Morphometric analysis of axonal ultrastructure: Coordinated scaling of organelles and myelin

The endoplasmic reticulum (ER) is a crucial neuronal organelle involved in protein synthesis, calcium homeostasis, and metabolic support, essential for neuronal function and plasticity. Understanding its three-dimensional (3D) architecture is key to elucidating functional organization. Using SBF-SEM and AI-assisted segmentation, we established a quantitative framework to characterize ER and mitochondrial scaling within 35 peripheral nervous system (PNS) myelinated axons. Analysis of individual organelle morphometrics revealed a strong power-law relationship between surface area and volume for both mitochondria (R2 = 0.949) and ER (R2 = 0.949). The resulting exponents were super-isometric (kMito = 0.85, kER = 0.73), suggesting structural plasticity that prioritizes membrane surface expansion. A key finding was the distinction between size and number regulation: mitochondrial and ER volumes were negligibly correlated (r approximate to 0.03), implying independent size regulation. However, organelle abundance (counts) showed a strong positive correlation (r = 0.79), maintaining an extremely low Bonferroni-adjusted Q value (8.1 x10- 9), suggesting coordinated control of organelle number in response to axonal size. Axonal populations were heterogeneous, with larger axons consistently containing more ER elements (r = 0.59) and mitochondria (r = 0.69). Furthermore, a low correlation of axon length with organelle content supports the idea that regulation is primarily a local phenomenon tied to cross-sectional size. These findings provide a quantitative basis for understanding how ER and mitochondria structurally adapt to axonal size, laying the groundwork for future research into how these scaling relationships influence neuronal metabolic health and contribute to neurological disease.