Abstract
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 this work, we combined whole-mount immunostaining for tyrosine hydroxylase (TH), oxytocin (OXT) and arginine vasopressin (AVP), with iDISCO+ clearing method, light-sheet microscopy and semi automated counting of 3D-labelled neurons to obtain a 3D distribution of these neuronal populations in a 5-day postnatal (P5) mouse brain. Segmentation procedure and 3D reconstruction allowed us to map with high resolution TH staining the various catecholaminergic cell groups and their ascending and descending fiber pathways. We show that TH pathways are present in the whole P5 mouse brain, similar to what was observed in the adult rat brain.
We also provide here new information on the postnatal distribution of OXT and AVP immunoreactive cells in the mouse hypothalamus, and show that, compared to AVP neurons, OXT neurons 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 reveales that OXT cell bodies are more numerous than AVP neurons but their immunoreactive soma have a volume half smaller. More AVP nerve fibers as compared to OXT were observed in the PVN and the retrochiasmatic area.
In conclusion, the present work describes 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.
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