Development of a Novel FIJI-Based Method to Investigate Neuronal Circuitry in Neonatal Mice

The emergence of systems neuroscience tools requires parallel generation of objective analytical workflows for experimental neuropathology. We developed an objective analytical workflow that we used to determine how specific autonomic neural lineages change during postnatal development. While a wealth of knowledge exists regarding postnatal alterations in respiratory neural function, how these neural circuits change and develop in the weeks following birth remains less clear. In this study, we developed our workflow by combining genetic mouse modeling and quantitative immunofluorescent confocal microscopy and used this to examine the postnatal development of neural circuits derived from the transcription factors NKX2.2 and OLIG3 into three medullary respiratory nuclei. Our automated FIJI-based image analysis workflow rapidly and objectively quantified synaptic puncta in user-defined anatomic regions. Using our objective workflow, we found that the density and estimated total number of Nkx2.2-derived afferents into the pre-Bötzinger Complex significantly decreased with postnatal age during the first three weeks of postnatal life. These data indicate that Nkx2.2-derived structures differentially influence pre-Bötzinger Complex respiratory oscillations at different stages of postnatal development.