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

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.

Keywords: automatic segmentation; brain-wide dataset; corticotropin-releasing hormone; image processing; neuron; optical imaging; three-dimensional reconstruction.

FIGURE 1

Specific EYFP-expression of CRH-IRES-Cre;Ai3 mouse brain. (A–J) Representative photomicrographs depicting CRH neurons expressing EYFP (green), CRH immunostaining (red) and their merged images in MOB, CEA, PVH, B, ECU, IO, MPN, PVT, PG, and TRN, respectively, in a CRH-IRES-Cre;Ai3 mouse brain. The arrowheads show EYFP and CRH double-positive neurons. Scale bar: 40 μm. Abbreviations: main olfactory bulb (MOB), central amygdalar nucleus (CEA), paraventricular hypothalamic nucleus (PVH), Barrington’s nucleus (B), external cuneate nucleus (ECU), inferior olivary complex (IO), medial preoptic nucleus (MPN), paraventricular nucleus of the thalamus (PVT), pontine gray (PG), and tegmental reticular nucleus (TRN).

FIGURE 2

The brain-wide distribution of CRH neurons imaged by BPS. (A) Three-dimensional rendering of EYFP-labeled CRH neurons. Resampling at 2 μm × 2 μm × 2 μm. (B) Sagittal reconstruction of the maximum intensity projection at the location indicated by a dot dash line in (A) in the CRH-IRES-Cre;Ai3 mouse brain. Projection thickness was 100 μm. (C) The 50-μm-thick maximum intensity projections of the coronal slices indicated by dash lines in (B), respectively. (D) Enlarged views of EYFP-labeled CRH neurons of the area indicated with text annotations and white boxes in (C). Images of MOs and HPF were the maximum intensity projections of 50 μm, reflecting the sparse distribution of CRH neurons. Images of PIR, ACB, PVH, B, and IO were original data of a single slice. (E) Original images of EYFP-labeled CRH neurons, PI-stained cytoarchitecture and the merge of enlarged views of the area indicated by a red arrowhead and a white box in CEA in (c3). Scale bars: (A,B) 1 mm, (C) 1 mm, and (D) 30 μm. Secondary motor area (MOs), piriform area (PIR), nucleus accumbens (ACB), PVH, CEA, hippocampal formation (HPF), B, and IO.

FIGURE 3

Locating EYFP-labeled CRH neurons in the whole CRH-IRES-Cre;Ai3 mouse brain. (A) Locating neurons in 100 μm typical image stacks, presented in maximum intensity projection. (B) Three-dimensional reconstruction and (C,D) projection views of the data cubes indicated as white boxes in (A), respectively. Gray and green signals represent CRH neuronal somas. Red dots represent the recognized centers of CRH neurons. The white, red, blue and orange arrowheads indicated the axial-overlapping neurons, low intensity neurons, error recognition neurons and misidentification neurons, respectively. (E) Counting accuracy. Blue and orange represent the recall and precision (n = 12), respectively. Each data cube was randomly selected at a size of 300 μm × 300 μm × 300 μm. Scale bars: (A) 1 mm and (C,D) 50 μm.

FIGURE 4

The segmentation of anatomical regions. (A) A 10-μm maximum intensity projection of representative PI-stained coronal slices in the same mouse brain. The cyan lines represent the segmented results using the ANTS tool. (B) EYFP-labeled CRH neurons and PI-stained cytoarchitecture of the enlarged view of the area indicated by a green rectangular box in (A). Green represents the 100 μm maximum intensity projection of EYFP-labeled CRH neurons. Red represents the 2 μm original data of PI-stained cytoarchitecture. White lines represent the laminated contours of the cortex. (C) Quantitative statistics of EYFP-labeled CRH neurons in different layers in MOp, MOs, VIS, AUD, SSp and SSs. Scale bar: (A) 1 mm, (B) 100 μm. Abbreviations: olfactory areas (OLF), pallidum (PAL), striatum (STR), hypothalamus (HY), cortical subplate (CTXsp), hippocampal formation (HPF), thalamus (TH), midbrain (MB), pons (P), medulla (MY), cerebellum (CB), primary motor area (MOp), secondary motor area (MOs), visual area (VIS), auditory area (AUD), primary somatosensory area (SSp), and supplemental somatosensory area (SSs).

FIGURE 5

Quantitative statistics of the distribution of EYFP-labeled CRH neurons in the CRH-IRES-Cre;Ai3 mouse brain. (A) Recognized positions of EYFP-labeled neurons in a CRH-IRES-Cre;Ai3 mouse brain. Different color points represent the soma centers of CRH neurons in different regions. (B) Cell densities of EYFP-labeled CRH neurons in 12 brain regions. Color definitions are the same as those shown in (A). (C) Cell density and nuclei distance of EYFP-labeled CRH neurons in the sub-regions of the above brain regions.

FIGURE 6

Analyzing soma morphology of EYFP-labeled CRH neurons in eleven discrete brain areas. (A) EYFP-labeled CRH neurons in 12 discrete brain areas. Each data cube was randomly selected at the size of 200 μm × 200 μm × 200 μm. Scale bar: 50 μm. (B) Extracting the morphological features of CRH neuronal somas using PCA transformation. Two normalized main features were acquired after PCA on the six morphological parameters. The cyan lines surrounding the most concentrated neurons with similar soma morphology account for 80% of the total neurons in each region. (C) SDs of both main features in the main regions except CB. (D) An ANOVA was conducted to analyze both of the main features. The light and dark colors represent P < 0.05 and P ≥ 0.05, respectively. Primary somatosensory area (SSp), MOB, Ammon’s horn (CA), endopiriform nucleus, dorsal part (EPd), CEA, bed nuclei of the stria terminalis (BST), central medial nucleus of the thalamus (CM), PVH, periaqueductal gray (PAG), PG, IO, and hemispheric regions (HEM).