All images were collected on a Leica confocal imaging system (TCS SP8) with a 20× or 63× oil immersion objective, Leica Thunder imaging system (DMi8) with a 20× objective, and Spinning Disk confocal imaging system (Nikon Ti2/Yokogawa CSU-W1) with 60× and 100× oil immersion objectives. For the quantification of recombined tdTom-positive cells with specific markers, serial sections starting from the beginning of the DG were used. Quantification of tdTom-positive and Ki67-positive cell labeling with different markers, DCX and NeuN, was performed using the software Fiji (ImageJ 2.0.0). To confirm Ki67, DCX, and NeuN colocalization with tdTom, single optical sections at 63× magnification were used.
For MPC1 quantification in tissue, z-stacks of single Nestin-GFP cells positive or negative for Ki67 (63× objective) were acquired. Quantification of MPC1 and GFP signal was performed using IMARIS software (Bitplane 9.9.1). First, the GFP volume reconstruction was performed using surface plugin. To confirm the signal of MPC1 in GFP-expressing cells, MPC1 signal was masked in GFP-positive cells using mask plugin. Last, MPC1 volume reconstruction of masked signal was performed using surface plugin. Eighteen to 26 GFP-positive cells were analyzed per group.
For Sholl analysis, the Cre virus–transfected GFP and tdTom double-positive newborn neurons were imaged with a 63× [0.75 numerical aperture (NA)] objective. Z-stacks were taken at 1-μm intervals, and dendrites were traced using the Neurolucida software (version 10, mbs Bioscience). Fifteen to 18 neurons from four mice per group were analyzed. Dendritic spine density and spine morphology were assessed as previously described (69 , 70 (link)) with little adjustments. Briefly, dendrites of 20 to 30 Cre virus–transfected GFP and tdTom double-positive newborn neurons were imaged using a 63× (2.5 NA) objective. The dendritic length and the number of spines were analyzed using the software Fiji (ImageJ 2.0.0). Spine density was expressed as the number of the spines divided by dendritic length. Spine morphology was classified in two groups on the basis of the maximal diameter of the spine head, as measured on maximal projections with Fiji (ImageJ 2.0.0). Immature spines (thin spines) were defined as 0.25 to 0.6 μm and mature spines (mushroom) >0.6 μm. The percentage of each type of dendritic spine was then expressed by neuron for each mouse (20 to 30 neurons per mouse, four mice per group). The data were expressed as the ratio between immature spines and mature spines. All images were analyzed in a blinded manner.
For MPC1 quantification in tissue, z-stacks of single Nestin-GFP cells positive or negative for Ki67 (63× objective) were acquired. Quantification of MPC1 and GFP signal was performed using IMARIS software (Bitplane 9.9.1). First, the GFP volume reconstruction was performed using surface plugin. To confirm the signal of MPC1 in GFP-expressing cells, MPC1 signal was masked in GFP-positive cells using mask plugin. Last, MPC1 volume reconstruction of masked signal was performed using surface plugin. Eighteen to 26 GFP-positive cells were analyzed per group.
For Sholl analysis, the Cre virus–transfected GFP and tdTom double-positive newborn neurons were imaged with a 63× [0.75 numerical aperture (NA)] objective. Z-stacks were taken at 1-μm intervals, and dendrites were traced using the Neurolucida software (version 10, mbs Bioscience). Fifteen to 18 neurons from four mice per group were analyzed. Dendritic spine density and spine morphology were assessed as previously described (69 , 70 (link)) with little adjustments. Briefly, dendrites of 20 to 30 Cre virus–transfected GFP and tdTom double-positive newborn neurons were imaged using a 63× (2.5 NA) objective. The dendritic length and the number of spines were analyzed using the software Fiji (ImageJ 2.0.0). Spine density was expressed as the number of the spines divided by dendritic length. Spine morphology was classified in two groups on the basis of the maximal diameter of the spine head, as measured on maximal projections with Fiji (ImageJ 2.0.0). Immature spines (thin spines) were defined as 0.25 to 0.6 μm and mature spines (mushroom) >0.6 μm. The percentage of each type of dendritic spine was then expressed by neuron for each mouse (20 to 30 neurons per mouse, four mice per group). The data were expressed as the ratio between immature spines and mature spines. All images were analyzed in a blinded manner.
Free full text: Click here
