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Fig. 2. Purinergic receptors regulate axon and dendrite growth in cultured hippocampal neurons. (A) Hippocampal neurons were cultured for 3 DIV in the presence or absence of the P2X antagonists BBG (5 µM), Ip5I (1 µM) and PPADS (30 µM). Neurons were stained with an anti-tubulin antibody to observe their morphology and axons were morphologically identified as the longest processes. Scale bar: 100 µm. (B) Quantification of axon length and the number of axon ramifications from the experiments shown in A. The length of the axon is represented as the total length of the axon including its ramifications. Secondary and tertiary ramifications represent first-order and second-order ramifications, respectively. The ratio between ramifications and axon length was calculated for each neuron and the graph represents the means of these ratios. The data represent the mean ± s.d. obtained from three independent experiments each involving at least 50 neurons. Statistical differences were analyzed using a paired t-test (**P<0.01, ****P<0.0001 versus control). (C) Hippocampal neurons cultured for 3 DIV in the presence or absence of the P2X-receptor antagonist KN-62 (50 nM). Neurons were stained with an anti-tubulin antibody (green) and phalloidin (red) to identify neuronal morphology. Neurons treated with KN-62 present the same axon morphology when compared with those treated with BBG. Scale bar: 100 µm. (D) The graphs represent the mean ± s.e.m. of the axon length, the axon ramifications and the ratio between axon length and axonal ramifications, obtained from three different experiments (n=150). Representative images are shown in C. The statistical differences were analyzed using a paired t-test (**P<0.01, ****P<0.0001 versus control).
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