Immunohistochemical Detection of the Sodium‐Calcium Exchanger in Rat Hippocampus Cultures Using Subtype‐Specific Antibodies

Abstract

All of the known Na+/Ca2+ exchanger subtypes, NCX1‐3, are expressed in the brain, albeit with marked regional differences. On the mRNA level, overall expression seems most prominent for NCX2, intermediate for NCX1, and, except for a few regions, low for NCX3. Using three subtype‐specific antibodies, we have now studied the cellular expression of the NCX subtypes in rat hippocampus cultures by immunohistochemical techniques. Our results provide evidence for a highly cell‐specific expression pattern of NCX subtypes and show surprisingly little colocalization. NCX1 and NCX3 are both primarily expressed in neuronal cells. While NCX1 is found in the large majority of neurons, NCX3 expression was restricted to a small minority of cells. By contrast, NCX2 was almost exclusively present in glial cells. The NCX2 antibody, a IgM, stained glial cell membranes as well as an intermediate fibrillar system. In spite of extensive screening, the nature of this fiber system has not yet been identified.

Differential distribution of NCX1 contributes to spine–dendrite compartmentalization in CA1 pyramidal cells

“…The antibody recognizes two intracellular epitopes located within the amino acid sequences 560-629 and 649-705, covering all NCX1 splice variants. The R3F1 antibody proved to be specific for NCX1 on Western blot (19) experiments. To further test the specificity of the antibody in brain slices, another mouse monoclonal antibody (MA3-926) raised against a different intracellular epitope (371-525) was used.…”

“…Available immunocytochemical data with the same antibody (R3F1) in hippocampal cell cultures are a bit confusing. Some studies suggest dominant somatic or somatodendritic localization (11,19), whereas others emphasize preferential NCX1 presence in presynaptic terminals (21,26) in pyramidal cells. Similarly, astrocytes have been shown either to have (11) or to lack (19) NCX1 labeling.…”

“…Hippocampal pyramidal cells and astrocytes were shown to express NCX1 and NCX2 mRNAs (16)(17)(18), but they seem to have differences at the protein level. By using monoclonal antibodies, the NCX1 protein was localized to pyramidal cells, whereas NCX2 was predominantly found on glial cell plasma membranes in cell culture (19). Owing to the lack of selective NCX subtype inhibitors, pharmacology is insufficient to study the contribution of different NCX subtypes to Ca 2ϩ homeostasis, especially in cells with complex dendritic structures, such as pyramidal cells.…”

Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor-mediated neurodegeneration via calpain activation in hippocampal neurons

“…Rabbit polyclonal anti-N SBDP was raised against end residues Gln-Gln-Glu-Val-Tyr of the aminoterminal spectrin fragment produced by calpain I. 38 Antibodies selective for NCX1 (clone R3F1) and NCX3 were developed by Dr Kenneth Philipson, 39 and the antibody selective for NCX2 (clone W1C3) was developed by Dr Harmut Porzig. 39 Anti-mouse IgG labelled with Alexa Fluor 488 or 594 and anti-rabbit IgG labelled with Alexa Fluor 488 or 594 were purchased from Molecular Probes (Leiden, The Netherlands).…”

“…38 Antibodies selective for NCX1 (clone R3F1) and NCX3 were developed by Dr Kenneth Philipson, 39 and the antibody selective for NCX2 (clone W1C3) was developed by Dr Harmut Porzig. 39 Anti-mouse IgG labelled with Alexa Fluor 488 or 594 and anti-rabbit IgG labelled with Alexa Fluor 488 or 594 were purchased from Molecular Probes (Leiden, The Netherlands). Polyvinylidene difluoride (PVDF) membranes, alkaline phosphatase-linked anti-mouse secondary antibodies, 45 Ca 2 þ and the Enhanced Chemifluorescence (ECF) reagent were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK).…”