Fig. 1: Cholinergic stimulation of astrocytes potentiates synaptic structure formation.

Astrocytes were treated with increasing concentrations of carbachol (0, 0.01, 0.10, and 1 mM) for 24 hours. After treatment washout, primary hippocampal neurons, grown on glass coverslips, were inverted over astrocytes and co-cultured for 24 hours. Neurons were immunocytochemically labeled for the pre and postsynaptic proteins synaptophysin and PSD-95 and imaged using confocal microscopy. Synaptic structures were assessed using three-dimensional object analysis. (A) Representative deconvolved images of hippocampal neurons after co-culture for 24 hours with astrocytes pretreated with 1 mM carbachol or with control astrocytes. Synaptophysin is shown in green pseudocolor; PSD-95 is shown in red; overlapping puncta are shown in yellow (scale bar = 10 μm). Quantification of the number of synaptophysin puncta (B), PSD-95 puncta (C), and overlapping puncta (D) in neurons exposed for 24 hours to control astrocytes or astrocytes pre-treated with increasing concentrations of carbachol. Control and 1 mM treatments: n= 55–66 neurons from 6 independent experiments; 0.010 and 0.100 mM treatments: n = 21–34 neurons from 3 independent experiments. The bold, dashed line represents the median; the upper and lower dotted lines represent the 75^th and 25^th percentile, respectively. One-way ANOVA followed by Dunnett’s Multiple Comparison test was used for statistical comparison of the means. *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs control.