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.

Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
National Institute on Drug Abuse
Zip Code
Diaz-Ruiz, Oscar; Zhang, Yajun; Shan, Lufei et al. (2012) Attenuated response to methamphetamine sensitization and deficits in motor learning and memory after selective deletion of ýý-catenin in dopamine neurons. Learn Mem 19:341-50
Cifani, Carlo; Koya, Eisuke; Navarre, Brittany M et al. (2012) Medial prefrontal cortex neuronal activation and synaptic alterations after stress-induced reinstatement of palatable food seeking: a study using c-fos-GFP transgenic female rats. J Neurosci 32:8480-90
Fitzgerald, Megan L; Chan, June; Mackie, Kenneth et al. (2012) Altered dendritic distribution of dopamine D2 receptors and reduction in mitochondrial number in parvalbumin-containing interneurons in the medial prefrontal cortex of cannabinoid-1 (CB1) receptor knockout mice. J Comp Neurol 520:4013-31
Sterky, Fredrik H; Hoffman, Alexander F; Milenkovic, Dusanka et al. (2012) Altered dopamine metabolism and increased vulnerability to MPTP in mice with partial deficiency of mitochondrial complex I in dopamine neurons. Hum Mol Genet 21:1078-89
Spivak, Charles E; Kim, Wook; Liu, Qing-Rong et al. (2012) Blockade of ?-cell K(ATP) channels by the endocannabinoid, 2-arachidonoylglycerol. Biochem Biophys Res Commun 423:13-8
Fitzgerald, Megan L; Lupica, Carl R; Pickel, Virginia M (2011) Decreased parvalbumin immunoreactivity in the cortex and striatum of mice lacking the CB1 receptor. Synapse 65:827-31
Luo, Alice H; Tahsili-Fahadan, Pouya; Wise, Roy A et al. (2011) Linking context with reward: a functional circuit from hippocampal CA3 to ventral tegmental area. Science 333:353-7
Good, Cameron H; Hoffman, Alexander F; Hoffer, Barry J et al. (2011) Impaired nigrostriatal function precedes behavioral deficits in a genetic mitochondrial model of Parkinson's disease. FASEB J 25:1333-44