Figure 4.

Analysis of CB₁ downregulation from the surface of primary cultured rat spinal cord neurons during prolonged treatment with WIN (1.5 μm). A surface-labeling protocol was used to assess surface expression of CB₁ (TRITC) and stained cultures were fixed and immunostained with an anti-neurofilament 200 (NF200) antibody (Cy5), as an internal control. All images were taken in the same imaging conditions, and all signals were below saturation limits. A, Typical examples of confocal images acquired in the sequential mode representing a high level of CB₁ expression at the surface of spinal neurites in the naive state (basal) and WIN-induced decrease in surface CB₁ expression (12, 24, and 48 h). Line profiles on the right demonstrate the intensity of the signals (solid lines) and background (dashed lines). B, Quantitative estimation of CB₁ expression at the surface of spinal neurites or soma as a fraction of the corresponding neurofilament 200 expression. Significant downregulation is observed after WIN treatment for 24 or 48 h (*p < 0.05, ANOVA, post hoc Fisher's test). n = 40 data points each per group. Scale bar, 40 μm.