Frontiers in Neuroanatomy (Apr 2024)

Unambiguous identification of asymmetric and symmetric synapses using volume electron microscopy

  • Nicolás Cano-Astorga,
  • Nicolás Cano-Astorga,
  • Nicolás Cano-Astorga,
  • Nicolás Cano-Astorga,
  • Sergio Plaza-Alonso,
  • Sergio Plaza-Alonso,
  • Sergio Plaza-Alonso,
  • Marta Turegano-Lopez,
  • Marta Turegano-Lopez,
  • Marta Turegano-Lopez,
  • José Rodrigo-Rodríguez,
  • José Rodrigo-Rodríguez,
  • José Rodrigo-Rodríguez,
  • Angel Merchan-Perez,
  • Angel Merchan-Perez,
  • Angel Merchan-Perez,
  • Javier DeFelipe,
  • Javier DeFelipe,
  • Javier DeFelipe

DOI
https://doi.org/10.3389/fnana.2024.1348032
Journal volume & issue
Vol. 18

Abstract

Read online

The brain contains thousands of millions of synapses, exhibiting diverse structural, molecular, and functional characteristics. However, synapses can be classified into two primary morphological types: Gray’s type I and type II, corresponding to Colonnier’s asymmetric (AS) and symmetric (SS) synapses, respectively. AS and SS have a thick and thin postsynaptic density, respectively. In the cerebral cortex, since most AS are excitatory (glutamatergic), and SS are inhibitory (GABAergic), determining the distribution, size, density, and proportion of the two major cortical types of synapses is critical, not only to better understand synaptic organization in terms of connectivity, but also from a functional perspective. However, several technical challenges complicate the study of synapses. Potassium ferrocyanide has been utilized in recent volume electron microscope studies to enhance electron density in cellular membranes. However, identifying synaptic junctions, especially SS, becomes more challenging as the postsynaptic densities become thinner with increasing concentrations of potassium ferrocyanide. Here we describe a protocol employing Focused Ion Beam Milling and Scanning Electron Microscopy for studying brain tissue. The focus is on the unequivocal identification of AS and SS types. To validate SS observed using this protocol as GABAergic, experiments with immunocytochemistry for the vesicular GABA transporter were conducted on fixed mouse brain tissue sections. This material was processed with different concentrations of potassium ferrocyanide, aiming to determine its optimal concentration. We demonstrate that using a low concentration of potassium ferrocyanide (0.1%) improves membrane visualization while allowing unequivocal identification of synapses as AS or SS.

Keywords