Astrocytes can release a variety of transmitters, including glutamate and ATP, in response to stimuli that induce increases in intracellular Ca(2+) levels. This release occurs via a regulated, exocytotic pathway. As evidence of this, astrocytes express protein components of the vesicular secretory apparatus, including synaptobrevin 2, syntaxin, and SNAP-23. Additionally, astrocytes possess vesicular organelles, the essential morphological elements required for regulated Ca(2+)-dependent transmitter release. The location of specific exocytotic sites on these cells, however, remains to be unequivocally determined.

Astrocytes can release the excitatory transmitter glutamate which is capable of modulating activity in nearby neurons. This astrocytic glutamate release can occur through six known mechanisms: (i) reversal of uptake by glutamate (ii) anion channel opening induced by cell swelling, (iii) Ca 2+ -dependent exocytosis, (iv) glutamate exchange via the cystine-glutamate antiporter, (v) release through ionotropic purinergic receptors and (vi) functional unpaired connexons, 'hemichannels', on the cell surface. Although these various pathways have been defined, it is not clear how often and to what extent astrocytes employ different mechanisms. It will be necessary to determine whether the same glutamate release mechanisms that operate under physiological conditions operate during pathological conditions or whether there are specific release mechanisms that operate under particular conditions.

Astrocytes can modulate synaptic transmission by releasing glutamate in a Ca(2+)-dependent manner. Although the internal Ca(2+) stores have been implicated as the predominant source of Ca(2+) necessary for this glutamate release, the contribution of different classes of these stores is still not well defined. To address this issue, we cultured purified solitary cortical astrocytes and monitored changes in their internal Ca(2+) levels and glutamate release into the extracellular space. Ca(2+) levels were monitored by using the Ca(2+) indicator fluo-3 and quantitative fluorescence microscopy. Glutamate release was monitored by an L-glutamate dehydrogenase-linked detection system. Astrocytes were mechanically stimulated with a glass pipette, which reliably caused an increase in internal Ca(2+) levels and glutamate release into the extracellular space. Although we find that the presence of extracellular Cd(2+), a Ca(2+) channel blocker, significantly reduces mechanically induced glutamate release from astrocytes, we confirm that internal Ca(2+) stores are the predominant source of Ca(2+) necessary for this glutamate release. To test the involvement of different classes of internal Ca(2+) stores, we used a pharmacological approach. We found that diphenylboric acid 2-aminoethyl ester, a cell-permeable inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist, greatly reduced mechanically induced glutamate release. Additionally, the preincubation of astrocytes with caffeine or ryanodine also reduced glutamate release. Taken together, our data are consistent with dual IP(3)- and caffeine/ryanodine-sensitive Ca(2+) stores functioning in the control of glutamate release from astrocytes.

Due to their electrical, chemical, mechanical and thermal properties, carbon nanotubes are one of the most promising materials for the electronics, computer and aerospace industries. Here, we discuss their properties in the context of future applications in biotechnology and biomedicine. The purification and chemical modification of carbon nanotubes with organic, polymeric and biological molecules are discussed. Additionally we review their uses in biosensors, assembly of structures and devices, scanning probe microscopy and as substrates for neuronal growth. We note that additional toxicity studies of carbon nanotubes are necessary so that exposure guidelines and safety regulations can be established in a timely manner. KeywordsCarbon Nanotubes; Structure; Purification; Modification; Biosensors; Assembly of Structures and Devices; Scanning Probe Tips; Nanosurgery; Substrates for Neuronal Growth Although nanomaterials have existed in nature long before mankind was able to identify forms at the nanoscale level, advances in synthetic chemistry have been one of the driving forces in the development of a biological nanotechnology. Nanomaterials have been designed for a variety of biomedical and biotechnological applications, including bone growth, 1 enzyme encapsulation, 2 biosensors 3, 4 and as vesicles for DNA delivery into living cells. 5,6 Whereas nanotechnology may provide novel materials which can result in revolutionary new structures and devices, biotechnology already offers extremely sophisticated tools to precisely position molecules and assemble hierarchal structures and devices. The application of the principles of biology to nanotechnology provides a valuable route for further miniaturization and performance improvement of artificial devices. The feasibility of the bottom-up approach which is based on molecular recognition and self-assembly properties of proteins has already been proved in many inorganic-organic hybrid systems and devices. Nanodevices with biorecognition properties provide tools at a scale, which offers a tremendous opportunity to study biochemical processes and to manipulate living cells at the single molecule level. The synergetic future of nano-and bio-technologies holds great promise for further advancement in tissue engineering, prostheses, genomics, pharmacogenomics, drug delivery, surgery and general medicine.*Author to whom correspondence should be addressed. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptEver since Edison discovered that carbon changes its resistance with pressure and a carbon filament glows when an electric current is passed through it, the unique properties of carbon have intrigued scientists. After more than a century of interest, carbon has found its apogee in the fullerenes and carbon nanotubes (CNTs)-arguably the most promising of all nanomaterials. Because of their unique quasi one-dimensional structure and fascinating mechanical and electronic properties, CNTs have captured the attention of physicists...

Astrocytes can respond to a variety of stimuli by elevating their cytoplasmic Ca2+ concentration and can in turn release glutamate to signal adjacent neurons. The majority of this Ca2+ is derived from internal stores while a portion also comes from outside of the cell. Astrocytes use Ca2+ entry through store-operated Ca2+ channels to refill their internal stores. Therefore, we investigated what role this store-operated Ca2+ entry plays in astrocytic Ca2+ responses and subsequent glutamate release. Astrocytes express canonical transient receptor potential (TRPC) channels that have been implicated in mediating store-operated Ca2+ entry. Here, we show that astrocytes in culture and freshly isolated astrocytes from visual cortex express TRPC1, TRPC4, and TRPC5. Indirect immunocytochemistry reveals that these proteins are present throughout the cell; the predominant expression of functionally tested TRPC1, however, is on the plasma membrane. Labeling in freshly isolated astrocytes reveals changes in TRPC expression throughout development. Using an antibody against TRPC1 we were able to block the function of TRPC1 channels and determine their involvement in mechanically and agonist-evoked Ca2+ entry in cultured astrocytes. Blocking TRPC1 was also found to reduce mechanically induced Ca2+-dependent glutamate release. These data indicate that Ca2+ entry through TRPC1 channels contributes to Ca2+ signaling in astrocytes and the consequent glutamate release from these cells.