Rapid golgi

Mouse proteins were prepared as previously described (Velazquez, Shaw, Caccamo & Oddo, 2016; Velazquez et al., 2017). One hemisphere of the brain was postfixed in 4% paraformaldehyde for 48 hr while the other hemisphere had the hippocampus and cortex isolated, flash‐frozen in dry ice, and stored at −80°C. A subset of hemispheres were dropped in Golgi–Cox solution following the manufacturer protocol (Rapid Golgi; FD NeuroTechnologies). The frozen brain regions were homogenized in ice‐cold T‐PER protein extraction buffer (Thermo Fisher Scientific) containing complete protease inhibitor (Roche Applied Science) and phosphatase inhibitor (Life Technologies). The homogenized mixtures were centrifuged at 100,000 g for 1 hr at 4°C. The resulting supernatant was recovered and stored at −80°C and used for Western blots, which were performed under reducing conditions using precast Novex gels (Life Technologies). Quantitative analyses of the Western blots were obtained by normalizing the intensity of the protein of interest with its loading control, β‐actin.

Mice were deeply anesthetized by intraperitoneal injection of 0.7% sodium pentobarbital solution. The brain was immediately removed, rinsed in PBS, and stained using Golgi-Cox method, according to the manufacturer’s instructions (Rapid Golgi; FD NeuroTechnologies). Briefly, brain tissue was stored at room temperature for 8 days in the dark, before coronal Sects. (150 µm) were cut using a Leica CM1900 cryostat (Wetzlar, Germany). Images (Z stacks at 1 µm intervals) were acquired on a Zeiss Pascal laser scanning microscope (Jena, Germany) with a 63× oil-immersion Neofluor objective (N.A. = 1.4). Secondary basal dendritic segments from hippocampal CA1 and cortical S1 regions were imaged. “n” presents the number of neurons and at least three mice were analyzed per experimental condition. Dendrite length was measured using ImageProPlus software (MediaCybernetics). Spine densities were calculated as the mean number of spines per micrometer dendrite. All images were analyzed blinded to the experimental condition and as it were acquired. For Golgi staining example images, bright-field Z-stack images were projected at minimal intensity and inverted, background was then subtracted, followed by brightness/contrast adjustment within linear ranges, using ImageJ (NIH).

To visualize dendrites and spines of pyramidal neurons, one hemisphere per animal was processed using the commercially available Golgi–Cox kit as described by the manufacturer (Rapid Golgi; FD NeuroTechnologies). Coronal sections (240 μm) were obtained using a freezing–sliding microtome, mounted on 2% gelatin‐coated glass slides, and stored in the dark at room temperature. After 2 days of drying, sections were rinsed, dehydrated, cleared with xylene, and coverslipped. Pyramidal neurons impregnated with the Golgi solution were readily identified in the dorsal hippocampal region by their characteristic triangular soma shape and numerous dendritic spines. CA1 neurons (three brains per group, four neurons per brain) were three‐dimensionally reconstructed by NeuroLucida Version 2017 Software (MicroBrightField). We quantified at least three basal and three apical dendrites per neuron. We used a 100× oil‐immersion objective to identify spines in dendrites longer than 200 μm. We calculated spine densities as mean numbers of spines per micrometer.