Fig. 8

Differentiated CTX0E16 neurons display hallmarks of putative synapses and respond to activity-dependent stimulation. a-c Representative confocal images of differentiation day (DD) 35 CTX0E16 neurons immunostained for MAP2, PSD-95 and either bassoon (a), synapsin 1 (b) or VGAT (c). As previously observed, all synaptic proteins display punctate distribution along dendrites. In addition, a subset of PSD-95 puncta co-localised with all three pre-synaptic proteins (red arrow), indicating the presence of putative synapses; co-localisation is seen as white puncta. Not all pre-synaptic puncta co-localised with PSD-95 (white open arrowheads), suggesting that synaptogenesis was ongoing. d, e Representative confocal images of a distal dendrite of green fluorescent protein (GFP)-expressing ) CTX0E16 neurons (DD 35). Examination of dendrites revealed the presence of filopodia and dendritic spine-like structures, indicative of ongoing synaptogenesis. Yellow boxes indicate region used in high magnification in inset. f Western blotting of CTX0E16 neurons at different stages of differentiation demonstrate an increase in PSD-95 with maturation, consistent with an increase in synaptogenesis. Rat ctx, whole-cell lysate taken from rat cortex. g Representative binary images of GFP-expressing CTX0E16 neurons (DD 15) following treatment with control conditions (vehicle or osmotic control (30 nM NaCl)) or activity-dependent stimulation with 30 nM KCl for 7 hours. h Measurement of average neurite length in response to stimulation demonstrates that activity-dependent stimulation results in an increase in average neurite length; n = 14–22 neurons from 3–5 independent experiments carried out in triplicate; error bars represent SEM; *p <0.05 (one way analysis of variance with Tukey post hoc analysis). Scale bars for a-e are 5 μm; d and e inset, 1 μm; g, 50 μm