Babu et al. Synaptic activity induces neurogenesis Ctr, 1.0; glycine, 1.84 ± 0.22; glycine+APV, 1.27 ± 0.05). NMDA receptor-mediated Ca2+ inﬂux is known to activate CaMK-IIα, which is a key enzyme in regulating the induction of LTP (Silva et al., 1992; Wu et al., 2006). Thus, if LTP-inducing synaptic response in the neuronal network was responsible for the increase in neu- ronal differentiation, blocking CaMK-IIα during the induction of LTP should prevent the induction of neuronal differentiation and recruitment into the neural network. When KN-93, a potent blocker of CaMK-IIα activity, was added to the cultures during the induction of LTP-like synaptic activity the cultures showed a sig- niﬁcant reduction in the number of new neurons (Ctr, 1.0; glycine, 1.84 ± 0.22; glycine + KN-93, 0.64 ± 0.03; Figure 3F). BDNF REGULATES THE INCREASE IN NEURONAL DIFFERENTIATION FROM NEURAL PRECURSOR CELLS As direct action of the same experimental stimuli to the precursor cells did not produce identical or even similar effects than in the co-culture situation, it seemed plausible that a mediating factor secreted by the active neurons might underlie the induction of neu- ronal differentiation in response to LTP-like synaptic activity. The most plausible candidates for such mediators are neurotrophins, in particular BDNF, that are secreted by neurons and have been linked to a wide variety of plastic responses (Figurov et al., 1996; Patterson et al., 2001). Several in vivo paradigms that increase adult hippocampal neurogenesis in vivo also show an increase in the transcription of neurotrophins (Sairanen et al., 2005; Rossi et al., 2006). We thus wondered whether neurotrophins would regulate neurogenesis in our paradigm. We ﬁrst investigated whether induction of synap- tic plasticity in our culture model would also lead to changes in neurotrophin levels. Primary neurons were subjected to LTP-like synaptic activity and the culture medium was harvested at several time points thereafter (Figure 4A). Release of BDNF and NT3 into the medium was assessed by ELISA. As hypothesized, LTP-induced cultures had signiﬁcantly higher levels of BDNF at 1h after the glycine stimulus and remained elevated for at least 6 h (Figure 4B). This increase was seen only in the case of BDNF, whereas NT3 (acting via a different receptor) did not show changes in response to LTP-like synaptic activity (Figure 4C). This suggested that the substrate neurons in our culture are a source for BDNF, whose secretion is regulated by synaptic activity. Detection of BDNF by the precursor cells would foremost require the presence of the appropriate trkB receptor. Mice lack- ing trkB receptor have been shown to have impaired adult hip- pocampal neurogenesis (Bergami et al., 2008; Li et al., 2008). RNA from the precursor cells was therefore assessed for the presence of trkB- and also trkC-transcripts. Both trkB- and trkC-transcripts were expressed in precursor cells, however it seems that only the trkB-transcript increased its expression 1 day after the start of the differentiation (Figure 4D). We also detected trkB recep- tor protein as revealed by Western blot analysis (Figure 4E). We stimulated precursor cells with BDNF and performed Western blot analyses on the cell lysates to test for trkB receptor phosphoryla- tion. BDNF induced an increase in the phosphorylation of trkB receptors suggesting that functional trkB receptors are present on the precursor cells (Figure 4E). If thus BDNF was indeed the key FIGURE 4 | Glycine induced neuronal differentiation is mediated by the neurotrophin BDNF. (A)Time line for the experiments where glycine induced synaptic plasticity was induced and media collected from hippocampal neuronal cultures at set time points. (B,C) Glycine induced potentiation causes increased release of BDNF from the hippocampal neurons. But NT3 a closely related neurotrophin is not regulated by glycine (C). (D) RT-PCR analysis conﬁrms expression of trkB- and trkC-receptors in neural precursor cells.The trkB receptor is upregulated early upon differentiation whereas the trkC receptor remains unchanged. (E) Precursor cells were cultured under differentiation conditions for 24 h, followed by stimulation with BDNF (50 ng/ ml) for 15 min. Immunoblots of equal amounts of cell lysates were probed with anti phospho-TrkB antibody. (F,G) Neutralizing the neurotrophins released by the neurons results in a strong decrease in neuronal differentiation from the precursor cells. Each experiment was performed three times. to the difference in the regulation of neurogenesis then blocking BDNF action should reduce neuronal differentiation after LTP- like synaptic activity induction. Indeed, scavenging secreted BDNF with bio-neutralizing antibodies led to a decrease in βIII- tubulin/MAP2ab-positive neurons (∼90% P < 0.005, Figure 4F). In addition, BDNF scavenging slightly decreased cell survival Frontiers in Neuroscience | Neurogenesis September 2009 | Volume 3 | Article 49 | 8