NMDA receptors mediate calcium accumulation in myelin during chemical ischaemia.

Central nervous system myelin is a specialized structure produced by oligodendrocytes that ensheaths axons, allowing rapid and efficient saltatory conduction of action potentials. Many disorders promote damage to and eventual loss of the myelin sheath, which often results in significant neurological morbidity. However, little is known about the fundamental mechanisms that initiate myelin damage, with the assumption being that its fate follows that of the parent oligodendrocyte. Here we show that NMDA (N-methyl-d-aspartate) glutamate receptors mediate Ca2+ accumulation in central myelin in response to chemical ischaemia in vitro. Using two-photon microscopy, we imaged fluorescence of the Ca2+ indicator X-rhod-1 loaded into oligodendrocytes and the cytoplasmic compartment of the myelin sheath in adult rat optic nerves. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor antagonist NBQX completely blocked the ischaemic Ca2+ increase in oligodendroglial cell bodies, but only modestly reduced the Ca2+ increase in myelin. In contrast, the Ca2+ increase in myelin was abolished by broad-spectrum NMDA receptor antagonists (MK-801, 7-chlorokynurenic acid, d-AP5), but not by more selective blockers of NR2A and NR2B subunit-containing receptors (NVP-AAM077 and ifenprodil). In vitro ischaemia causes ultrastructural damage to both axon cylinders and myelin. NMDA receptor antagonism greatly reduced the damage to myelin. NR1, NR2 and NR3 subunits were detected in myelin by immunohistochemistry and immunoprecipitation, indicating that all necessary subunits are present for the formation of functional NMDA receptors. Our data show that the mature myelin sheath can respond independently to injurious stimuli. Given that axons are known to release glutamate, our finding that the Ca2+ increase was mediated in large part by activation of myelinic NMDA receptors suggests a new mechanism of axo-myelinic signalling. Such a mechanism may represent a potentially important therapeutic target in disorders in which demyelination is a prominent feature, such as multiple sclerosis, neurotrauma, infections (for example, HIV encephalomyelopathy) and aspects of ischaemic brain injury.

Calcium imaging in live rat optic nerve myelinated axons in vitro using confocal laser microscopy.

Intracellular Ca(2+) plays a major role in the physiological responses of excitable cells, and excessive accumulation of internal Ca(2+) is a key determinant of cell injury and death. Many studies have been carried out on the internal Ca(2+) dynamics in neurons. In constrast, there is virtually no such information for mammalian central myelinated axons, due in large part to technical difficulty with dye loading and imaging such fine myelinated structures. We developed a technique to allow imaging of ionized Ca(2+) in live rat optic nerve axons with simultaneous electrophysiological recording in vitro at 37 degrees C using confocal microscopy. The K(+) salt of the Ca(2+)-sensitive indicator Oregon Green 488 BAPTA-2 and the Ca(2+)-insensitive reference dye Sulforhodamine 101 were loaded together into rat optic nerves using a low-Ca(2+)/low-Na(+) solution. Axonal profiles, confirmed immunohistochemically by double staining with neurofilament-160 antibodies, were clearly visualized by S101 fluorescence up to 800 microm from the cut ends. The Ca(2+) signal was very low at rest, just above the background fluorescence intensity, indicating healthy tissue, and increased significantly after caffeine (20 mM) exposure designed to release internal Ca(2+) stores. The health of imaged regions was further confirmed by a virtual absence of spectrin breakdown, which is induced by calpain activation in damaged CNS tissue. Red and green fluorescence decayed to no less than 70% of control after 60 min of recording at 37 degrees C, with the green:red fluorescence ratio increasing slightly by 21% after 60 min. Electrophysiological responses recorded simultaneously with confocal images remained largely stable as well.

Kinetic hole burning, hole filling, and conformational relaxation in heme proteins: direct evidence for the functional significance of a hierarchy of dynamical processes.

Band III is a disorder and conformation-sensitive near-infrared (approximately 760 nm) charge transfer absorption band characteristic of equilibrium and nonequilibrium five coordinate ferrous high-spin hemes. The time evolution of this absorption band subsequent to photodissociation of six coordinate ferrous hemoglobin or myoglobin can provide detailed information regarding conformational relaxation, including the thermally driven fluctuations that result in the transition from inhomogeneous to homogeneous ligand rebinding kinetic. Such time-resolved measurements over a range of temperatures are difficult due to long sample recovery times at cryogenic temperatures. A new restoring technique that allows for the rapid movement of a large optically accessible cryostat is used in combination with nanosecond time-resolved near-infrared absorption spectroscopy to generate band III as a function of time for the photoproducts of the carbon monoxide derivative of adult human hemoglobin (COHbA) and, to a more limited extent, horse myoglobin (COMb). The measurements are made over a wide range of temperatures extending from well below the solvent (75% glycerol:water) glass transition at approximately 180 K to ambient temperatures. Three temperature- and/or viscosity-dependent phenomena are observed. At the highest temperatures, only conformational relaxation is observed for the 75% glycerol sample. At very high viscosity (> or = 400 cp), conformational relaxation slows dramatically, and both kinetic hole burning followed by the filling in of the "hole" (dynamic hole filling) are observed. As the temperature is lowered, conformational relaxation slows and finally ceases. Kinetic hole burning and dynamic hole filling as well as additional broadening of band III are observed down to 140 K. The observation of kinetic hole burning (KHB) is indicative of the sample being inhomogeneous on the time scale of the ligand rebinding giving rise to KHB. The onset of hole filling is a direct manifestation of the thermal homogenization of the initial inhomogeneous distribution of conformational substates responsible for KHB. The observed dynamics are used to explain the inverse temperature effect associated with the non-Arrhenius slow down of geminate rebinding above approximately 180 K. The inverse temperature effect appears to arise not only from the onset of conformational relaxation but also from the increase in the rate on thermal averaging of the initial inhomogeneous distribution of conformational substates.

Golgi apparatus is involved in intracellular Ca2+ regulation in epithelial LLC-PK1 cells.

Several lines of evidence suggest that the Golgi apparatus is involved in Ca2+ regulation in renal epithelial LLC-PK1 cells. Laser scanning confocal microscopy (LSCM) was employed to establish that a prominent perinuclear region is occupied mainly by the Golgi apparatus in this cell line. LSCM measurements in individual cells with the ionized Ca2+ indicator calcium green revealed that stimulation of LLC-PK1 cells with arginine vasopressin (AVP) resulted in the elevation of ionized Ca2+ levels. However, the vasopressin-induced rise in ionized Ca2+ was attenuated if the Golgi apparatus was disassembled by pretreating the cells with brefeldin A (BFA). Subcellular measurements of total Ca2+ with ion microscopy in cryogenically prepared cells indicated that 1) within 1 min of AVP treatment significant quantities of sequestered Ca2+ were released from the perinuclear Golgi region and 2) the BFA treatment reduced the total Ca2+ stored in the Golgi region. These observations indicate that the Golgi apparatus is sensitive to hormonal stimulation and may play important roles in intracellular Ca2+ regulation in LLC-PK1 cells.