Activity and metabolism-related Ca2+ and mitochondrial dynamics in co-cultured human fetal cortical neurons and astrocytes

Abstract

Neurons and neighboring astrocytic glia are mostly studied in nervous tissues from rodents whereas less is known on their properties and interactions in the human brain. Here, confocal/multiphoton fluorescence imaging for several hours revealed that co-cultured fetal human cortical neurons and astrocytes show pronounced spontaneous rises of cytosolic Ca(2+) which last for up to several minutes without concomitant changes in either movements or membrane potential of mitochondria. Similar Ca(2+) rises were evoked mainly in neurons by bath-applied glutamate or γ-aminobutyric acid (GABA) acting via N-methyl-d-aspartate (NMDA)+AMPA/Kainate and GABAA receptors, respectively. Predominantly in astrocytes, Ca(2+) baseline was elevated by adenosine diphosphate (ADP) and adenosine triphosphate (ATP) acting via P2Y1 and P2X7 receptors, likely causing the release of glutamate and glutamine. Mainly astrocytes responded to histamine, whereas the activation of muscarinic acetylcholine (ACh) receptors raised Ca(2+) in both cell types. Evoked neuronal and astrocytic Ca(2+) rises could last for several minutes without affecting mitochondrial movements or membrane potential. In contrast, reversible depolarization of mitochondrial membrane potential accompanied neuronal Ca(2+) rises induced by cyanide-evoked chemical anoxia or the uncoupling of mitochondrial respiration with carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP). During such metabolic perturbation, mitochondrial depolarization also occurred in astrocytes, whereas Ca(2+) was largely unaffected. In summary, fetal human cortical neurons and astrocytes show distinct patterns of neuro/glio-transmitter- and metabolically-evoked Ca(2+) rises and possess active mitochondria. One aspect of our discussion deals with the question of whether the functional mitochondria contribute to cellular Ca(2+) homeostasis that seems to be already well-developed in fetal human cortical brain cells.

Keywords: (S)-3,5-dihydroxy-phenylglycine; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 4′,6-diamidino-2-phenylindole; 6-cyano-7-nitroquinoxaline-2,3-dione; ACh; ANOVA; APV; CNQX; DAPI; DHPG; DMSO; FBS; FCCP; GFAP; HEPES; HPLC; MAP2; MEM; N-methyl-d-aspartate; NMDA; ROIs; Rh-123; Rhodamine-123; TMRM; acetylcholine; analysis of variance; anoxia; calcium; carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone; dimethyl sulfoxide; dl-2-amino-5-phosphonovalerate; fetal bovine serum; fluorescence imaging; glial fibrillary acidic protein; high-performance liquid chromatography; hypoxia; mGluR; membrane-associated protein-2; metabotropic glutamate receptor; minimum essential medium; mitochondrial potential; regions of interest; tACPD; tetramethyl-rhodamine methyl ester; trans-1-amino-cyclopentane-1,3-dicarboxylic acid.