Figure 6

Activation of CB₁-Rs evokes Ca²⁺ transients in the processes of cultured mouse spinal astrocytes. (a–c) Micrographs showing the fluorescence intensity of a Fluo-8 loaded astrocytic process (green arrowhead) and astrocytic cell body (orange arrowhead) before (a) and after the application of 10 µM WIN (b) and 180 µM ATP (c). (d) Graphical representation of changes in fluorescence intensities of the process (green trace) and the cell body (orange trace) which are labeled with green and orange arrowheads on (a–c) respectively, before and after the application of 10 µM WIN and 180 µM ATP. Note that both the process and cell body responded to ATP, whereas only the process showed activity following the administration of WIN. (e–f) Individual (gray) and average (black) Ca²⁺ transients in cultured astrocyte processes following the application of 10 µM WIN alone (e) and after a pretreatment with 5 µM AM251. In both cases, cell viability was tested by the application of 180 µM ATP. Only those processes that exhibited Ca²⁺ transients in response to the application of ATP were considered alive and included in the consecutive statistical analysis. (g) Box plot illustrating the area under the curve (AUC) values (normalized to ATP response) of Ca²⁺ transients recorded from processes of cultured astrocytes under control conditions, as well as following the administration of WIN without and with a preincubation of the cells with AM251. Asterisks indicate significant differences between AUC values of calcium transients recorded in the different experimental conditions (p = 8.81 × 10⁻³¹, control vs WIN; p < 0.001, the Mann-Whitney test provided the value of 0, WIN vs AM + WIN).