PMC

12 mo-old male mice were used in this study. (A) Representative sagittal PET images averaged over a period 2–10 min. (B) Brain regional time-uptake curves in AD (red) and control (green) mice. Data: mean SUV ± SD (n = 3 per genotype) (C) Averaged SUV from the same studies. (# p ≤ 0.05, ANOVA). Abbreviations: CTX–cortex; CB–cerebellum, WB–whole brain.

12 mo-old male mice were used in this study. (A) Whole brain time-uptake curves. Insert: averaged SUV values (2–10 min). Red–baseline; black–blockade. (B) Sagittal baseline (top) and blockade (bottom) images (2–10 min). This study demonstrates that in vivo binding of [¹¹C]A836339 in AD mice is specifically mediated by CB2 receptors that is consistent with our previous [¹¹C]A836339 ex vivo studies in AD mice [].

Representative confocal images of cortical neurons (upper panels) and neurons of the motor trigeminal nucleus (lower panels) with double immunostaining using NeuN (green; A) and CB2 R (red, H60: B) primary antibodies. Scale is 15 μm. C. Example of neuronal bodies visualized by NeuN (green). The neuron outlines were transferred to CB2 channel (red). Note cytoplasmic granules (pseudo-yellow) with autofluorescence visible on DAPI channel (not shown). The areas occupied by such granules were not considered in the analyses of CB2 immunoreactivity. Scale is 5 μm. D. Quantification of CB2 densities (integrated intensities/area) in NeuN-positive and –negative areas. Signal intensities were averaged across 30 non-overlapping fields (n = 2 mice). * and ** indicate significant differences in CB2 signal between NeuN-positive areas and background, p<0.01 and 0.001, respectively (ANOVA).

(A) Representative confocal images of double immunostaining using DAPI (blue) and CB2 R antibody (H60, Santa Cruz) (red). CA3 area of the hippocampus is shown. To check for specificity of H60 antibody J20 mice were used that either have both (left) or no copies (right) of a CNR2 gene that encodes for CB2 receptor. (B) Confocal images of CB2 staining (b1: red, H60) in neurons of the hippocampus from postmortem brain of a patient with AD. Blue channel was used for detection of DAPI staining (b2: blue). Note the presence of some fluorescent aggregates localized in perinuclear areas of big neurons (shown as pseudo green). These aggregates were visible through different channels (blue, green and far red) suggesting that autofluorescence could be a possible source of these signals. Green and far red channels were used to detect these non-specific signals (b2). b3 shows the composite of b1 and b2. b4 shows the composite of the four channels for an image stained only with secondary antibody. Note the presence of fluorescent granules positive on all channels (white color). Scale in A and B is 10 μm.

(A) A confocal image of CB2 (green) and CD68 staining (red) centered at the core of an Aβ plaque (marked by an asterisk). Note a region of high intensity for CB2 and CD68 staining around the plaque implying that CB2 receptors are localized in microglial processes surrounding the plaque. Scale 15 μm. (B) A magnification of a microglia cell body (red) and its process (red) forming an engulfment synapse on a dense core amyloid plaque (marked by an asterisk, DAPI). Note CB2 receptor staining along the edge of CD68-positive staining and at the engulfment synapse. White boxes indicate areas used for quantifications in C-D. (C-D) Quantification of CB2, CD68, and DAPI signals from the image of a microglia cell body (C) and engulfment synapse (D). The quantification was done using a Plot profile analysis tool (Fiji). Signals were averaged along short axes of boxes shown in B and normalized to a max value (100%) for each channel. Units of X axis are pixels, scale: 11.1 pixels/ μm. (E) 3D reconstruction of the engulfment synapse shown in B, D. A z stack of 0.31 μm slices (n = 29) was processed by using a background subtraction function and normalization for each of the channels. Surfaces for the plaque (DAPI), microglia process (CD68), and CB2 signal were created by arbitrary thresholding at an upper third of intensity distributions. Note high intensities of CB2 signals are located between CD68 and DAPI surfaces. Insert shows orientation of a 3D window as related to a position of the brain slide.

A representative confocal image of staining for CB2 receptors (green, H60 antibody) in the cortex of 12 mo-old transgenic mice. (B) An overlap of red (CD68) and blue (DAPI) channels for the image shown in A. Note a characteristic gathering of activated microglia around an amyloid plaque (marked by an asterisk). (C) An overlap of channels shown in A-B. Note that areas with high CB2 intensities overlap with CD68-positive areas. White rectangle shows an example of areas used for quantifications presented in E-F. Scale bar is 15 μm. (D) Quantification of CB2 densities (integrated intensities/area) in CD68-positive and –negative areas. 26 areas like that shown in A-C were used for the quantification (n = 2 transgenic mice). Asterisk indicates a significant difference between CD68+ and CD68- areas (one-way ANOVA, p<0.0001). (E) A scatterplot of CB2 and DAPI intensities as a function of distance from the center of an Aβ plaque with radius ~10 μm. Note low CB2 signal in the core of the plaque. CB2 and DAPI intensities were normalized (%) to a maximum signal on each channel. An example of an area used for calculations is shown by a white rectangle in C. (F) Quantification of CB2 signal at different distances from a plaque center. 4–6 slices of z stacks from five plaques (range of radiuses 7–15 μm) were used in one-way ANOVA. Asterisks indicate a significant increase (p<0.0001, post-hoc test) in CB2 intensities as compared to the core of plaques (radius ≤ 7 μm).

(A) Representative confocal images from the cortex of 12 mo-old non-transgenic (NTG) and APPswe/PS1ΔE9 transgenic (AD) mice stained with a CB2 receptor antibody (H60sc; left columns; green) and markers for neurons (NeuN, far red), activated microglia (CD68, red), and astrocytes (GFAP, far red). Brain slides were counterstained with DAPI shown with a grey pseudo color. Note substantial micro- and astro-gliosis in the cortex of the AD mouse brain. In the NTG mice, CD68+ and/or GFAP+ areas were rare (indicated in the upper right panel by an arrowhead and arrow, respectively). (B) Quantification of densities (+-SEM) for CB2 receptor immunoreactivity (integrated intensities/area) in areas positive for NeuN, CD68, and GFAP markers. Densities were averaged over 22 (AD) and 14 (NTG) images of the cortex as shown in A (n = 2 mice per genotype). Single and double asterisks indicate a significant difference between NTG and AD groups as a result of LSD post-hoc test with p levels <0.01 and 0.0001, respectively. Arcs indicate non-significant (NS) differences. Single and double pound signs (p levels <0.05 and 0.001) indicate markers that correspond to the highest CB2 density in the NTG (blue sign) or AD (red sign) groups (LSD post-hoc test). Solid black line at the level of 4,930 shows average densities for the background. (C) An example of NeuN (blue), CD68 (red), and GFAP (green) masks from the AD image shown in A. NeuN masks were drawn by hand as shown in ; masks for CD68 and GFAP were created by a threshold function. Black area represents background. Scale is 15 μm.