ABSTRACT
NMDA receptors (NMDARs) are involved in synaptic transmission and synaptic plasticity in different brain regions, and they modulate glutamate release at different presynaptic sites. Here, we studied whether non-postsynaptic NMDARs, putatively presynaptic (preNMDARs), are tonically active at hippocampal CA3-CA1 synapses, and if they modulate glutamate release. We found that when postsynaptic NMDARs are blocked by MK801, D-AP5 depresses evoked and spontaneous excitatory synaptic transmission, indicating that preNMDARs are tonically active at CA3-CA1 synapses, facilitating glutamate release. The subunit composition of these NMDARs was determined by studying evoked and spontaneous excitatory synaptic transmission in the presence of Zn2+, Ro 25-6981, and PPDA, antagonists of NMDARs containing GluN2A, GluN2B, and GluN2C/D, respectively. We found that evoked and spontaneous release decreased when the activity of NMDARs containing GluN2B and GluN2C/D subunits but not GluN2A was impeded. In addition, we found that the increase in glutamate release mediated by these NMDARs requires protein kinase A (PKA) activation. We conclude that preNMDARs that contain GluN2B and GluN2C/2D subunits facilitate glutamate release at hippocampal CA3-CA1 synapses through a mechanism that involves PKA.
Subject(s)
Glutamic Acid/metabolism , Hippocampus/metabolism , Protein Subunits/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Synapses/metabolism , Synaptic Transmission/physiology , Animals , Excitatory Postsynaptic Potentials/physiology , Mice , Patch-Clamp TechniquesABSTRACT
Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. By using a cocktail composed of taxol (a microtubule stabilizer), Zn(2+) (a caspase inhibitor) and coenzyme Q10 (a lipid antioxidant), we were able to stabilize H460 apoptotic cells in cell cultures for at least 72 h, preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cell characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy.
