Analyzing the Role of NCKX2 in Hippocampal Long Term Potentiation

Abstract

Na+/Ca2+ exchangers control clearance of cytosolic Ca2+ following a signaling event when Ca2+ is elevated due to their low-affinity/high capacity transport properties. NCKX2 is a member of the family of K+-dependent Na+/Ca2+-exchangers, and is abundant in brain neurons. Targeted knockout of the NCKX2 gene results in a profound loss of long term potentiation and an increase in long term depression at hippocampal Schaffer/CA1 synapses. We examined the expression of a variety of synaptic molecules when NCKX2 is knocked out to disclose the role of NCKX2 in hippocampal plasticity. Co -localization experiments using confocal and electron microscopy were conducted to define the location of NCKX2 in hippocampus. Immunoblot results from whole brain lysates showed that GluR2/3 and NMDA-receptor 1 were reduced in NCKX2 knockout mice. There was no statistical difference in immunoblot for CamKII, PSD95, or GluR1 between wild type and the knockout. In hippocampus phospho-CamKII was upregulated, and small but insignificant changes were found in the expression of other synaptic marker proteins. Co-localization analyses showed minimal overlap of NCKX2 with either the post synaptic marker CamKII or the presynaptic markers synapsin, synaptophysin or VGLUT-1. Immunocytochemistry revealed a decrease in net MAP-2 label in the CA3 region of knockout animals that correlates to a reduction in the number of neuronal profiles and increase in DAPI-labeled nuclei, suggesting neuronal loss and glial cell proliferation. From the above experiments, we can for first time approach the idea of the exact location of NCKX2. Expression change for various synaptic molecules in hippocampus is not obvious, but rather loss of CA3 neurons and the associated Schaeffer collateral projection to CA1, potentially resulting from changes in calcium dynamics when NCKX2 is knocked out.