Large Field of View Scanning Electron Microscopy of the Paraventricular Nucleus of the Hypothalamus During Diet‐Induced Obesity

• Published in: The FASEB journal 2021-05, Vol.35 (S1), p.n/a, Article fasebj.2021.35.S1.03830
• Main Authors: Arestakesyan, Hovhannes, Blackmore, Katherine, Smith, Hannah, Popratiloff, Anastas, Young, Colin
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.2021.35.S1.03830

The paraventricular nucleus of the hypothalamus (PVN) is critical in autonomic and endocrine control. Previous work has indicated that PVN dysregulation is associated with a variety of cardiometabolic diseases including those related to obesity. However, investigations have focused primarily on molecular alterations. Importantly, changes in the anatomical structure of brain networks also leads to functional alterations. While electron microscopy (EM) can provide nanometer resolution of normal/abnormal brain structures, an inherent limitation of traditional transmission EM is the random sampling of an object within a large region of interest (i.e. PVN). To overcome the inherent limitations of EM, we introduce an approach that utilizes large field of view high‐resolution focused ion beam scanning electron microscopy (SEM) of the PVN. Six week old male C57Bl/6J mice were fed a high fat diet (HFD) or normal chow for 10 weeks (n=4/group), and brains prepared for SEM imaging. Due to the COVID‐19 pandemic, which required social distancing, SEM imaging and analysis was conducted remotely (i.e. at home) using a virtual network computing approach. We first acquired interactive, zoomable maps of the PVN at a low magnification. This image was then used to navigate precisely to the PVN and acquire a high‐resolution image encompassing ~30‐40% of the nucleus (Figure). With this approach, we could rapidly scan for any visually apparent anatomical alterations. This initial evaluation revealed more electron dense regions within PVN neuronal nuclei, potentially indicative of heterochromatin clusters, in HFD mice relative to normal chow counterparts. Thus, we performed subsequent analysis on 80‐120 neurons from each diet group by outlining neuronal nuclear envelopes and quantifying pixel distribution. This provided an indirect estimate of heterochromatin (i.e. dark pixels) and euchromatin (lighter pixels), with a shift towards darker pixels suggestive of increased chromatin “clustering”. The area and perimeter of analyzed PVN neuronal nuclei between groups was similar (e.g. area: 4.0±2.9 vs 3.7±1.4 µm2x106, normal chow vs HFD, p