John C. Ashton

The cannabinoid CB2 receptor as a target for inflammation-dependent neurodegeneration.

Endocannabinoids are released following brain injury and may protect against excitotoxic damage during the acute stage of injury. Brain injury also activates microglia in a secondary inflammatory phase of more widespread damage. Most drugs targeting the acute stage are not effective if administered more than 6 hours after injury. Therefore, drugs targeting microglia later in the neurodegenerative cascade are desirable. We have found that cannabinoid CB2 receptors are up-regulated during the activation of microglia following brain injury. Specifically, CB2-positive cells appear in the rat brain following both hypoxia-ischemia (HI) and middle cerebral artery occlusion (MCAO). This may regulate post-injury microglial activation and inflammatory functions. In this paper we review in vivo and in vitro studies of CB2 receptors in microglia, including our results on CB2 expression post-injury. Taken together, studies show that CB2 is up-regulated during a process in which microglia become primed to proliferate, and then become fully reactive. In addition, CB2 activation appears to prevent or decrease microglial activation. In a rodent model of Alzheimers disease microglial activation was completely prevented by administration of a selective CB2 agonist. The presence of CB2 receptors in microglia in the human Alzheimers diseased brain suggests that CB2 may provide a novel target for a range of neuropathologies. We conclude that the administration of CB2 agonists and antagonists may differentially alter microglia-dependent neuroinflammation. CB2 specific compounds have considerable therapeutic appeal over CB1 compounds, as the exclusive expression of CB2 on immune cells within the brain provides a highly specialised target, without the psychoactivity that plagues CB1 directed therapies.

Expression of the cannabinoid CB2 receptor in the rat cerebellum: An immunohistochemical study

Reports of cannabinoid CB2 receptor protein in the brain have been ambiguous. We therefore tested for CB2 immunoreactivity in the rat brain using immunofluorescence. We detected CB2 labeling in fine fibers in the granule layer. This CB2 labeling did not co-localise with the astrocyte marker glial fibrillary acidic protein (GFAP) and, therefore, the CB2-positive fibers were not astrocytes and were possibly microglial or neuronal. Additionally, strong CB2 labeling was detected in capillary endothelia in the granule, Purkinje cell, and molecular layers. Our results suggest that the role of CB2 receptors in the brain may have been previously underestimated.

Cerebral hypoxia-ischemia and middle cerebral artery occlusion induce expression of the cannabinoid CB2 receptor in the brain

Until recently the cannabinoid CB2 receptor was believed to be absent from the central nervous system. In this study we have identified CB2 expressing cells that appear in the rat brain following stroke and hypoxic-ischemia. At 3 days following surgery CB2-positive macrophages, deriving from resident microglia and/or invading monocytes appear on the lesioned side of the brain. By day 7, a mixed population of CB2-positive cells is present. Microglia-derived macrophages are the key cells in the first stages of brain inflammation, and a pivotal step in the neurodegeneration that follows the acute stage of injury. Thus, CB2 may be important in the brain during injury, and in inflammatory neurodegenerative disorders. The presence of CB2-positive cells in the brain following stroke may provide a novel strategy for cannabinoid-mediated intervention into stroke induced neurodegeneration without the psychoactive effects of CB1 receptor stimulation.

Cannabinoid receptors: a brief history and "what's hot".

Our understanding of the complexity of the endocannabinoid system has evolved considerably since the cloning of the receptors in the early 1990s. Since then several endogenous ligands have been identified and their respective biosynthetic pathways unravelled. This research has revealed the involvement of the cannabinoid system in a number of important physiological processes including the regulation of neurotransmitter release, pain and analgesia, energy homeostasis, and control of immune cell function. All of these events are mediated by two similar receptors, CB1 and CB2, which were initially thought to possess mutually exclusive expression profiles. Recent advances have begun to dissolve such absolutes with the discovery of CB2 in brain tissue and identification of a range of functions for CB1 in peripheral tissues. With improved understanding of the cannabinoid system comes the illumination of various roles in disease pathologies and identification of potential therapeutic targets. This review provides an overview of the current understanding of the endocannabinoid system, and then focuses on recent discoveries that we believe are likely to shape the future directions of the field.

