Quantitative reconstruction of neuronal mitochondrial network in neurites and somata in rat hippocampus and prefrontal cortex

Introduction: Mitochondrial networks exhibit striking heterogeneity in their morphology and distribution across different neuronal compartments, reflecting the diverse metabolic demands of these structures. Methods: In this study, we used automated tape-collecting ultramicrotome scanning electron microscopy (ATUM-SEM) to reconstruct and quantify mitochondrial networks in the somata and neurites of neurons in the rat prefrontal cortex (PFC) and hippocampus (HPC; CA1 stratum radiatum). We developed an automated segmentation pipeline based on an attention-enhanced 3D U-Net to extract all mitochondria from volumetric EM data. Results: Our quantitative analyses revealed pronounced regional and subcellular heterogeneity. In the PFC, the mitochondrial volume fraction was higher in neurites (7.2%) than in somata (2.9%; 7.1% when nucleus was excluded). Mean individual mitochondrial volume was 0.11 μm³ for neuritic and 0.33 μm³ for somatic mitochondria in the PFC, with similar results observed in the HPC (0.13 μm³ in neurites, 0.31 μm³ in somata). In both regions, the vast majority of mitochondria (~91%) assumed an oval or rod shape, with few displaying branched or donut-shaped structures (~1%). Notably, elongated linear mitochondria (~8%) were mostly confined to neurites, and approximately 90% of these comprised up to 120 nanotunnels-thin segments (<220 nm) connecting enlarged, oval-shaped structures (>350 nm) in tandem. Conclusion: These data provide a detailed quantitative characterization of mitochondrial network architecture in the adult rat cortex and hippocampus, revealing significant regional and subcellular differences in mitochondrial morphology and distribution.