Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities.

Abstract

Postsynaptic densities (PSDs) have been isolated from cerebral cortex, midbrain, cerebellum, and brain stem by the Triton X-100 method previously used in the isolation of cerebral PSDs (Cohen et al ., 1977, 1. Cell Biol. 74 :181) . These PSDs have been compared in protein composition, protein phosphorylation, and morphology . Thin-section electron microscopy revealed that cerebral cortex and midbrain PSDs were identical, being^-57 nm thick and composed of apparent aggregates 20-30 nm in diameter . Isolated cerebellar PSDs appeared thinner (33 nm) than cerebral cortex PSDs and lacked the apparent 20-to 30-nm aggregates, but had a latticelike structure. In unidirectional and rotary-shadowed replicas, the cerebrum and midbrain PSDs were circular in shape with a large central perforation or hole in the center of them . Cerebellum PSDs did not have a large perforation, but did have numerous smaller perforations in a lattice like structure. Filaments (6-9 nm) were observed connecting possible 20-to 30-nm aggregates in cerebrum PSDs and were also observed radiating from one side of the PSD. Both cerebral cortex and midbrain PSDs exhibited identical protein patterns on SDS gel electrophoresis. In comparison, cerebellar PSDs (a) lacked the major 51,000 Mr protein, (b) contained two times less calmodulin, and (c) contained a unique protein at 73,000 Mr. Calcium plus calmodulin stimulated the phosphorylation of the 51,000 and 62,000 Mr bands in both cerebral cortex and midbrain PSDs . In cerebellar PSDs, only the 58,000 and 62,000 M r bands were phosphorylated . In the PSDs from all brain regions, CAMP stimulated the phosphorylation of Protein la (73,000 Mr), Protein Ib (68,000 Mr), and a 60,000 Mr protein, although cerebrum and midbrain PSDs contained very much higher levels of phosphorylated protein than did the cerebellum .On the basis of the morphological criteria, it is possible that PSDs isolated from cerebrum and midbrain were derived from the Gray type I, or asymmetric, synapses, whereas cerebellum PSDs were derived from the Gray type II, or symmetric, synapses, Since there is some evidence that the type I synapses are involved in excitatory mechanisms while he type II are involved in inhibitory mechanisms, the role of the PSD and of some of its proteins in these synaptic responses is discussed .

Postmortem Brain: An Underutilized Substrate for Studying Severe Mental Illness

“…Synaptosomes are small lipid vesicles that form from synapses and astrocytic process following mechanical disruption of brain tissue using a blender or homogenizer (Whittaker et al, 1963). Synaptosomes may contain pre-and postsynaptic elements; this is a potentially confounding problem, as many of the proteins that facilitate receptor localization, anchoring, and signaling may be present in both compartments (Carlin et al, 1980;Morciano et al, 2005). One recent study has made strides to address this problem.…”

αCaMKII autophosphorylation levels differ depending on subcellular localization

“…In order to separate different parts of the neuron, a modified subcellular fractionation protocol was used (Carlin et al, 1980;Huttner et al, 1983;Cho et al, 1992;Goebel et al, 2005;Smith et al, 2006). This protocol, which consisted of differential centrifugation combined with a detergent extraction using the non-ionic detergent Triton X-100, yielded a PSD-enriched fraction which we termed the TxP, an extra-synaptic membrane-enriched fraction which we termed the TxS, a microsome-enriched fraction which we termed the P3, and a cytosolic/ soluble fraction which we termed the S3.…”

“…Following harvesting, the slices were immediately homogenized in a glass grinding vessel by a Teflon pestle rotating at 1000 RPM. Subsequently, a modified subcellular fraction protocol was employed (Carlin et al, 1980;Huttner et al, 1983;Cho et al, 1992;Goebel et al, 2005;Smith et al, 2006). Due to the limited amount of tissue, the commonly used sucrose density gradient centrifugation step(s), which are utilized to obtain a more pure synaptic fraction, were omitted from our fractionation protocol.…”

Relative Quantification of Stable Isotope Labeled Peptides Using a Linear Ion Trap-Orbitrap Hybrid Mass Spectrometer

“…Each developmental brain homogenate was mixed in a 1:1 ratio with 15 N labeled rat brain homogenate and synaptosomes were isolated as previously described 12 . The synaptosome enriched samples was digested with proteinase K as previously described 13 .…”