Vectashild h 1000

We used microinjection and confocal microscope (Dumitriu et al. 2011) to visualize and quantify the neural morphology at 24 h and 48 h post-stimulation. The sections were selected within the distance of approximately ±0.4 mm anterior-posterior (AP) away from the optic fiber. The injection was done only into the pyramidal neurons from layer 2 -3 of ACC (24a/24b) from both hemispheres. The injection pipettes were pulled from glass capillaries with filament, with a final resistance around 150 MΩ. We filled the pipette with red fluorescent dye solution Alexa 568 hydrazide (#A10441, Thermo Fisher, USA) in filtered 1× PBS (1 : 40). We performed microinjection under the microscope of a patch-clamp set-up. During injection, we penetrated the pipette tip into the soma and switched on the current to -20 pA to drive the dye diffusion for 20 min. Later we switched off the current but left the pipette tip inside the soma for another 5 min to fill the dendrite and spines. All the sections were retrieved and covered with Vectashild H-1000 (Vector Laboratories, Germany) for confocal microscope imaging. We checked all the injected neurons for YFP signal; only neurons with YFP signal were identified as pyramidal neurons and selected for further analysis.