Cm10 tem

Four samples were fixed by chemical fixation or high pressure freezing and freeze substitution as previously described ^{49 (link)}. Ultrathin sections were cut using a RMC Powertome XL, collected onto grids, and imaged using either a Philips CM10 TEM or Zeiss Supra 40 FE-SEM. Sections were elastically aligned ⁵⁰ and volumetrically reconstructed using TrakEM2 ^{51 (link)} . The MCM cell bodies were identified in the EM sections based on position and morphology and in comparison to similar hermaphrodite sections. This identity was further confirmed by the identification of a posterior projection, as no other cell with its soma in the same region is known to project posteriorly. This was followed by serial tracing of the projections to establish their morphology and connectivity. Synaptic connectivity and skeleton diagrams were determined using Elegance ^{52 (link)} . Circuit diagrams of connectivity were generated using Cytoscape ^{53 (link)}. We estimated the anatomical strength of synaptic connectivity between two neurons by summing the number of serial sections where we observed the ultrastructural morphology presynaptic components using the same criteria as ¹⁴