Golgi staining and Sholl analysis

The fresh brains were removed and immediately processed using the FD Rapid Golgi Stain Kit (FD Neurotechnologies, Columbia, SC, USA) according to the manufacturer’s protocol. The different regions of brain tissues were immersed in the 1:1 mixture of solutions A and B and stored at room temperature avoiding light for 1 day, followed by replacement with fresh solution and storage for another 2 weeks. Under the same conditions, the brain tissues were then transferred into solution C for 1 day, followed by incubation for another 3 days in fresh solution C. After this process, the brain tissues were cut into 120 μm slices, rinsed two times in Milli-Q water for 4 min, and then placed into a mixed solution composed of solution D, solution E and Milli-Q water at a ratio of 1:1:2 for 10 min. The brain slices were rinsed twice with Milli-Q water for 4 min. Next, the brain slices were placed in a series of 50%, 75% and 95% ethanol for 4 min each and rinsed four times with absolute ethanol for 4 min for dehydration. Finally, slices were placed in xylene for 4 min and washed three times for clearing and cover-slipping with Permount. For image collection, bright-field microscopy was used with an ×10 or ×63 objectives using a Zeiss Axio Scope A1 microscope. Neurons in the CA1 and DG regions of the hippocampi and in layer II/III of the prefrontal cortices in each group were randomly selected from four brains, and 3 slices/brain were quantified. For dendritic spine analysis, dendrites from 2 cells/slice randomly observed in 3 slices/brain were included in each group. Dendritic spines were counted using the following criteria: the primary basal dendrites were defined at the middle between the soma and distal termination, and the secondary apical dendrites were sampled from the midpieces of dendritic ramifications from the primary dendrites. For each selected sample, the analyzed branch length was at least 10 μm. The spine number was counted within the corresponding segment dendritic branch. The dendritic spine density was evaluated as the ratio of the spine number/the length of the dendritic branch. The selected neurons were traced and reconstructed after switching to 8 bit using Image-Pro Plus software. The dendritic complexity was determined by Sholl analysis, in which, with the neuronal body as the origin, concentric circles 10 μm apart were drawn until reaching the end of the farthest dendrite and the dendrite crossings on every circle were then recorded (Fig. 2h).

a Representative photomicrograph (left) and tracing image (right) of pyramidal neurons in the hippocampal CA1 from the sham control group (upper), OSA group (middle) and OSA treated with metoprolol group (below), scale bar = 100 μm. b−g Quantification of total dendrite length (b), total dendrite number (c), apical dendrite length (d), basal dendrite length (e), apical dendrite number (f), and basal dendrite number (g) in hippocampal pyramidal neurons. h Schematic experimental diagram of Sholl analysis. i−k Sholl analysis of the intersection number of total dendrites (10–200 μm) (i), apical dendrites (10–200 μm) (j) and basal dendrites (10–120 μm) (k). *P < 0.05, **P < 0.01, ***P < 0.001 vs. sham control group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. OSA group, values are expressed as the mean ± SEM.