Using the mitochondrial potential (ΔΨm) marker JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) and high-resolution imaging, we functionally analyzed mitochondria in cultured rat hippocampal astrocytes. Ratiometric detection of JC-1 fluorescence identified mitochondria with high and low ΔΨm. Mitochondrial density was highest in the perinuclear region, whereas ΔΨm tended to be higher in peripheral mitochondria. Spontaneous ΔΨm fluctuations, representing episodes of increased energization, appeared in individual mitochondria or synchronized in mitochondrial clusters. They continued upon withdrawal of extracellular Ca2+, but were antagonized by dantrolene or 2-aminoethoxydiphenylborate (2-APB). Fluo-3 imaging revealed local cytosolic Ca2+ transients with similar kinetics that also were depressed by dantrolene and 2-APB. Massive cellular Ca2+ load or metabolic impairment abolished ΔΨm fluctuations, occasionally evoking heterogeneous mitochondrial depolarizations. The detected diversity and ΔΨm heterogeneity of mitochondria confirms that even in less structurally polarized cells, such as astrocytes, specialized mitochondrial subpopulations coexist. We conclude that ΔΨm fluctuations are an indication of mitochondrial viability and are triggered by local Ca2+ release from the endoplasmic reticulum. This spatially confined organelle crosstalk contributes to the functional heterogeneity of mitochondria and may serve to adapt the metabolism of glial cells to the activity and metabolic demand of complex neuronal networks. The established ratiometric JC-1 imaging—especially combined with two-photon microscopy—enables quantitative functional analyses of individual mitochondria as well as the comparison of mitochondrial heterogeneity in different preparations and/or treatment conditions.

Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ATP adenosine-5'-triphosphate BAPTA 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) Ca 2+ calcium CNS central nervous system CNcyanide DAPI 4'-6-Diamidino-2-phenylindole DMSO dimethyl sufoxide DIC days in culture  m mitochondrial transmembrane potential ER endoplasmic reticulum FADH 2 flavin adenine dinucleotide FCCP carbonyl cyanide 4-trifluoro-methoxyphenylhydrazone Fluo-3 visible light calcium indicator GTP guanidine triphosphate GFAP glial fibrillary acidic protein IP 3 inositol triphosphate JC-1 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide K + potassium K ir inwardly rectifying potassium channel NADH/H + nicotinamide adenine dinucleotide NMDA N-methyl-D-aspartic acid pO 2 partial oxygen pressure Rho123 rhodamine 123 ROI region of interest ROS reactive oxygen species RyR ryanodine receptor TCA tricarboxylic acid TPELSM two-photon excitation laser scanning microscope UV ultraviolet (light of 10 -400 nm range) IV 1.2. The hippocampus -a versatile structure in the CNS The hippocampus was possibly given its name by the Italian anatomist G C Aranzi in the 16 th century referring to its stretched and wound up shape in the medial temporal lobe. It represents a locally eccentric and phylogenetically old Perpendicular to its allocortical trilayer the hippocampus is subdivided into the subfields CA1 1 , CA2, CA3 and CA4, forming the central hippocampus, and adjacent structures named dentate gyrus and subiculum. The assembly is oftenThe energy liberated in this redox reaction is used to pump protons from the mitochondrial matrix to the intermembrane space across the inner membrane from a compartment of relatively negative charge and low proton concentration to the intermembrane space -a positively charged region of already high proton concentration. The resulting proton-motive force provides the energy for ATP generation executed by the ATP synthase (or F o F 1 ATPase or complex V).High glutamate concentrations may also elevate Ca 2+ in at least two ways: by ionotropic AMPA receptors, that trigger depolarization via cationic ion influx and via G-protein coupled metabotropic receptors that lead to a rise in IP 3 . IP 3 has receptors on the ER of the astrocyte causing Ca 2+ release into the cytosol. Ca 2+