Rapid golgi staining kit

The half brain of each mouse was rapidly processed in accordance with the instructions of a rapid Golgi staining kit (FD NeuroTechnologies, Ellicott city, MD) after the behavioral tests. The entire hippocampus (1.72–6.72 mm from the bregma) was cut into 100 μm potions of each slice (Xu et al., 2009 (link)). The procedure was followed by previously established methods in our lab that successfully stained hippocampal pyramidal cells (Xu et al., 2009 (link)). Sections were coded during processing and decoded on the completion of analysis. One of every ten slices was taken out for the Golgi staining assay, so a total of five slices of each mouse, i.e., 40 slices for each group, were analyzed. For the statistical analysis, cells were chosen based on the following criteria: the cell was located in the CA1 of hippocampus and relatively isolated from surrounding neurons. The average values of five pyramidal neurons from each mouse were treated as one sample, six samples (30 neurons totally) were analyzed in each group for morphological quantification. The number of dendrites and total dendritic length were quantified from a distance range of 50–400 µm from the soma (Vyas et al., 2002 (link)). To calculate the number of spines per 10 µm (spine density), the exact length of the dendritic segment was calculated, and the number of spines along the length was counted.

Golgi staining was performed using a rapid Golgi staining kit (FD NeuroTechnologies, MD). Briefly, 200 µm acute hippocampal slices were prepared as described above. Slices were quickly rinsed with distilled water and prepared for Golgi staining by following the manufacturer’s manual. Images of dendritic spines were acquired using a 100 × objective. For each hippocampal CA1 pyramidal neuron, the spines in the principal apical dendrite were counted in 50–100μm segments that were at least 50μm away from the cell body of the neuron, and a 30μm segment of secondary apical dendrite. Spines were counted only if they had both a head and visible neck. Analyses were performed in a blinded manner. A subset of neurons was counted by two different investigators to ensure consistency of counting. No significant differences were found when the same segment was counted by different investigators.

Golgi-Cox impregnation was performed on brain slices using the Rapid Golgi staining kit (FD Neurotechnologies) following the manufacturer's instructions. Secondary or tertiary dendrites from the striatum radiatum of the CA1 region were photographed from coronal sections of the hippocampus using a MSHOT camera (Digital Microscope Camera MD-90) mounted over an Olympus CX31 microscope. Images had 3,488 × 2,616 pixels and were taken at 100x magnification. The images were processed as previously described to obtain the digital skeleton of the dendrites (Orlowski and Bjarkam, 2012 (link)). Dendritic spines were counted using the imageJ software and the skeleton analysis function. Dendritic spine density was estimated from 3 animals (8 week-old) per condition in 20–30 dendrites per mouse.

Mice brains were quickly taken out and processed according to the protocols of the rapid Golgi staining kit (FD NeuroTechnologies, Ellicott City, MD). Briefly, serial sections (100 μm, 1 in 9 series) were obtained through the whole hippocampus (−1.4 to −2.4 mm from the bregma) on a freezing microtome (Paxinos and Franklin, 2001 ). Brain sections were dehydrated in alcohol, cleared in xylene, and mounted in neutral balsam. For morphological analysis of hippocampal neurons, 5 pyramidal neurons from each mouse (4 mice/group, 20 brain sections from each group) were calculated from area CA1 of the hippocampus. A camera lucida drawing tube attached to an Olympus microscope BX51 (Olympus, Tokyo, Japan) was applied to find selected neurons (400 ×) for computerized image analysis. The center of the soma was as the reference dot, the total dendritic length and the number of dendrites were measured every 50 μm. Meanwhile, the spine density (per 10 μm distances) was quantified (Shankaranarayana Rao et al., 2001 (link); Vyas et al., 2002 (link)).

The spine density of different neurons was assessed by a rapid Golgi staining kit (FD Neurotechnologies). Briefly, the brains were collected and stored in the mixed Golgi solution in the dark for 21 days. The brains were transferred to a cryoprotection solution for 3 days and sectioned horizontally with a vibratome (150 μm). The sections were placed on clean gelatin-coated microscope slides and stained with a certain solution. Then, the sections were rinsed with distilled water and dehydrated with successive baths of alcohol and mounted on glass cover-slips. The sections were imaged with a Slice Scanner (FV1000, Olympus). The images were quantitatively analyzed by Image J software. Spines on the secondary dendrites of PGCs, GCs, MCs, and TCs were involved in the analysis.