Korean J Physiol Pharmacol.  2012 Dec;16(6):423-429. 10.4196/kjpp.2012.16.6.423.

Oxygen/Glucose Deprivation and Reperfusion Cause Modifications of Postsynaptic Morphology and Activity in the CA3 Area of Organotypic Hippocampal Slice Cultures

• Jung YJ
• Suh EC
• Lee KE

• KMID: 2285451
• DOI: http://doi.org/10.4196/kjpp.2012.16.6.423

Abstract

Brain ischemia leads to overstimulation of N-methyl-D-aspartate (NMDA) receptors, referred as excitotoxicity, which mediates neuronal cell death. However, less attention has been paid to changes in synaptic activity and morphology that could have an important impact on cell function and survival following ischemic insult. In this study, we investigated the effects of reperfusion after oxygen/glucose deprivation (OGD) not only upon neuronal cell death, but also on ultrastructural and biochemical characteristics of postsynaptic density (PSD) protein, in the stratum lucidum of the CA3 area in organotypic hippocampal slice cultures. After OGD/reperfusion, neurons were found to be damaged; the organelles such as mitochondria, endoplasmic reticulum, dendrites, and synaptic terminals were swollen; and the PSD became thicker and irregular. Ethanolic phosphotungstic acid staining showed that the density of PSD was significantly decreased, and the thickness and length of the PSD were significantly increased in the OGD/reperfusion group compared to the control. The levels of PSD proteins, including PSD-95, NMDA receptor 1, NMDA receptor 2B, and calcium/calmodulin-dependent protein kinase II, were significantly decreased following OGD/reperfusion. These results suggest that OGD/reperfusion induces significant modifications to PSDs in the CA3 area of organotypic hippocampal slice cultures, both morphologically and biochemically, and this may contribute to neuronal cell death and synaptic dysfunction after OGD/reperfusion.

Keyword

Neuronal cell death; NMDAR; OGD/reperfusion; Organotypic hippocampal slice cultures; Postsynaptic density

MeSH Terms

Brain Ischemia
Cell Death
Dendrites
Endoplasmic Reticulum
Ethanol
Mitochondria
N-Methylaspartate
Neurons
Organelles
Phosphotungstic Acid
Post-Synaptic Density
Presynaptic Terminals
Protein Kinases
Proteins
Receptors, N-Methyl-D-Aspartate
Reperfusion
Ethanol
N-Methylaspartate
Phosphotungstic Acid
Protein Kinases
Proteins
Receptors, N-Methyl-D-Aspartate

Figure

• 

  Fig. 1 Light micrographs of the CA3 pyramidal cell layer of organotypic hippocampal slice cultures, stained with toluidine blue, after OGD/reperfusion. (A) Control, (B) OGD for 30 min, (C) reperfusion for 6 h after OGD, (D) reperfusion for 24 h after OGD. Neuronal cell death was induced after OGD/reperfusion. Black arrows indicate condensed pyknotic nuclei, and white arrows indicate cells stained dark, with triangular shrunken perikaryon. Scale bar=100 µm.

• 

  Fig. 2 Electron micrographs of osmiumuranium-lead-stained stratum lucidum in the CA3 area of organotypic hippocampal slice cultures after OGD/reperfusion. (A, E) control, (B, F) OGD for 30 min, (C, G) reperfusion for 6 h after OGD, (D, H) reperfusion for 24 h after OGD. (E~H) higher magnification images. In control slices, typical intact synapses with a clear narrow synaptic cleft can be clearly seen (arrowhead). Mitochondria, ER, dendrites, and synaptic terminals were swollen, and the thickness of the PSD in many synapses became thicker (black arrows) in the OGD/reperfusion group than in the controls. Scale bar=2 µm.

• 

  Fig. 3 Electron micrographs of EPTA staining of stratum lucidum in the CA3 area of organotypic hippocampal slice cultures after OGD/reperfusion. (A) Control, (B) reperfusion for 24 h after OGD. (C) Synapse density/100 µm2, (D) length of a PSD, (E) minimum and maximum thickness of a PSD, (F) area of a PSD. In the control slices, many PSDs appeared as thin and compact lines. At 24 h after OGD, the density of PSDs was significantly decreased, and the PSDs became thicker, longer, and more irregular. Insert images are high magnification of the synapses. Scale bar=1 µm. Data are expressed as mean±SEM. *p<0.05 compared to control.

• 

  Fig. 4 Representative western blots for PSD-95, NR, NR2B, and CaMKII after OGD/reperfusion. (A) At 24 h after OGD, the levels of all the PSD complex proteins were decreased. (B) Data are expressed as mean±SEM. *p<0.05 vs. the control. n=3.

• 

  Fig. 5 Aschematic illustration of the interaction of NMDARs with PSD-95 and CaMKII.

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