Abstract
Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA) receptor (NMDAR) is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF) labeling in rat cultured cortical astrocytes (rCCA) of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa2+ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(-)-2-amino-5-phosphonopentanoic acid (APV) and Kynurenic acid (KYNA) and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa2+ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca2+ depletion, indicating flux-independent NMDAR function. In contrast, the IP3 receptor (IP3R) inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa2+ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ) measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa2+ increase.
Highlights
The role of astrocytes in the CNS had been circumscribed to structural cells supporting neuronal functions, the cells in charge of carrying out the integration, processing and storage of information
Kynurenic acid (KYNA) significantly reduced by 80% the rat cultured cortical astrocytes (rCCA) response to N-methyl D-aspartate (NMDA) with only 8.9% of cells above the RTV (Table 1; Fig 4F). Taken together these results demonstrated that rCCA express functional N-methyl-D-aspartate receptor (NMDAR) that, after ligand binding, mediate iCa2+ increase, and they rule out unspecific effects associated with the NMDA concentration employed
Whether functional NMDARs are present in cultured astrocytes is still a matter of debate due to contradictory findings
Summary
The role of astrocytes in the CNS had been circumscribed to structural cells supporting neuronal functions, the cells in charge of carrying out the integration, processing and storage of information. Astrocytes are known to secrete gliotransmitters, to express neurotransmitter receptors and more importantly, to form functional networks in which intracellular Ca2+ (iCa2+) is critical for cell communication [1,2,3,4,5,6,7,8]. These properties support the tripartite synapse hypothesis [9]. Astrocytes’ role within the CNS is still a matter of debate [14]
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