Figure 3

Differentiated neurons originating from hippocampal precursor cells showed features of dentate gyrus granule cells. After differentiation the cells were fixed and stained for markers characteristic of dentate gyrus granule cells. A; β-III-tubulin (green), Prox1(red). B; β-III-tubulin (green), Calbindin (Red). C; β-III-tubulin (Green), Synaptoporin (Red). D; β-III-tubulin (Green), NeuN (Red). E; Differentiation led to up-regulation of molecular neuronal markers and down-regultion of precursor cell markers. Proliferating cultures (day 0) were allowed to differentiate and mRNA was extracted after different times. Differentiation led to up-regulation of NeuroD1, mash1, VGLUT1 transcripts, whereas Nestin was down-regulated. F; Up-regulation of GAD67 by differentiated neurons. Neurons differentiated for 10–12 days were washed once with medium and subjected to a 2.5 h stimulation of kainate (KA, 10 µM) or rhBDNF (100 ng/ml) in fresh medium and subsequently fixed and evaluated. GAD67 (red) was upregulated in β-III-tubulin-positive cells (green), a property associated with granule neurons in vivo. The images were captured with identical laser settings at the confocal microscope. G; Electrical properties of cultured dentate gyrus precursor cells and differentiated neurons. Membrane properties of cultured dentate gyrus precursor cells and neurons derived from them were measured while voltage-clamped to −60mV. G, H, J, K; Typical membrane currents recorded by series of depolarizing and hyperpolarizing voltage steps ranging from −120 to+80mV, with 10-mV increments (only first 7 voltage steps are shown). G; On depolarizing steps in voltage-clamps, cells showed typical features of precursor cells with outward rectifying Potassium channels. H; differentiated neurons showed properties consistent with sodium currents. I; In current-clamp, single action potentials were elicited in differentiated neurons by injecting current pulses in 20mV-increments for 100 ms. K; Current voltage curves of the fast inward currents recorded from differentiated neurons with and without TTX were constructed. Mean±SEM values averaged from day 28 old cultures are shown (•; n = 11). Such currents were completely blocked by 1 µM TTX (n = 3).