Three-dimensional distribution of tyrosine hydroxylase, vasopressin and oxytocin neurones in the transparent postnatal mouse brain.

Over the years, advances in immunohistochemistry techniques have been a critical step in detecting and mapping neuromodulatory substances in the central nervous system. The better quality and specificity of primary antibodies, new staining procedures and the spectacular development of imaging technologies have allowed such progress. Very recently, new methods permitting tissue transparency have been successfully used on brain tissues. In the present study, we combined whole-mount immunostaining for tyrosine hydroxylase (TH), oxytocin (OXT) and arginine vasopressin (AVP), with the iDISCO+ clearing method, light-sheet microscopy and semi-automated counting of three-dimensionally-labelled neurones to obtain a (3D) distribution of these neuronal populations in a 5-day postnatal (P5) mouse brain. Segmentation procedure and 3D reconstruction allowed us, with high resolution, to map TH staining of the various catecholaminergic cell groups and their ascending and descending fibre pathways. We show that TH pathways are present in the whole P5 mouse brain, similar to that observed in the adult rat brain. We also provide new information on the postnatal distribution of OXT and AVP immunoreactive cells in the mouse hypothalamus, and show that, compared to AVP neurones, OXT neurones in the supraoptic (SON) and paraventricular (PVN) nuclei are not yet mature in the early postnatal period. 3D semi-automatic quantitative analysis of the PVN reveals that OXT cell bodies are more numerous than AVP neurones, although their immunoreactive soma have a volume half smaller. More AVP nerve fibres compared to OXT were observed in the PVN and the retrochiasmatic area. In conclusion, the results of the present study demonstrate the utility and the potency of imaging large brain tissues with clearing procedures coupled to novel 3D imaging technologies to study, localise and quantify neurotransmitter substances involved in brain and neuroendocrine functions.