Figure 1
From: Adenosine A2A receptors control synaptic remodeling in the adult brain
Pharmacological activation of A2ARs induces the formation of abnormal secondary axons in cultured hippocampal neurons. (a) Representative images of E18 rat-derived hippocampal neurons cultured in the absence and in the presence of the selective agonist of A2ARs, CGS21680 (30 nM), from DIV0, double immunolabeled with SMI31 (axonal marker; yellow) and βIII-tubulin (neuronal marker; blue) antibodies at DIV 3 (scale bar, 30 µm), showing that (b) the exposure to CGS21680 increased the number of axons per neuron (upper graph), reflecting an increased percentage of neurons with multiple axons (lower graph). The selective antagonist of A2ARs, SCH58261 (50 nM), did not modify the average number of axons per neuron, but prevented the ability of CGS21680 to modify the number of axons per neuron. (c) CGS21680 increased axonal length (in those cells displaying more than one axon, it was counted the longest axon), whereas SCH58261 was devoid of effects. In the presence of SCH58261, CGS21680 did not modify axonal length. Neither CGS21680 nor SCH58261 significantly modified axonal branching. Data are mean ± SEM quantified from 6 independent cultures, analyzing a minimum of 100 cells per culture and condition. (d) In cells electroporated at DIV0 with either shRNA-Control (shCTR) or shRNA-A2AR (shA2AR) (EGFP+), CGS21680 increased the number of axons per neuron and axonal length in cells transfected with shCTR but not in cells transfected with shA2AR. Scale bar, 10 μm. Data are mean ± SEM quantified from 7 independent cultures, analyzing a minimum of 25 transfected cells per culture and condition. **P < 0.01 and ***P < 0.001. (b upper; c lower) one-way ANOVA with Dunnet’s post-hoc test; (b lower; d left) two-way ANOVA with Sidak’s post-hoc test; (c upper; d right) one-sample t-test vs. hypothetical value of 1. (e) Immunocytochemical analysis of A2AR immunoreactivity (yellow) in a non-polarized neuron and in an axonal growth cone labelled with phalloidin (blue). Scale bar for upper and middle image, 10 μm; scale bar for the lower image, 5 μm.
