances more) days in culture, the cells were ﬁxed and evaluated for neuronal differentiation with antibod- ies against pan-neuronal marker, βIII-tubulin, and for relatively more mature neurons in vitro MAP2ab (Dehmelt and Halpain, 2005), together with EGFP (Figure 1E). Co-cultured cells positive for EGFP and MAP2ab simultaneously have been shown to exhibit mature neuronal electrophysiological characteristics including syn- apse formation and network integration (Song et al., 2002). After 6 days in culture about 12% of the precursor cells had differen- tiated into neurons. These cells showed characteristic neuronal morphologies with more than two ﬁne processes and stained for βIII-tubulin and MAP2ab. Some cells that differentiated into neu- rons consistently showed a lower intensity of EGFP labeling than non-neuronal cells. The example in Figure 1E strongly expresses EGFP. Generally, transgene downregulation has been reported pre- viously and depends on the site of viral integration into the genome host cell (Xu et al., 1989). When the cells were cultured for more than a week the EGFP- labeled βIII-tubulin-positive neurons had formed synapses, which were positive for synaptophysin (Figure 1F), a synaptic vesicle pro- tein that identiﬁes presynaptic compartments adjacent to dendritic spines and expressed in maturing neurons at the site of synapses (Calakos and Scheller, 1994; Becher et al., 1999a,b; Hannah et al., 1999). The characteristic immunoreaction was found along the EGFP-labeled dendrites. Since spines represent the major domain for excitatory synaptic input, its distribution throughout the den- dritic tree suggests integration of the neuron into the existing neural network. STIMULI INDUCING NEURONAL SYNAPTIC PLASTICITY INCREASE NEURONAL DIFFERENTIATION IN PRECURSOR CELLS Neuronal activity can induce neuronal differentiation from neural precursor cells in co-culture with hippocampal neurons (Deisseroth et al., 2004). However, it remains unknown, which kind of physiological neuronal activity patterns would be responsible for regulating adult neurogenesis. We addressed this question in our co-culture system, where complex neuronal activity such as synchronization of neuronal activity and synaptic plasticity can be imposed on neuronal networks and studied in vitro. In the hippocampus in vivo, synchronized bursts co-exist with popula- tion oscillation and are involved in information coding (Lisman, 1997; Bacci et al., 1999; Opitz et al., 2002; Izhikevich et al., 2003; Arnold et al., 2005). NMDA-dependent synaptic activity has been observed in hippocampal neurons and leads to changes in synaptic strengths resulting in the induction of LTP (Malenka, 2003). We evoked enhanced synaptic activity in the co-culture model with Mg2+-free solution containing glycine to allow for maxi- mal NMDA receptor activation. Extracellular Ca2+ enters via the NMDA receptors and activates the downstream signaling cascades within the hippocampal neurons. This model is considered to represent LTP and has been used with preparations of hippocam- pal neurons similar to ours and to study the physiological basis of learning and memory on the cellular level (Lu et al., 2001; Malenka, 2003). We ﬁrst performed intracellular calcium imaging with the Ca2+ sensitive ﬂuorescent dye Fura-2. We found a synchronous rise in the intracellular Ca2+ concentration within the substrate neurons when the Mg2+-free solution with added glycine was applied to the bath (Figures 2A,B). This increase was not a bulk rise in Ca2+ but rather appeared as periodic network activity that was present as long as glycine was applied. This was investigated by simultaneously record- ing the Ca2+ concentration in pairs of cells as shown in [Figures 2B,C (red and blue traces)]. The change in Ca2+ as calculated by the ﬁrst derivative over time [Figure 2C (middle traces)] shows that the Ca2+ inﬂux in neighboring neurons occurs in a synchronous man- ner. To quantify coherence of synchronous activity we calculated the cross-correlation coefﬁcient between the Ca2+ changes in cell pairs and found a mean peak value of 0.74 ± 0.05 at a time interval of 6 ± 28 ms (n = 11 pairs; Figure 2D). These data suggest that the neurons perform relative synchronous AP ﬁring during the stimula- tion period leading to a periodic Ca2+ inﬂux followed by subsequent extrusion similar to what was reported previously (Watt et al., 2004). On average, the intracellular Ca2+ concentration increased from a basal level of 87 ± 11 nM to a mean concentration of 456 ± 52 nM (n = 22 cells) during the stimulation period (3–5 min Figure 2E). To study the effect of glycine on synaptic transmission between neurons, we performed whole-cell voltage-clamp recordings in the substrate neurons (Figure 3A). We applied glycine to cultured neurons and simultaneously recorded the postsynaptic currents at a holding potential of −80 mV. The mEPSC were recorded in the presence of 1 µM TTX and 20 µM Bicuculline to block spon- taneous APs and inhibitory GABAergic responses (Figure 3B), before and after application of the glycine solution. We detected a robust increase in mEPSC frequency in the cultured neurons after stimulation as compared to the control period (Figure 3C). This increase was observed as early as 5 min after glycine withdrawal and synaptic currents remained potentiated throughout the rest of the recording period with an average increase of the mEPSC fre- quency to 158 ± 24% (n = 4) of control. In some cases we recorded enhanced mEPSC frequencies as long as 40 min after stimulation. By contrast, the amplitude of mEPSCs was not signiﬁcantly differ- ent from control (95 ± 14% of control; P > 0.5; n = 4). These data are consistent with an enhanced synaptic release probability and recruitment of silent synapses as typically reported for synaptic long-term potentiation (Malenka, 2003). www.frontiersin.org September 2009 | Volume 3 | Article 49 | 5