Golgi-Cox Staining and Morphological Analysis

Mice were transcardially perfused with 0.9% saline under anesthesia (pentobarbital sodium, 200 mg/kg, i.p.). The brains were quickly removed and immersed in Golgi-Cox solution for 2 weeks in the dark at room temperature. Then the brains were rinsed in 20 mL ddH₂O, transferred to 20 mL 30% sucrose, and left in the dark for 5 days. Serial coronal sections were cut at 120 μm on a Microm HM vibratome (Thermo Scientific, Walldorf, Germany) and collected in 6% sucrose in ddH₂O at 4 °C. The sections were then mounted on Superfrost plus slides (Thermo Scientific) and pre-processed according to a previously-described protocol [17, 32].

Pyramidal neurons in superficial (II/III) and deep (V) layers of the mPFC, fully impregnated with Golgi stain and minimally overlapped with other stained cells, were used for morphological analyses (6–8 neurons from each layer in each subregion per mouse; the number of neurons was chosen based on previous studies [16, 32]). For each neuron, the circumference of the cell body and the dendritic branches in the x, y, and z directions were manually traced at ×400 with Neurolucida software (MicroBrightField Bioscience, Williston, VT).

Sholl analysis was used to assess the total dendritic length and dendritic complexity using NeuroExplorer software. For each neuron, the dendritic length was measured as the sum of the lengths of the apical main dendrite and branches. The apical main dendrite refers to the large apical dendrite that connects the soma to distal tuft dendrites. The apical branches included all the oblique dendrites that emanated from the apical main dendrite. Dendritic complexity was measured as the number of intersections per concentric circle (20 μm) of increasing distance from the cell body.

For dendritic spine analysis of mPFC pyramidal neurons, the inclusion criteria for segments to be analyzed were as follows [32]: (1) segments of apical main or oblique dendrites initiated at 120 µm from the soma; (2) segment length >  30 µm; (3) comparable segment diameter for each dendritic domain; and (4) no overlap with other segments, to prevent possible confusion in spine visualization. Bright-field z-series images of dendritic segments (6–8 segments from each dendritic domain per animal) were digitized at ×1000 using a CoolSNAP MP5 CCD camera (Roper Scientific, Tucson, AZ) mounted to an Olympus BX51 microscope (Tokyo, Japan). Dendritic spines in the mPFC were classified as thin, stubby, or mushroom types according to the criteria in the literature [33], and counted using NIH ImageJ software. Spine density is expressed as the number of spines per 10 µm of dendrite.