Large-field-of-view scanning electron microscopy of the paraventricular nucleus of the hypothalamus during diet-induced obesity

Dysregulation in the paraventricular nucleus of the hypothalamus (PVN) is associated with a variety of diseases including those related to obesity. Although most investigations have focused on molecular changes, structural alterations in PVN neurons can reveal underlying functional disruptions. Although electron microscopy (EM) can provide nanometer resolution of brain structures, an inherent limitation of traditional transmission EM is the single field of view nature of data collection. To overcome this, we used large-field-of-view high-resolution backscatter scanning electron microscopy (bSEM) of the PVN. By stitching high-resolution bSEM images, taken from normal chow and high-fat diet mice, we achieved interactive, zoomable maps that allow for low-magnification screening of the entire PVN and high-resolution analyses of ultrastructure at the level of the smallest cellular organelle. Using this approach, quantitative analysis across the PVN revealed marked electron-dense regions within neuronal nucleoplasm following high-fat diet feeding, with an increase in kurtosis, indicative of a shift away from a normal distribution. Furthermore, measures of skewness indicated a shift toward darker clustered electron-dense regions, potentially indicative of heterochromatin clusters. We further demonstrate the utility to map out healthy and altered neurons throughout the PVN and the ability to remotely perform bSEM imaging in situations that require social distancing, such as the COVID-19 pandemic. Collectively, these findings present an approach that allows for the precise placement of PVN cells within an overall structural and functional map of the PVN. Moreover, they suggest that obesity may disrupt PVN neuronal chromatin structure.