研究目的
Investigating the synaptic specializations of melanopsin-expressing retinal ganglion cells (mRGCs) in multiple brain regions to understand how these cells facilitate diverse visual functions.
研究成果
The study reveals that ipRGCs exhibit region-specific synaptic specializations in different brain regions, which correspond to the functional characteristics of each target region. A subset of SCN neurons forms a network through dendrodendritic chemical synapses (DDCSs), with ipRGCs preferentially synapsing on these DDCS-linked cells. The methods developed for this study can propel other CLEM studies of long-distance brain circuits at high resolution.
研究不足
The study is limited by the technical challenges of SBEM, including the inability to genetically label specific cell types in large tissue volumes and the computational resources required for data analysis. The automatic segmentation algorithm had limitations in distinguishing between myelinated non-ipRGC axons and miniSOG+ ipRGC axons.
1：Experimental Design and Method Selection:
Utilized a genetically encoded electron microscopy tag (miniSOG) expressed in mRGCs for correlated light and electron microscopy (CLEM). Employed serial block-face electron microscopy (SBEM) to analyze the optic nerve and synaptic neuropil in five different brain regions.
2：Sample Selection and Data Sources:
Used Opn4Cre/+ mice for specific expression of miniSOG in mRGCs. Collected tissues from the retina, optic nerve, and five brain regions (SCN, OPN, dLGN, vLGN, IGL).
3：List of Experimental Equipment and Materials:
AAV2-EF1a-DIO-miniSOG-f virus for genetic labeling, confocal microscopy for light microscopy, SBEM for electron microscopy.
4：Experimental Procedures and Operational Workflow:
Intravitreal injection of AAV vectors, photo-oxidation of miniSOG, tissue processing for SBEM, manual and automatic segmentation of SBEM data.
5：Data Analysis Methods:
Quantitative analysis of synaptic boutons, axon arborization, and synaptic density using IMOD software and custom algorithms.
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