Figure 4. Cocaine alters intensity of GABAergic (GAD65/67) puncta near PV neurons surrounded by PNNs in the prelimbic PFC. Cells were visualized with confocal microscopy and analyzed using Imaris segmentation tools. A, Representative PV neuron (red) surrounded by a PNN labeled with WFA (blue). B, The PV neuron is traced and a surface is created. C, The Mask Channel function was used to isolate GAD65/67 labeling to the area outside of the PV neuronal surface, and GAD65/67-labeled puncta (green arrowheads) were identified. D, GAD65/67-labeled puncta (green arrowheads) were then thresholded using the Imaris Spots tool. Spots that met the size criteria and were located adjacent to the PV neuron surface were included in the analysis (green arrows). E, F, VGluT1-labeled puncta (magenta arrowheads) were segmented separately using the same protocol (E), and VGluT1 spots meeting our size and location criteria were included in the analysis (magenta arrows; F). G–I, One day of cocaine exposure increases GAD 65/67 puncta (saline, N = 3, 45 cells; cocaine, N = 3, 49 cells; G), no change in VGluT1 puncta (H), and a trend toward an increase in the ratio of GAD65/67 to VGluT1 puncta (I). J, One day of cocaine exposure decreases PV cell volume. K–M, Five days of cocaine exposure increases GAD 65/67 puncta (saline, N = 4, 49 cells; cocaine, N = 6, 78 cells; K), yields no change in VGluT1 puncta (L), and increases the ratio of GAD65/67 to VGluT1 puncta (M). N, Five days of cocaine exposure does not alter PV cell volume. Data from bar graphs are the mean ± SEM. Scale bar, 3 µm. *p < 0.05 and #p < 0.1, compared with saline controls.