Neuroimaging and immunofluorescence of the Pseudopus apodus brain: unraveling its structural complexity

The present study provides an in-depth neuroanatomical characterization of the brain of Pseudopus apodus , combining magnetic resonance imaging (MRI) with histological analysis by immunofluorescence. In the telencephalon, the pallial regions showed distinct anatomical features, including a cortical structure, a dorsal ventricular ridge and the spherical nucleus, but prominent layering patterns, observable on histological slides, were not fully resolved by MRI. Subpallial structures, such as the nucleus accumbens and the basal ganglia, were delineated with histological clarity and further supported by MRI. In the hypothalamic and diencephalic regions, the dense and complex cellular composition made precise delineation of individual nuclei difficult by MRI, in contrast to the histological accuracy, however by MRI the identification of the major tracts running through these domains are clearly identifiable. Mesencephalic and rhombencephalic structures, including the optic tectum, isthmic nuclei, cerebellum, and reticular groups, were systematically described using a combination of histological and MRI techniques. In addition, immunofluorescence analysis of specific markers, such as Calretinin, ChAT, Isl1, Satb1, Serotonin and Tyrosine Hydroxylase, provided higher resolution of functional sub-regions, allowing precise identification of boundaries and facilitating comprehensive regional mapping, showing complex organizational arrangements, both in rostral regions, such as the dorsal ventricular crest, and in caudal regions, within the tegmental and posterior nuclei of the brain, including the ventral tegmental area, substantia nigra and raphe nuclei. These findings establish a robust neuroanatomical framework for Pseudopus apodus , contributing significantly to the understanding of reptile brain organization and providing valuable insights into the evolutionary adaptations underlying a limbless lizard neuroanatomy.