In the continuing quest to measure reliable calcium signals from astrocytes, most of our efforts this year have utilized the transgenic mouse line that expresses green fluorescent protein in a construct driven by the astrocyte-specific promoter for glial fibrillary acidic protein (GFAP-GFP mice). The astrocytes from these animals give strong fluorescent signals that are microanatomically correct for astrocytes. Bulk loading of brain slices with the calcium-sensitive fluorophore Rhod-2 AM gave signals for calcium under electrical and chemical stimulation. Three limitations to this approach were noted: (1) the signals occurred in the cell bodies and were rarely if ever seen in the finer processes of the astrocytes where other groups have shown activity in """"""""microdomains"""""""";(2) the responding signals were only seen in the uppermost 30 micrometers of tissue, where damage and possible gliosis would be expected from tissue slicing;(3) bulk loading is only effective in animals less than 30 days old, when developmental changes in the astrocyte population are still in progress. Two approaches are in progress to overcome these limitations. In the first, astrocytes identified in GFAP-GFP mice by green fluorescence undergo whole cell patch clamp using a patch pipette loaded with the calcium-sensitive dye Rhod-2. While under voltage clamp, the identity of the astrocyte is confirmed by electrophysiological characteristics (resting membrane potential, capacitance, and non-rectifying current- voltage curve). Then the cell is allowed to passively fill with Rhod-2 for about 10 minutes, monitoring the loading throughout by confocal microscopy. Upon withdrawing the patch pipette, the cell is then stimulated both electrically and chemically to observe activity in the microdomains of cells 40 micrometers or more beneath the cut surface of the slice. To date, no robust signals have been observed in either the nucleus accumbens or in the hippocampus by this method. The second approaches use transgenic mice. The first line was the cross GCaMP3 X GFAP-Cre/ERT2 line, in which the fluorescent calcium reporter protein, GCaMP3, is expressed when the fusion protein Cre/ERT2 is driven into the nucleus conditionally, that is, when the estrogen receptor ERT2 is occupied by the estrogen antagonist Tamoxifen. In six such transgenic mice treated with high doses of Tamoxifen for 5 days, the calcium signal was absent or too weak to be useful. Immunohistochemistry showed that expression of GCaMP3 was highly variable between animals and always incomplete (i.e. only a fraction of astrocytes expressed it). A second line of transgenic mice will be tried next. These are GFAP-Cre mice that will express GCaMP5 after being infected stereotaxically in the nucleus accumbens with the virus AAV-DIO- GCaMP5. The first litter of these mice will be ready for testing in coming weeks..

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