First attempt to measure adult neurogenesis in a lophotrocozoan (Octopus vulgaris) brain using flow-cytometry technique

In the past decades, researchers provided an amount of significant information about the anatomical, molecular and functional mechanisms underlying neurogenesis in the adult brain. Adult neurogenesis consists in proliferation, migration and differentiation of newborn cells that will be functionally integrated into the existing neural circuitry of adult brain. This process plays a crucial role in adaptation to the environmental challenges. It occurs in animals with complex and centralized nervous system exhibiting cognitive capabilities and sophisticated behavioral repertoires, such as mammals, including humans, non-mammals vertebrates and, among invertebrates, it has been demonstrated in ecdisozoan taxa such as insects and crustaceans too. Octopus vulgaris is considered an “advanced invertebrate” for the size of its brain, the largest of any invertebrates. Evolved from the basal molluscan plan of tetraneury and characterized by a hierarchical organization. Octopus central nervous system is located around the esophagus, in a cartilaginous “cranium” between the eyes, and consists in a supra-esophageal and sub-esophageal masses connected to two optic lobes. Octopus shows complex behaviours and unusual cognitive skills, as learning and memory, problem solving, individual personality and capabilities to play. For these reasons, it seems to be the most likely candidate for the neurogenic process among lophotrocozoans. In our previous works, we found cell proliferation in specific areas of octopus brain involved in learning, memory and processing sensory information. Moreover, we demonstrated, using specific markers as PCNA, PARP1 and Oct-elav1 gene, that enriched environment increases proliferation and synaptogenesis. Given that, we developed a protocol for flow-cytometry analysis to measure the proliferative activity in a faster and more reliable manner then the classical immunohistochemistry. Dissociated cells from proliferating areas of octopus brain, vertical frontal system and optic-olfactory lobes were exposed to BrdU and subjected to PI staining and FACS analysis. Bivariate distributions of BrdU content vs DNA content were analyzed and the G1/S subpopulation was determined. Univariate analysis of DNA content was used to determine the percentage of G2/S cells. Using this technique, we accurately quantified the effective number of proliferating cells in each areas, in order to determine the magnitude of the neurogenic process in octopus brain.