Synaptic Network Activity Induces Neuronal Differentiation of Adult Hippocampal Precursor Cells through BDNF Signaling

Abstract

Adult hippocampal neurogenesis is regulated by activity. But how do neural precursor cells in the hippocampus respond to surrounding network activity and translate increased neural activity into a developmental program? Here we show that long-term potentiation (LTP)-like synaptic activity within a cellular network of mature hippocampal neurons promotes neuronal differentiation of newly generated cells. In co-cultures of precursor cells with primary hippocampal neurons, LTP-like synaptic plasticity induced by addition of glycine in Mg(2+)-free media for 5 min, produced synchronous network activity and subsequently increased synaptic strength between neurons. Furthermore, this synchronous network activity led to a significant increase in neuronal differentiation from the co-cultured neural precursor cells. When applied directly to precursor cells, glycine- and Mg(2+)-free solution did not induce neuronal differentiation. Synaptic plasticity-induced neuronal differentiation of precursor cells was observed in the presence of GABAergic neurotransmission blockers but was dependent on NMDA-mediated Ca(2+) influx. Most importantly, neuronal differentiation required the release of brain-derived neurotrophic factor (BDNF) from the underlying substrate hippocampal neurons as well as TrkB receptor phosphorylation in precursor cells. This suggests that activity-dependent stem cell differentiation within the hippocampal network is mediated via synaptically evoked BDNF signaling.

Keywords: adult neurogenesis; hippocampus; long-term potentiation; network oscillation; neurotrophins; precursor cell.

Figure 1

Neural precursor cells in their local microenvironment in vivo and in vitro. (A) Immunohistochemical analysis of a hippocampal section from mouse showing the close association of neural precursor cells with neurons. Confocal image (optical section of 1 μm thickness). Green: Nestin-GFP; Red: Dcx, Blue: NeuN; Scale bar, 25 μm. (B) Confocal projections (56×) of the dentate gyrus (z-stack of 11 optical sections with 2 μm thickness); Green: Nestin-GFP; Red: Dcx; Blue: NeuN; one square in the underlying grid equals 16.4 μm. (C) Calretinin-immunohistochemistry highlights the inner molecular layer. Radial glia-like type-1 cells (Green) reach into this area, which receives the input from the entorhinal cortex. Green: NestinEGFP; Red: Calretinin; Blue: NeuN. (D) Newborn neurons in the adult dentate gyrus are in close neighborhood with neurites in the mouse hippocampus; Green: NestinEGFP, Red: MAP2ab, Blue: Dcx. (E) Representative image of precursor cells differentiated into neurons in co-culture of precursor cells with primary mouse hippocampal neurons. EGFP cells differentiated into neurons and also non-neuronal cells types, presumably glial cell, in co-culture. Green: EGFP (precursor cells); Red: βIII-tubulin (neurons). (F) The neurons generated from precursor cells in co-culture integrate into the underlying neuronal circuit as suggested by the presence of synapses (arrows) onto it indicated by the synaptophysin staining. Green: EGFP; Red: βIII-tubulin, Blue: synaptophysin. Scale bar [in (F) for (C–F)]: C, 250 μm; D, 200 μm; E, 25 μm; F, 50 μm.

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.

Figure 3

Glycine-induced synaptic plasticity changes are associated with neuronal differentiation from neural precursor cells. (A) Experimental time line for glycine induced synaptic plasticity changes and associated neuronal differentiation. (B) Glycine induced an increase in the frequency of mEPSCs in hippocampal neurons [158 ± 24% (n = 4) compared to the controls]. The recordings were performed before and after the addition of glycine. There was no change in the amplitude of the mEPSCs. The experiments were performed on neurons that were 12 days old. (C) This increase in the frequency of mEPSCs was blocked by the inhibition of NMDA receptors by APV and also by the chelating intracellular Ca²⁺ with EGTA. (D,E) Glycine induced increased neuronal differentiation from GFP-labeled neural precursor cells in co-culture with primary hippocampal neurons (Ctr; 1.0, LTP; 1.79 ± 0.22, P < 0.01). The co-cultures were stimulated with Mg²⁺-free glycine solution the day after the co-cultures were started as shown in (A). The number of cells per field of view remained unchanged in between the experimental conditions. There was no change in neuronal differentiation when precursor cells were stimulated with glycine in Mg²⁺-free media in the absence of hippocampal neurons. Experiments were performed at least three times in duplicates. (F) The increase in neuronal differentiation is not due to activation of GABAergic receptors on precursor cells by released GABA from neurons. Inhibiting GABA receptors with SR95531 paradoxically increases neuronal differentiation compared to control possibly by blocking synaptic GABA receptors (Ctr, 1.0; SR95531, 1.63 ± 0.20; P < 0.03). Experiments were done at least three times in duplicates. (G) The increase in neuronal differentiation is blocked by NMDA inhibitor APV and CaMK inhibitor KN-93. Each experiment was done at least three times.

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.