Antibody testing for brain immunohistochemistry: Brain immunolabeling for the cannabinoid CB2 receptor

The question of whether cannabinoid CB₂ receptors are expressed on neurons in the brain and under what circumstances they are expressed is controversial in cannabinoid neuropharmacology. While some studies have reported that CB₂ receptors are not detectable on neurons under normal circumstances, other studies have reported abundant neuronal expression. One reason for these apparent discrepancies is the reliance on incompletely validated CB₂ receptor antibodies and immunohistochemical procedures. In this study, we demonstrate some of the methodological problems encountered using three different commercial CB₂ receptor antibodies. We show that (1) the commonly used antibodies that were confirmed by many of the tests used for antibody validation still failed when examined using the knockout control test; (2) the coherence between the labeling patterns provided by two antibodies for the same protein at different epitopes may be misleading and must be validated using both low- and high-magnification microscopy; and (3) although CB₂ receptor antibodies may label neurons in the brain, the protein that the antibodies are labeling is not necessarily CB₂. These results showed that great caution needs to be exercised when interpreting the results of brain immunohistochemistry using CB₂ receptor antibodies and that, in general, none of the tests for antibody validity that have been proposed, apart from the knockout control test, are reliable.

Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity.

Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest—in this case CB2—but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.

Spinal cannabinoid CB2 receptors as a target for neuropathic pain: an investigation using chronic constriction injury

Agonists for the cannabinoid CB2 receptor are antinociceptive in several rodent models and several reports have suggested that the target for these drugs is CB2 expressed in the spinal cord pain pathway. After confirming the efficacy of a systemically delivered CB2-selective agonist, GW405833, we tested this hypothesis by administering the CB2 agonists GW405833 and JWH-133, via intrathecal cannulation, to the lumbar spinal cord of rats that had undergone chronic constriction injury to induce mechanical allodynia. We found that although the non-selective CB1/CB2 cannabinoid receptor agonist WIN55,212-2 reversed mechanical allodynia in both ipsilateral and contralateral hind paws, neither GW405833 nor JWH-133 reversed mechanical allodynia. In addition, we investigated the expression of CB2 receptors in the neuropathic spinal cord using immunohistochemistry, Western blot and CB2 agonist stimulated [(35)S]GTPγS binding. Although protein-based analysis of CB2 partially matched the results of earlier studies using the same antibody, we found evidence that this antibody may be insufficiently specific for the detection of CB2 in native tissue. Using [(35)S]GTPγS binding assays, we found no evidence of functional CB2 in the spinal cord, in sham or surgery-treated tissue. However, WIN55,212-2 stimulated [(35)S]GTPγS binding showed clear evidence of functional CB1 receptors consistent with the known distribution of elements of the pain pathway, and we concluded that spinal CB2 receptors are not a likely target for cannabinoid-mediated antinociception in this model.

Immunohistochemical characterisation and localisation of cannabinoid CB1 receptor protein in the rat vestibular nucleus complex and the effects of unilateral vestibular deafferentation

CB1 receptor expression has been reported to be low in the brainstem compared with the forebrain, and low in the vestibular nucleus complex (VNC) compared with other regions in the brainstem. However, a frequent effect of cannabis is dizziness and loss of balance. This may be due to the activation of cannabinoid receptors in the central vestibular pathways. We used immunohistochemistry to study the distribution of CB1 receptor protein in the VNC, and Western blotting to measure CB1 receptor expression in the VNC following unilateral vestibular deafferentation (UVD); the hippocampal CA1, CA2/3 and dentate gyrus (DG) regions were also analysed for comparison. This study confirms a previous electrophysiological demonstration that CB1 receptors exist in significant densities in the VNC and are likely to contribute to the neurochemical control of the vestibular reflexes. Nonetheless, CB1 receptor expression did not change significantly in the VNC during vestibular compensation. In addition, despite some small but significant changes in CB1 receptor expression in the CA2/3 and the DG following UVD, in no case were these differences statistically significant in comparison to both control groups.

Drug combination studies and their synergy quantification using the Chou-Talalay method--letter.

Advances in the theory of combination therapy ([1][1]) and development of new targeted drugs are leading to a new emphasis on drug combination experiments. Many studies continue to use the median-effect method of Chou–Talalay ([2][2]). However, the median-effect method follows a similar logic to

The use of knockout mice to test the specificity of antibodies for cannabinoid receptors.

