Morphometric analysis of hippocampal neurons

Hippocampal neurons were transfected with plasmids coding for shRNAs against RagA or RagB, or mCherry as a control, on DIV 8 using Lipofectamine 2000, as described⁷⁵. All constructs carry an additional expression cassette for GFP which allows the monitoring and assessment of neuronal structure. After transfection, neurons were maintained at 37 °C and 5% CO₂ in standard medium consisting of a 9:1 mixture of buffered saline solution and MEM, as described above, and placed in the IncuCyte Live-Cell Analysis System to monitor neurite outgrowth using a 20× objective to acquire images of the entire surface of the well every 6 h. Twenty-four hours after transfection, neurons were treated with 50 μM bicuculline to induce activity-dependent dendritic remodelling and imaged for 48 h. Neurite length of GFP-expressing neurons was analysed using IncuCyte NeuroTrack Software Module. The neurite length values were plotted over time, and the area under the curve was computed to extrapolate one value per experimental condition. Values derived from bicuculline-treated samples were then normalized to the respective control-treated samples to yield activity-dependent dendritogenesis.