Three-dimensional mapping of tyrosine hydroxylase in the transparent brain and adrenal of prenatal and pre-weaning mice: Comprehensive methodological flowchart and quantitative aspects of 3D mapping

•A 3D atlas of TH immunoreactivity in the mouse brain and adrenal from embryonic to pre-weaning stages is provided.•A flowchart for generating a 3D atlas of central and peripheral TH immunostaining is described.•Quantitative and qualitative aspects of 3D mapping of developing neuronal populations and pathways are reported. Tyrosine hydroxylase (TH) catalyzes the rate-limiting step for the biosynthesis of the catecholamines dopamine, noradrenaline and adrenaline. Although its distribution in different organs, species and stages of development has been the subject of numerous studies, the recent emergence of 3D imaging techniques has created the potential to shed new light on the dynamics of TH expression during the development of the mammalian central and peripheral nervous systems. Here, we describe a flowchart summarizing different protocols adapted to developmental stage-specific tissues to generate a 3D atlas of the catecholaminergic system in the brain and peripheral nervous system in mice from embryonic to pre-weaning stages. The procedures described allowed a quantitative assessment of developing TH-positive neuronal populations and pathways, previously understudied due to dimensional limitations. Our approach allowed us to reveal in 3D the dynamics of the onset and the establishment of the catecholaminergic system in embryonic and developing central and peripheral nervous system. Quantitative analyses applied to 3D images yielded accurate measurements of neuron population volumes and numbers, and tract pathway dimensions for selected TH-positive brain structures. We applied a set of different protocols to yield a comprehensive flowchart for 3D imaging and a precise quantitative assessment of specific neuronal populations during the course of their development up to adulthood in mice. The procedures described and the extensive 3D mapping of TH immunoreactivity at early embryonic and postnatal stages provide a comprehensive view of the onset and development of the catecholaminergic system in the mouse brain and sympathoadrenal nervous system.