A 3D atlas of the trigeminal nerve and its relevance for comparative studies of the masticatory apparatus in rodents

The trigeminal nerve (CN V) is a key cranial nerve responsible for sensory input from the face and motor control of the masticatory muscles, playing a central role in mandibular movement and oral cavity function across tetrapods. Despite its importance and its link with the masticatory musculature, no comprehensive comparative study of the trigeminal nerve across rodent groups has yet been conducted. This is partly due to the difficulty of visualizing nerves using conventional dissection techniques, and partly due to the complex, three-dimensional organization of the nervous system. Here, we combined diffusible iodine-based contrast-enhanced CT imaging (diceCT) with traditional dissection to characterize the trigeminal nerve and its relationship to masticatory muscles in three representative rodents: the rat, the red squirrel, and the Guinea pig. Our findings demonstrate that the broad architecture of the nervous system is highly constrained but exhibits fine-scale plasticity in nerve branching and muscle innervation. While the gross nerve organization is largely conserved, we identified species-specific differences, particularly in Guinea pigs, linked to skeletal and muscular modifications. The study underscores the value of integrating 3D imaging with dissection to accurately capture nerve morphology and discuss muscle homologies, with implications for comparative anatomy, functional morphology, and paleontological reconstruction.