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. 2006 Mar 22;26(12):3141–3153. doi: 10.1523/JNEUROSCI.5437-05.2006

Figure 4.

Figure 4.

Somatodendritic trafficking of endogenous CB1R after pharmacological treatments. A, Hippocampal neurons at DIV 9 are incubated for 3 h with either 0.2% DMSO as vehicle (Aa, Ab), 1 μm agonist WIN (Ac, Ad), or 10 μm inverse agonist AM281 (Ae, Af). Anti-CB1R N-Ter feeding plus C-Ter immunolabeling followed by immunoperoxidase staining is performed to maximize sensitivity. Shown are representative somas and proximal dendrites for the three conditions. The right panels in Aa, Ac, and Ae are detailed images from the boxed areas in the left panels. Scale bars: somas, Aa, Ac, Ae, 2 μm; details, Ab, Ad, Af, 0.5 μm. For the right column, the S/N ratio of this individual cell is indicated in the lower right corner, referring to the quantification in B. In control conditions (Aa, Ab), receptors are mostly found in somatic endosomes (Aa, filled arrows), with sparse labeling of the somatodendritic plasma membrane (Aa, right, empty arrow). CB1R-positive en passant axons exhibit a dense surface staining (Ab, arrowhead). Treatment with agonist WIN downregulates somatodendritic surface CB1Rs and upregulates CB1R-positive endosomes (Ac, filled arrows). Note that remaining surface CB1Rs are now found inside pits (Ad, top, empty arrow) and that axonal surface labeling is still partially present (Ad, bottom, arrowhead). Treatment with inverse agonist AM281 upregulates somatodendritic surface receptors that often appear as patches on the plasma membrane (Ae, empty arrows). Axonal labeling is not significantly different from control condition (Af, arrowhead), as seen on CB1R-positive axons passing along CB1R-positive somas. B, Quantification of CB1R translocation for endogenous CB1R (corresponding to images shown in A). S/N ratio represents the ratio between mean plasma membrane intensity (with eventual CB1R staining) and nuclear membrane intensity (used as control). Five neurons are quantified for each condition. WIN-treated neurons exhibit a drop in S/N ratio, showing downregulation of plasma membrane CB1Rs, whereas AM281-treated neurons exhibit higher S/N ratios, with enhanced plasma membrane labeling. The bars show the mean, the error bars indicate the SEM, and the asterisks indicate significance between the vehicle and treated conditions, as defined in Materials and Methods.