Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC.

Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca2+ (Kd approximately 20 microM, 6 microM and 12 microM respectively) and selectivity over Mg2+ (Kd approximately 2 mM for mag-Fura-2, > 10 mM for Fura-2FF and BTC). Among the tested indicators, BTC was limited by a modest dynamic range upon Ca2+ binding, susceptibility to photodamage, and sensitivity to alterations in pH. All three indicators bound other metal ions including Zn2+, Cd2+ and Gd3+. Interestingly, only in the case of BTC were spectral differences apparent between Ca2+ and other metal ions. For example, the presence of Zn2+ increased BTC fluorescence 6-fold at the Ca2+ isosbestic point, suggesting that this dye may be used as a fluorescent Zn2+ indicator. Fura-2FF has high specificity, wide dynamic range, and low pH sensitivity, and is an optimal low-affinity Ca2+ indicator for most imaging applications. BTC may be useful if experimental conditions require visible wavelength excitation or sensitivity to other metal ions including Zn2+.

Oligodendrocytes from forebrain are highly vulnerable to AMPA/kainate receptor-mediated excitotoxicity.

Little is known of the molecular mechanisms that trigger oligodendrocyte death and demyelination in many acute central nervous system insults. Since oligodendrocytes express functional alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-type glutamate receptors, we examined the possibility that oligodendrocyte death can be mediated by glutamate receptor overactivation. Oligodendrocytes in primary cultures from mouse forebrain were selectively killed by low concentrations of AMPA, kainate or glutamate, or by deprivation of oxygen and glucose. This toxicity could be blocked by the AMPA/kainate receptor antagonist 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX). In vivo, differentiated oligodendrocytes in subcortical white matter expressed AMPA receptors and were selectively injured by microstereotaxic injection of AMPA but not NMDA. These data suggest that oligodendrocytes share with neurons a high vulnerability to AMPA/kainate receptor-mediated death, a mechanism that may contribute to white matter injury in CNS disease.

Distinct roles for sodium, chloride, and calcium in excitotoxic dendritic injury and recovery.

The postsynaptic neuronal dendrite is selectively vulnerable to hypoxic-ischemic brain injury and glutamate receptor overactivation. We explored the glutamate receptor pharmacology and ionic basis of rapid, reversible alterations in dendritic shape which occur in cultured neurons exposed to glutamate. Dendrite morphology was assessed with the fluorescent membrane tracer, DiI, or immunofluorescence labeling of the somatodendritic protein, MAP2. Cortical cultures derived from 15-day-old mouse embryos underwent segmental dendritic beading when exposed to NMDA, AMPA, or kainate, but not to metabotropic glutamate receptor agonists. Varicosity formation in response to NMDA or kainate application was substantially attenuated in reduced sodium buffer (substituted with N-methyl-D-glucamine). Furthermore, veratridine-induced sodium entry mimicked excitotoxic alterations in dendrites and additionally caused varicosity formation in axons. Solutions deficient in chloride (substituted with Na methylsulfate) and antagonists of chloride-permeable GABA/glycine receptors reduced NMDA- or kainate-induced varicosity formation. An increase in dendrite volume was observed as varicosities formed, and varicosity formation was attenuated in sucrose-supplemented hypertonic media. Despite marked structural changes affecting virtually all neurons, dendrite shape returned to normal within 2 h of terminating glutamate receptor agonist application. Neurons exposed to kainate recovered more rapidly than those exposed to NMDA, and neurons exposed to NMDA in calcium-free buffer recovered more rapidly than cells treated with NMDA in normal buffer. While sodium, chloride, and water entry contribute to excitotoxic dendritic injury acutely, calcium entry through NMDA receptors results in lasting structural changes in damaged dendrites.

Neuronal free calcium measurement using BTC/AM, a low affinity calcium indicator.

BTC is a low affinity calcium indicator (Kd approximately 7-26 microM) featuring many desirable properties for cellular calcium imaging, including long excitation wavelengths (400/485 nm), low sensitivity to Mg2+, and accuracy of ratiometric measurement [Iatridou H., Foukaraki E., Kuhn M.A., Marcus E.M., Haugland R.P., Katerinopoulos H.E. The development of a new family of intracellular calcium probes. Cell Calcium 1994; 15: 190-198]. To assess the usefulness of this indicator in cultured neurons, we examined properties of BTC and its acetoxymethyl ester, BTC/AM. BTC/AM had substantial calcium-independent fluorescence at all excitation wavelengths. BTC/AM was readily loaded into neurons and was rapidly hydrolysed. There was little dye compartmentalization, as assessed by digitonin lysis, Co2+ quenching of BTC fluorescence and by confocal microscopy. Despite adequate loading, BTC gradually became unresponsive to [Ca2+]i when cultures were examined under routine imaging conditions. This effect was a function of the cumulative fluorescence illumination and could be minimized by attenuating light intensity or duration. Ratio imaging after exposure of neuronal cultures to 1-50 microM ionomycin revealed distinct sensitivity ranges for BTC and Fura-2. BTC reported graded neuronal [Ca2+]i responses to glutamate receptor stimulation with N-methyl-D-aspartate in the range 10-50 microM, whereas Fura-2 did not distinguish between these stimuli. Under appropriate loading and illumination conditions, bath-loaded BTC/AM may be well suited for measurement of moderate to high calcium concentrations in cultured neurons.