Suarez et al. (2009) characterized immunolabeling in the brain for both cannabinoid CB1 and CB2 receptors. Various researchers have observed that obtaining robust results for immunohistochemistry has been challenging for G-protein couple receptors. Reliable antibodies for cannabinoid receptors have proven especially difficult to construct (Grimsey et al., 2008) and immunohistochemistry for the cannabinoid CB2 receptor has been the focus of recent controversy (Atwood and Mackie, 2010). Suarez et al. (2009) presented evidence for the specificity of the antibodies used in their study, using the method of testing the antibodies on sections taken from transgenic mice lacking either cannabinoid receptor. So called ‘‘knockout (KO) controls’’ are considered the best available test for antibody specificity (Lorincz and Zoltan, 2008). However, the evidence for the specificity of the CB2 antibody used by Suarez et al. (2009) may be questioned. When the images from Figure 1 in Suarez et al. (2009) were imported into Adobe Photoshop V5.0 (Abobe, USA) and the input levels of the knockout controls adjusted to match the mean and range of the output levels of the histogram for corresponding WT section, striking differences between the CB1 and CB2 KO controls emerged. This adjustment equalizes the mean and range of intensity of the KO image in the black channel with the WT section, so that the images only differ with respect to the pattern but not intensity of staining. This method determines whether immunolabeling patterns have been changed in the KO section (as expected by removal of the specific binding site) or simply reduced in intensity. This revealed that the CB2 antibody had a near identical pattern of staining in KO sections as in wild-type (WT) sections, but at reduced intensity. By contrast, the CB1 antibody gave a qualitatively different pattern of staining in the KO section compared to WT. Tellingly, this CB1 antibody has been extremely well validated is other assays, and is the only one of a large number of antibodies tested in a recent study of CB1 antibody specificity that yields a pattern of CB1 expression consistent with autoradiography (Grimsey et al., 2008). When level histograms for the equalized images are compared, it can be seen that the frequency distribution for the CB1 KO is of a different shape than for WT, as expected if specific binding sites are removed by the gene deletion (Fig. 1). However, the frequency distribution for the CB2 KO is very similar in shape to the distribution for the WT image. Therefore, both visual inspection and spectral analysis show that the CB2 KO image has the same pattern of staining as WT, but at reduced intensity. This result is incompatible with a high degree of specificity for the CB2 antibody. Immunolabeling in a knockout control section should demonstrate an absence (not merely a reduction in number or ‘‘knockdown’’) of the specific binding site. Therefore, background staining in KO sections should show a qualitatively different pattern than that found in WT sections. We propose three possible explanations for the results for the CB2 receptor in the KO test in Suarez at al. (2009). First, partial expression of the CB2 receptor may occur in the CB2 KO mouse. This might be explained by the recent discovery of isoforms of the CB2 receptor (Liu et al., 2009). However, the relevant C-terminus region of the mouse receptor is identical in the two known isoforms, and differences in the mice CB2 isoforms only exist in promoter regions of the gene, not in the protein coding regions. Therefore, the epitope for this Cterminus antibody should be absent from the C-terminus KO mouse for both known CB2 isoforms. Second, it is conceivable that the specific binding site for the CB2 antibody precisely overlaps with low-affinity nonspecific binding sites and with background staining, such that addition of specific binding merely intensifies the staining pattern for nonspecific staining. Arguing against this explanation is the qualitatively different pattern of nonspecific and background staining seen in the CB1 KO mouse for the CB1 antibody. A third possibility is that the CB2 KO and WT sections have been subject to slight differences in the DAB staining protocol due to small random variations in such parameters as ambient light, temperature, incubation time, and other factors that influence DAB staining. Peroxidase-based DAB staining is highly sensitive to small differences between protocols, and it is crucially important that multiple negative control sections are run at the same time as experimental sections. Irrespective of which of these explanations is the correct, the specificity of the CB2 receptor antibody used by Suarez et al. (2009) is open to doubt. Department of Pharmacology & Toxicology, Otago School of Medical Sciences, University of Otago, PO Box 913, Dunedin, New Zealand Correspondence to: John C. Ashton, Department of Pharmacology & Toxicology, Otago School of Medical Sciences, University of Otago, PO Box 913, Dunedin 9054, New Zealand. E-mail: john.ashton@otago.ac.nz Accepted for publication 17 February 2011 DOI 10.1002/hipo.20946 Published online 2 May 2011 in Wiley Online Library (wileyonlinelibrary.com). HIPPOCAMPUS 22:643–644 (2012)