(A) Projection of an EGFP-labeled MSN from a BAC D2 mouse. The cell was patched with a pipette containing Alexa 594 (50 μM) for visualization and Fluo-4 (200 μM) for measuring changes in intracellular Ca2+ (right). Cells were voltage clamped at −70 mV. A puffer pipette containing sphingosine 1-phosphate (S1P, 10 μM) was positioned near a dendrite, 60–80 μm from the soma (left/cartoon). (B) High magnification images of a dendritic segment (control, left panel) show an increase in Ca2+ associated with S1P application (S1P puff, center panel) that reversed with washing (wash, right panel). The change in Ca2+ was determined by calculating the percent change in fluorescence of Fluo-4 relative to that of Alexa 594 ( G/R). The blue circle in the first panel indicates the analyzed region of interest (ROI). (C) Time course showing the S1P induced increase in intracellular Ca2+ in the ROI from B (orange trace); similar recordings from BAC D1 MSNs (black trace) or 10 μM thapsigargin loaded BAC D2 MSNs (green trace) did not reveal any significant changes in dendritic Ca2+ levels with S1P application. (D) Box plot summarizing the S1P effects. Percent increase in fluorescence (ΔG/R) in BAC D2 MSNs (median=146%, range 44 to 294%, n=6); BAC D1 MSNs (median=17%, range 13 to 22%, n=4); and thapsigargin-loaded BAC D2 MSNs (median=4%, range –9 to 10%, n=4).