Abstract
The “connectome,” a comprehensive wiring diagram of synaptic connectivity, is achieved through volume electron microscopy (vEM) analysis of an entire nervous system and all associated non-neuronal tissues. White et al. (1986) pioneered the fully manual reconstruction of a connectome using Caenorhabditis elegans. Recent advances in vEM allow mapping new C. elegans connectomes with increased throughput, and reduced subjectivity. Current vEM studies aim to not only fill the remaining gaps in the original connectome, but also address fundamental questions including how the connectome changes during development, the nature of individuality, sexual dimorphism, and how genetic and environmental factors regulate connectivity. Here we describe our current vEM pipeline and projected improvements for the study of the C. elegans nervous system and beyond.
Highlights
We describe technical issues general to volume electron microscopy (vEM) studies and highlight key technical considerations for C. elegans
General Considerations for High-Pressure Freezing and Freeze Substitution For the original C. elegans wiring diagram reconstruction, animals were submerged in one or more chemical fixatives, either glutaraldehyde followed by osmium tetroxide, or osmium tetroxide alone (White et al, 1986)
Freeze Substitution With C. elegans Samples For morphological analyses, freeze substitution is performed in a programmable freeze substitution unit, where frozen samples are kept at −90◦C in the presence of tannic acid and glutaraldehyde, before being replaced by 2% OsO4, and brought to room temperature (Box 1; Weimer, 2006)
Summary
General Considerations for High-Pressure Freezing and Freeze Substitution For the original C. elegans wiring diagram reconstruction, animals were submerged in one or more chemical fixatives, either glutaraldehyde followed by osmium tetroxide, or osmium tetroxide alone (White et al, 1986). Freeze Substitution With C. elegans Samples For morphological analyses, freeze substitution is performed in a programmable freeze substitution unit, where frozen samples are kept at −90◦C in the presence of tannic acid and glutaraldehyde, before being replaced by 2% OsO4, and brought to room temperature (Box 1; Weimer, 2006). This protocol yields consistent results as long as samples are handled properly (see sections “General Considerations for High-Pressure Freezing and Freeze Substitution” and “High-Pressure Freezing of C. elegans”), and the high-pressure freezer is properly assembled and maintained.
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