Our efforts this year have been directed to exploring methods and working out procedures. As we are employing cytosolic calcium release as a measure of astrocyte activation, we have tested the calcium sensitive fluophores Fluo-3 AM, Fluo-4 AM, and calcium orange AM to examine the responses of striatal astrocytes in brain slices from juvenile rats. The detection system is a Zeiss 710 confocal/2-photon microscope. We have explored a variety of stimulation conditions including electrical impulses, ATP, and glutamate. For detection we have explored both conventional confocal as well as two- photon microscopy to settle on conditions that optimize light collection, contrast, depth of penetration into the slice, and speed of recording. We presume that the calcium """"""""flashes"""""""" we see originate from astrocytes because (1) they occur on the second, not millisecond time scale and (2) they originate from cell bodies about 8 micrometers in diameter. We found that the reputed fluorescent identifier of glia, sulforhodamine 101, was completely unreliable in predicting active astrocytes. Because dopamine is central to the drug abuse paradigm and because the only previous studies on dopamine activation were performed in cultured astrocytes, we focused on the astrocyte responses to this transmitter in striatal slices. Reliable responses were seen at concentrations of 30 to 100 micromolar (concentrations used in the published cultured astrocyte literature). However to test for possible cross-sensitivity of dopamine at norepinephrine receptors, we tested the latter and found strong responses even below one micromolar concentrations. To avoid the uncertainty in identifying astrocytes, we are now testing transgenic mice in which expression of green fluorescent protein is driven by the astrocyte-specific promoter for glial fibrillary acidic protein. These slices still have to be loaded with a calcium selective fluorophore. To avoid this second step, we are acquiring new transgenic mice that express in astrocytes the fusion protein GCaMP3, derived from green fluorescent protein, the calcium chelating protein calmodulin, and a helical peptide M13. In this way astrocyte selectivity and calcium-selective fluorescent signaling will both genetically encoded.
