Ultrastructural comparison of dendritic spine morphology preserved with cryo and chemical fixation

Abstract

Previously we showed that cryo fixation of adult mouse brain tissue gave a truer representation of brain ultrastructure in comparison with a standard chemical fixation method (Korogod et al 2005). Extracellular space matched physiological measurements, there were larger numbers of docked vesicles and less glial coverage of synapses and blood capillaries. Here, using the same preservation approaches we compared the morphology of dendritic spines. We show that the length of the spine and the volume of its head is unchanged, however, the spine neck width is thinner by more than 30 % after cryo fixation. In addition, the weak correlation between spine neck width and head volume seen after chemical fixation was not present in cryo-fixed spines. Our data suggest that spine neck geometry is independent of the spine head volume, with cryo fixation showing enhanced spine head compartmentalization and a higher predicted electrical resistance between spine head and parent dendrite.

Ultrastructural analysis of dendritic spine necks reveals a continuum of spine morphologies

“…Previous studies in living tissue have reported no correlation between spine head and neck parameters (Araya et al, 2014;Tønnesen et al, 2014). Studies in fixed tissue have found weak correlations between head volume and neck diameter in chemically fixed spines, but not in cryo-fixed spines (Arellano et al, 2007;Bartol et al, 2015;Tamada et al, 2020). We used our dataset and analysis pipeline to explore this issue with a larger number of ultrastructural reconstructed spines (Figure 5).…”

Quantification of Dendritic Spines Remodeling under Physiological Stimuli and in Pathological Conditions

“…The key element in dendritic spine staining is to apply an appropriate, gentle tissue fixation to assess the relevant effects on spine structure and eliminate cascades of biochemical processes affecting biophysical properties of neuronal membrane caused by cell death [ 32 , 96 , 98 , 99 ], therefore the vital condition of cells is an important factor in accurate spine analysis [ 97 , 100 ]. It is still not clear how in vitro, ex vivo, and in vivo models differ in speed of biochemical processes [ 97 , 100 , 101 , 102 ]. Therefore, the live cell imaging approach constitutes a suitable alternative, however, it requires analyzing more neurons, culture or animals per group due to technical limitations during live imaging.…”

“…Investigation of all the parameters including neck width is achievable also using serial section electron microscopy (EM), which enables a detailed morphometric analysis at the nanoscale [ 127 , 128 , 129 ]. However, in the EM imaging, a sample needs to be fixated, permeabilized, dehydrated, and also placed under high vacuum—these procedures include the type of sample fixation (cryo- or chemical- fixation) that frequently causes structural artifacts to disturb the morphometric parameters of the dendritic spines [ 101 , 130 , 131 ] (comparative morphological analysis of images from live imaging and their respective images obtained using EM, see [ 130 , 131 ]). Currently, the most popular method used for precise dendritic spines imaging is based on super-resolution fluorescent light microscopy (lateral resolution around 20 to 40 nm in fixed tissue) through an expression of fluorescent membrane proteins that reduce an impact of sample preparation [ 101 , 132 ].…”

“…However, in the EM imaging, a sample needs to be fixated, permeabilized, dehydrated, and also placed under high vacuum—these procedures include the type of sample fixation (cryo- or chemical- fixation) that frequently causes structural artifacts to disturb the morphometric parameters of the dendritic spines [ 101 , 130 , 131 ] (comparative morphological analysis of images from live imaging and their respective images obtained using EM, see [ 130 , 131 ]). Currently, the most popular method used for precise dendritic spines imaging is based on super-resolution fluorescent light microscopy (lateral resolution around 20 to 40 nm in fixed tissue) through an expression of fluorescent membrane proteins that reduce an impact of sample preparation [ 101 , 132 ]. In STED microscopy, the use of continuous-wave lasers requires higher depletion beam power than with pulsed lasers, resulting in more severe photobleaching of the sample.…”

Three-Dimensional Structure of Dendritic Spines Revealed by Volume Electron Microscopy Techniques

“…The ultrastructure of neurons also varies depending on the choice of fixative. Compared with rapid cryofixation of tissue, aldehyde-based chemical fixatives greatly reduce the space occupied by the extracellular matrix and increase the spine neck width (Korogod et al, 2015 ; Tamada et al, 2020 ). Therefore, great care should be taken in interpreting EM data and special caution should be applied when comparing data obtained from using different fixation conditions.…”