A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue

Area of Science:

• Neuroscience
• Developmental Biology
• Cell Biology

Background:

• Carbocyanine dyes are fluorescent lipophilic substances used for neuronal studies.
• Previous applications focused on live tissue for membrane potential and fluidity.
• Recent studies showed their utility for in vivo and in vitro neuronal staining.

Purpose of the Study:

• To investigate the utility of carbocyanine dyes diI and diO for labeling neurons in fixed embryonic brain tissue.
• To assess the quality and extent of neuronal labeling in fixed tissue.
• To apply this technique to study early optic fiber development in mouse embryos.

Main Methods:

• Utilized carbocyanine dyes diI and diO on fixed embryonic mouse and chicken brain tissue.
• Examined neuronal processes and perikarya labeling in anterograde and retrograde directions.
• Analyzed the staining progression along axons to understand the mechanism.

Main Results:

• DiI and diO effectively labeled neurons in aldehyde-fixed embryonic brain tissue.
• Staining was smooth, clear, and observed along considerable distances in both anterograde and retrograde directions.
• The technique allowed visualization of early optic fiber development, showing a predominantly crossed projection with some ipsilateral growth from the outset.

Conclusions:

• Carbocyanine dyes diI and diO offer a robust method for neuronal tracing in fixed embryonic tissues.
• This technique rivals established methods like HRP or Lucifer Yellow injections.
• Early optic projections in mouse embryos reveal that both crossed and uncrossed pathways originate from similar retinal regions, with laterality partitioning occurring subsequently.