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. 2019 Oct 24;10:1269. doi: 10.3389/fphys.2019.01269

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Copyright © 2019 Denver, D’Adamo, Hu, Zuo, Zhu, Okuma, Kim, Castro, Jones, Leal, Mekkittikul, Ghadishah, Teter, Vinters, Cole and Frautschy.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Neuron-specific calbindin is reduced in SHRSP/FAD rats, while caspase-cleaved actin is increased. (A) Micrographs depict calbindin staining in the hypothalamus and thalamus showing numerous calbindin-immunoreactive neurons. (B) Two-way ANOVA of percentage area stained (SHRSP × FAD) in the reticular thalamus showed no effect of sex, but a significant main effect of SHRSP (p < 0.0001), consistent with post hoc analysis, which showed that calbindin staining was reduced in SHRSP (p < 0.05) and SHRSP/FAD (p < 0.001) rats, compared to WKY. Furthermore, calbindin staining was significantly reduced in SHRSP/FAD rats, compared to FAD (p < 0.01), suggesting that FAD exacerbates neuron loss in SHRSP rats. Changes in calbindin staining in the hypothalamus were similar, and while there was a main SHRSP effect (p < 0.0001), there was an additional FAD main effect (p < 0.025). Post hoc analysis showed that calbindin staining was significantly reduced in the hypothalamus of SHRSP/FAD rats, compared to SHRSP (p < 0.0001), FAD (p < 0.01), and WKY (p < 0.0001) rats. (C) We also measured caspase-cleaved actin in the entorhinal cortex as an indication of caspase activity by western blot (lower panel). One-way ANOVA of relative optical density showed a significant strain effect (p = 0.045), and post hoc analysis showed that levels of caspase-cleaved actin were increased in SHRSP/FAD rats compared to WKY rats (p < 0.05). (D) Levels of post-synaptic NR2B were significantly different among strains. There were main effects of strain, and post hoc analysis showed a slight elevation of NR2B levels in FAD compared to WKY, but levels in SHRSP/FAD groups were higher than all other groups (p < 0.01). Data represent means ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^****p < 0.0001; one- and two-way ANOVA with LSD Fisher’s post hoc test.