Unambiguous identification of asymmetric and symmetric synapses using volume electron microscopy

Abstract

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.

Tracing nerve fibers with volume electron microscopy to quantitatively analyze brain connectivity

“…The study was performed in the neuropil, composed of axons, dendrites, and glial processes, so cell bodies or blood vessels were not present in the imaged volume of tissue. Synaptic junctions were identified based on several morphological features 11 , 24 . Presynaptic elements show an accumulation of synaptic vesicles close to the presynaptic membrane, which shows an electron-dense thickening.…”

“…The segmentation algorithm applies a gray-level threshold to extract the voxels of the pre- and postsynaptic densities that appear as dark, electron-dense structures under the electron microscope 77 . As previously described 11 , 24 , 78 , synaptic junctions with a prominent or thin postsynaptic density were classified as asymmetric or symmetric synaptic junctions, respectively 25 , 26 . The density of synapses was calculated by directly counting the number of segmented synapses inside a three-dimensional counting frame of known volume 11 .…”