A Quantitative Analysis of the Distribution of CRH Neurons in Whole Mouse Brain.

Jie Peng,Hong Ni,Xue Peng,Anan Li,Jing Yuan,Qingming Luo,Xiangning Li,Ben Long,Hui Gong

doi:10.3389/fnana.2017.00063

Abstract

Corticotropin-releasing hormone (CRH), with widespread expression in the brain, plays a key role in modulating a series of behaviors, including anxiety, arousal, motor function, learning and memory. Previous studies have focused on some brain regions with densely distributed CRH neurons such as paraventricular hypothalamic nucleus (PVH) and bed nuclei of the stria terminalis (BST) and revealed some basic structural and functional knowledge of CRH neurons. However, there is no systematic analysis of brain-wide distribution of CRH neurons. Here, we performed a comprehensive study of CRH neurons in CRH-IRES-Cre;Ai3 mice via automatic imaging and stereoscopic cell counting in a whole mouse brain. We acquired four datasets of the CRH distributions with co-localized cytoarchitecture at a voxel resolution of 0.32 μm × 0.32 μm × 2 μm using brain-wide positioning system (BPS). Next, we precisely located and counted the EYFP-labeled neurons in different regions according to propidium iodide counterstained anatomical reference using Neuronal Global Position System. In particular, dense EYFP expression was found in piriform area, BST, central amygdalar nucleus, PVH, Barrington’s nucleus, and inferior olivary complex. Considerable CRH neurons were also found in main olfactory bulb, medial preoptic nucleus, pontine gray, tegmental reticular nucleus, external cuneate nucleus, and midline thalamus. We reconstructed and compared the soma morphology of CRH neurons in 11 brain regions. The results demonstrated that CRH neurons had regional diversities of both cell distribution and soma morphology. This anatomical knowledge enhances the current understanding of the functions of CRH neurons. These results also demonstrated the ability of our platform to accurately orient, reconstruct and count neuronal somas in three-dimension for type-specific neurons in the whole brain, making it feasible to answer the fundamental neuroscience question of exact numbers of various neurons in the whole brain.

Highlights

Corticotropin-releasing hormone (CRH) is an important broadly expressed neuropeptide which has neuroendocrine and neurotransmitter properties (Vale et al, 1981; Young, 1992)
We systematically analyzed the co-localization of EYFP expression with CRH immunoreactivity in the brain regions which had considerable neurons to evaluate the accuracy of targeting CRH neurons
The majority (∼80%) of CRH stained neurons were YFP positive in the main olfactory bulb (MOB), paraventricular hypothalamic nucleus (PVH), B, external cuneate nucleus (ECU), inferior olivary complex (IO), paraventricular nucleus of the thalamus (PVT), pontine gray (PG), and tegmental reticular nucleus (TRN), while relatively lower (∼50%) co-labeling was observed in central amygdalar nucleus (CEA) and medial preoptic nucleus (MPN)

Summary

Introduction

Corticotropin-releasing hormone (CRH) is an important broadly expressed neuropeptide which has neuroendocrine and neurotransmitter properties (Vale et al, 1981; Young, 1992). Genetic techniques provide access for targeting specific neurons according to the molecular expression of CRH in the brain. Using these labeling methods, the distribution features of CRH in the rodent brain have been extensively explored in some brain regions and nuclei, such as the amygdala (De Francesco et al, 2015), olfactory bulb (Huang et al, 2013; Garcia et al, 2016) and bed nuclei of the stria terminalis (BST) (Nguyen et al, 2016). The expression patterns of CRH in whole mouse brains had been qualitatively described (Alon et al, 2009; Kono et al, 2016). These studies acquired data from images of histological sections and analyzed these images through two-dimensional cell counting. The comprehensive organization of brain-wide CRH network has not been systematically and quantitatively analyzed

Methods

Results

Conclusion