Application of three-dimensional electron microscopy to morphological study of neurons in brainstem cochlear nucleus

doi:10.3969/j.issn.1674-8115.2022.02.002

Abstract

Abstract: Objective

·To explore the feasibility of cross-scale neuroanatomy and connectomics of mouse cochlear nucleus (CN) by using a new three-dimensional (3D) electron microscopic imaging method.

Methods

·The intact CN was obtained after the brain tissue of adult CBA/Ca mice (2 months old) was fixed and dissected, stained with heavy metals (reducing osmium amplification method) , dehydrated with gradient ethanol and anhydrous acetone and embedded with low viscosity resin. The CN tissue was imaged by X-ray microscopy and 3D reconstruction, and its staining quality was evaluated. Then targeted subdivision was located according to the specific distribution of auditory nerve fibers. The low-resolution pre-scan of the CN tissue was performed by scanning electron microscopy, and compared with X-ray microscopic dataset. After the target location was determined, the volume of interest was mapped by serial block-face scanning electron microscopy (SBEM) for 3D reconstruction, and the ultrastructures of bushy cells and the synapses projecting on its surface were tracked, annotated and reconstructed.

Results

·3D electron microscopic samples of intact CN of CBA/Ca mice were successfully prepared. The 3D structure of CN with cell resolution was collected and reconstructed by X-ray microscopic imaging, and the anatomical localization of subregions of CN was achieved. The 3D electron microscopic data of bushy cells in the target area of CN were successfully collected by SBEM. The tracking, labeling and reconstruction of endbulb of Held synapses and other non-auditory synapses on the cell body of target bushy cells were completed. The data of 3D electron microscopy showed that there were 5 endbulb of Held synapses projecting to the surface of the target bushy cell, forming a total of 348 synaptic active zones, while there were 97 synapses from non-auditory nerve.

Conclusion

·It is feasible to prepare 3D electron microscopic samples of adult mouse intact CN by Osmium-based enbloc staining and resin embedding. X-ray microscopic imaging can be used for rapid and accurate localization of subregions and target volume of CN, and the acquired 3D electron microscopic data can be used to study the neuronal morphology and synaptic connections in the CN.

Key words: cochlear nucleus (CN), bushy cell, endbulb of Held synapase, serial block-face scanning electron microscopy (SBEM), X-ray microscopic imaging

CLC Number:

R764.4

Jialei ZHOU, Haibin SHENG, Haoyu WANG, Yan LU, Fangfang WANG, Hao WU, Yunfeng HUA. Application of three-dimensional electron microscopy to morphological study of neurons in brainstem cochlear nucleus[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2022, 42(2): 142-149.

Figures/Tables 5

TrendMD

References 39

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Item	Sample
Item	1^#	2^#	3^#	4^#	5^#
Mouse species	CBA/Ca	CBA/Ca	CBA/Ca	CBA/Ca	CBA/Ca
Gender	male	male	male	male	male
Age/week	7	7	7	8	8
Sample size/mm³	1.3×1.7×2.5	1.4×1.6×2.8	1.3×1.5×2.6	1.3×1.5×2.5	1.2×1.4×2.6
X-ray microscopic imaging resolution/μm³	1.6	1.5	0.7	1.4	2.8
SBEM imaging
Pixel/nm³	10×10×50	15×15×50	‒	12×12×35	15×15×50
Data size/μm³	114.3×105.0×105.0	754.6×192.2×258.1	‒	443.0×141.1×91.0	193.1×505.1×126.7
Accelerating voltage/kV	2.0	2.0	‒	2.0	2.0
Exposure time/μs	1.5	1.0	‒	1.0	1.5

Item	Sample
Item	1^#	2^#	3^#	4^#	5^#
Mouse species	CBA/Ca	CBA/Ca	CBA/Ca	CBA/Ca	CBA/Ca
Gender	male	male	male	male	male
Age/week	7	7	7	8	8
Sample size/mm³	1.3×1.7×2.5	1.4×1.6×2.8	1.3×1.5×2.6	1.3×1.5×2.5	1.2×1.4×2.6
X-ray microscopic imaging resolution/μm³	1.6	1.5	0.7	1.4	2.8
SBEM imaging
Pixel/nm³	10×10×50	15×15×50	‒	12×12×35	15×15×50
Data size/μm³	114.3×105.0×105.0	754.6×192.2×258.1	‒	443.0×141.1×91.0	193.1×505.1×126.7
Accelerating voltage/kV	2.0	2.0	‒	2.0	2.0
Exposure time/μs	1.5	1.0	‒	1.0	1.5

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