Structural changes at synapses after delayed perfusion fixation in different regions of the mouse brain

Abstract

We recently showed by electron microscopy that the postsynaptic density (PSD) from hippocampal cultures undergoes rapid structural changes after ischemia-like conditions. Here we report that similar structural changes occur after delay in transcardial perfusion fixation of the mouse brain. Delay in perfusion fixation, a condition that mimics ischemic stress, resulted in 70%, 90%, and 23% increases in the thickness of PSDs from the hippocampus (CA1), cerebral cortex (layer III), and cerebellar cortex (Purkinje spines), respectively. In step with PSD thickening, the amount of PSD-associated alpha-calcium calmodulin-dependent protein kinase II (alpha- CaMKII) label increased more in cerebral cortical spines than in Purkinje spines. Although the Purkinje PSDs thickened only slightly after delayed fixation, they became highly curved, and many formed sub-PSD spheres approximately 80 nm in diameter that labeled for CaMKII. Delayed perfusion fixation also produced more cytoplamic CaMKII clusters ( approximately 110 nm in diameter) in the somas of pyramidal cells (from hippocampus and cerebral cortex) than in Purkinje cells. Thus a short delay in perfusion fixation produces cell-specific structural changes at PSDs and neuronal somas. Purkinje cells respond somewhat differently to delayed perfusion fixation, perhaps owing to their lower levels of CaMKII, and CaMKII binding proteins at PSDs. We present here a catalogue of structural changes that signal a perfusion fixation delay, thereby providing criteria by which to assess perfusion fixation quality in experimental structural studies of brain and to shed light on the subtle changes that occur in intact brain following metabolic stress.

Organization, control and function of extrasynaptic NMDA receptors

“…25%) processes. These extrasynaptic sites spread to an average width of less than 100 nm, which means they are substantially narrower than synaptic sites that have an estimated width of 195 nm in the CA1 region [37]. Interestingly, extrasynaptic NMDAR clusters seem to colocalize with intercellular adhesion molecules such as b-catenins and some scaffolding proteins such as SAP102 and PSD95 in culture [22], although it is not clear in this work whether any specific MAGUKs are preferentially interacting with extrasynaptic NMDARs.…”

Degenerating Synaptic Boutons in Prion Disease

“…An increase in PSD curvature and thickness has been shown to occur after a delay in transcardial perfusion fixation of the mouse brain, a condition that induces ischemic stress. 21 However, the PSDs in ischemic tissue arched into the presynaptic terminal to form convex PSDs, while in ME7-animals the PSDs were strikingly concave both at 16 and 18 weeks p.i.…”

“…A slow cardiac perfusion (20 to 30 minutes) was performed with fixative containing 3.4% paraformaldehyde, 1.25% glutaraldehyde, 0.2% picric acid in 0.1 M/L sodium phosphate buffer (final pH 7.2 to 7.4) immediately after short (Ͻ1.5 minutes) perfusion with heparinized saline, to minimize synaptic and glial ultrastructural changes that could be caused by brain hypoxia. 21 After approximately 1 hour, the brains were dissected and postfixed in fresh fixative overnight at 4°C; 150 m thick coronal sections were cut on a vibrotome and the area of CA1 pyramidal layer and stratum radiatum was dissected out. Microdissected areas were washed in 0.1 M/L sodium phosphate buffer and postfixed at room temperature for 1 hour in 1% osmium tetroxide.…”

“…21 An angle of 180 deg denotes a flat PSD, which does not arc. PSDs that arch into the presynaptic terminals (convex) were given a negative value, however, these profiles were rarely detected.…”

Effect of atorvastatin on dendritic cells of tubulointerstitium in diabetic rats

“…We harvested the renal tissue until the kidney turned whiter and harder. The perfusate was 4% paraformaldehyde (13).…”