Supplementary Material for: Food for thought: The effects of feeding on neurogenesis in the ball python, Python regius

Introduction Pythons are a well-studied model of postprandial physiological plasticity. Consuming a meal evokes a suite of physiological changes in pythons including one of the largest documented increases in post-feeding metabolic rates relative to resting values. However, little is known about how this plasticity manifests in the brain. Previous work has shown that cell proliferation in the python brain increases six days following meal consumption. This study aimed to confirm these findings and build on them in the long term by tracking the survival and maturation of these newly created cells across a two-month period. Methods We investigated differences in neural cell proliferation in ball pythons six days after a meal with immunofluorescence using the cell-birth marker 5-bromo-12’-deoxyuridine (BrdU). We investigated differences in neural cell maturation in ball pythons two months after a meal using double-immunofluorescence for BrdU and a reptilian ortholog of the neuronal marker Fox3. Results We did not find significantly greater rates of cell proliferation in snakes six days after feeding, but we did observe more new cells in neurogenic regions in fed snakes two months after the meal. Feeding was not associated with higher rates of neurogenesis, but snakes that received a meal had higher numbers of newly created non-neuronal cells than fasted controls. We documented particularly high cell survival rates in the olfactory bulbs and lateral cortex. Conclusions Consuming a meal stimulates cell proliferation in the brains of ball pythons after digestion is complete, although this effect emerged at a later time point in this study than expected. Higher rates of proliferation partially account for greater numbers of newly created non-neuronal cells in the brains of fed snakes two months after the meal, but our results also suggest that feeding may have a mild neuroprotective effect. We captured a slight trend toward higher cell survival rates in fed snakes, and survival rates were particularly high in brain regions associated with olfactory perception and processing. These findings shed light on the relationship between energy balance and the creation of new neural cells in the brains of ball pythons.