Glutamate release through volume-activated channels during spreading depression.

Volume-sensitive organic anion channels (VSOACs) in astrocytes are activated by cell swelling and are permeable to organic anions, such as glutamate and taurine. We have examined the release of glutamate through VSOACs during the propagation of spreading depression (SD). SD was induced by bath application of ouabain in hippocampal brain slices and was monitored by imaging intrinsic optical signals, a technique that provides a measure of cellular swelling.

Imaging spreading depression and associated intracellular calcium waves in brain slices.

Spreading depression (SD) was analyzed in hippocampal and neocortical brain slices by imaging intrinsic optical signals in combination with either simultaneous electrophysiological recordings or imaging of intracellular calcium dynamics. The goal was to determine the roles of intracellular calcium (Ca2+int) waves in the generation and propagation of SD. Imaging of intrinsic optical signals in the hippocampus showed that ouabain consistently induced SD, which characteristically started in the CA1 region, propagated at 15-35 micrometer/sec, and traversed across the hippocampal fissure to the dentate gyrus.

Near-field confocal optical spectroscopy (NCOS): subdiffraction optical resolution for biological systems.

Optical resolution is limited by diffraction. However, in near-field microscopes sample illumination is provided through a subwavelength aperture to increase optical resolution. In this study we have evaluated the usefulness of this technique for living biological systems and report two significant improvements in this form of microscopy to enhance optical resolution for biological studies.

Expression of synaptobrevin II, cellubrevin and syntaxin but not SNAP-25 in cultured astrocytes.

Astrocytes, a sub-type of glial cell in the central nervous system, can release the excitatory transmitters glutamate and aspartate in response to elevated levels of internal calcium. To investigate potential release mechanisms that may be present in these cells we have determined whether protein components of the neuronal secretory apparatus are expressed in astrocytes. Western blots, immunocytochemistry and RT PCR demonstrate that astrocytes express cellubrevin, synaptobrevin II and syntaxin, proteins known to form a macromolecular fusion complex.

Dynamic imaging of purified individual synaptic vesicles.

The atomic force microscope (AFM) was used to directly image purified synaptic vesicles. Individual secretory vesicles (approximately 50 nm diameter) were resolved with the AFM when imaged either dry or in solution. Vesicles were observed repeatedly for periods of greater than 2 h.