Pannoni, K. E. | bioRxiv Science

bioRxiv2021

The mitochondrial calcium uniporter regulates mitochondrial mass and dendritic localization in distinct hippocampal circuits

CA2 is an understudied subregion of the hippocampus that is critical for social memory. Previous studies identified multiple components of the mitochondrial calcium uniporter (MCU) complex as selectively enriched in CA2, however the functional significance of this enrichment remains unclear. The MCU complex regulates calcium entry into mitochondria, which in turn regulates mitochondrial transport and localization to active synapses. We found that MCU is strikingly enriched in CA2 distal apical dendrites, precisely where CA2 neurons receive entorhinal cortical input carrying social information. Further, MCU-enriched mitochondria in CA2 distal dendrites are larger compared to mitochondria in CA2 proximal apical dendrites and neighboring CA1 apical dendrites, an effect also seen with genetically labeled mitochondria and electron microscopy. MCU overexpression in neighboring CA1 led to larger mitochondria preferentially in proximal dendrites compared to distal dendrites and controls, suggesting that MCU may act as a coincidence detector linking synaptic activity to mitochondrial morphology and function. Our findings demonstrate that mitochondria are molecularly and structurally diverse across hippocampal cell types and circuits, and implicate MCU expression in regulating mitochondrial mass and layer-specific dendritic localization. Functionally distinct mitochondria in CA2 distal dendrites may confer unique synaptic and circuit properties underlying CA2 function in social memory.

neuroscience↗

bioRxiv2020

Visualizing subcellular structures in neurons with expansion microscopy

Protein expansion microscopy (proExM) is a powerful technique that crosslinks proteins to a swellable hydrogel to physically expand and optically clear biological samples. The resulting increased resolution (~70 nm) and physical separation of labeled proteins make it an attractive tool for studying the localization of subcellular organelles in densely packed tissues, such as the brain. However, the digestion and expansion process greatly reduces fluorescence signals making it necessary to optimize ExM conditions per sample for specific end goals. Here we describe a proExM workflow optimized for resolving subcellular organelles (mitochondria and the Golgi apparatus) and reporter-labeled spines in fixed mouse brain tissue. By directly comparing proExM staining and digestion protocols, we found that immunostaining before proExM and using a proteinase K based digestion for 8 hours consistently resulted in the best fluorescence signal to resolve subcellular organelles while maintaining sufficient reporter labeling to visualize spines and trace individual neurons. With these methods, we more accurately quantified mitochondria size and number and better visualized Golgi ultrastructure in reconstructed CA2 neurons of the hippocampus.

neuroscience↗