Research ReportCannabinoid CB2 receptors: Immunohistochemical localization in rat brain
Introduction
Research on the molecular and neurobiological basis of the physiological and neurobehavioral effects of marijuana and cannabinoids lagged behind those of other natural addictive products like opium and tobacco because of lack of specific molecular tools and technology. Now, significant and rapid progress has transformed marijuana–cannabinoid research into mainstream science with the cloning of genes encoding cannabinoid receptors (Cnrs) and generation of cannabinoid receptor knockout mice. Furthermore, these advancements in marijuana and cannabinoid research indicate the existence of a previously unknown, but elaborate and ubiquitous endocannabinoid physiological control system (EPCS) in the human body and brain whose role is unfolding. This remarkable progress includes identification of genes encoding cannabinoid receptors (Cnrs) (Chakrabarti et al., 1995, Matsuda et al., 1990, Munro et al., 1993), isolation of endocannabinoids (eCBs) (Devane et al., 1992, Hanus et al., 2001, Mechoulam and Parker, 2003, Onaivi et al., in press, Porter et al., 2002, Sugiura et al., 1995, Tsou et al., 1998) and entourage ligands (Ben-Shabat et al., 1998), and functional identification of transporters and enzymes for the biosynthesis and degradation of these endogenous substances, which, thus, represent the EPCS (for a review, see Onaivi et al., 2002). While CB1 turned out to be one of the most abundant neuromodulatory receptors in the brain, both CB1 and CB2 receptors are widely distributed in peripheral tissues with CB2 cannabinoid receptors particularly enriched in immune tissues (Berdyshev, 2000, Suigiura and Waku, 2000, Wilson and Nicoll, 2001). Despite this wealth of information and major advances, little information is available about the CB2 cannabinoid receptors that have been generally referred to as peripheral Cnrs because CB2 receptor has been found primarily in cells of the immune system. While a number of laboratories have not been able to detect the presence of CB2 in healthy brains (Carlisle et al., 2002, Chakrabarti et al., 1995, Derocq et al., 1995, Galiegue et al., 1995, Griffin et al., 1999, Shatz et al., 1997, Sugiura et al., 2000), there has been demonstration of CB2 expression in rat microglial cells (Kearn and Hilliard, 1997), in cerebral granule cells (Skaper et al., 1996), in mast cells (Facci et al., 1995, Samson et al., 2003), in adult rat retina (Lu et al., 2000) and induction of CB2 receptor expression in the rat spinal cord of neuropathic but not inflammatory chronic pain models (Zhang et al., 2003). Additional reports indicate that CB2 receptors are expressed by a limited population of microglial cells in normal healthy and neuritic plaque-associated glia in Alzheimer's disease brain (Benito et al., 2003, Nunez et al., 2004, Pazos et al., 2004), human brain capillaries and microvessels (Golech et al., 2004), CB2 receptors expressed in the brains of macaque model of encephalitis induced by simian immunodeficiency virus (Benito et al., 2005), and human astrocytes expressed in both CB1 and CB2 cannabinoid receptors (Sheng et al., 2005). Despite these studies indicating that CB2 receptors might be present in the CNS, the expression of CB2 cannabinoid receptors in the brain has been much less well established and characterized in comparison to the expression of abundant brain CB1 receptors. However, our initial RT-PCR analyses of brain CB1 and CB2 mRNAs supporting (Onaivi et al., in press) brain expression of CB2 receptor gene transcripts prompted us to investigate the localization of CB2 cannabinoid receptors in rat brain slices using two CB2 affinity purified polyclonal antibodies raised in rabbits immunized with peptide conjugates that corresponded to amino acids 1–33 and 20–33. Our results indicate wide spread expression of CB2 receptors in the brain, and, in-vitro, CB2 immunoreactivity (iCB2) was detected in hippocampal NSE-positive neuronal cells. Immunohistochemistry localization revealed abundant CB2 immunostaining in apparent neuronal and glial processes in a number of brain areas. Cerebellar Purkinje cells and hippocampal pyramidal cells revealed substantial immunoreactivity that was absent when sections were stained with preadsorbed sera.
Section snippets
CB2 receptor immunohistochemical and gene expression analysis in the brain
Immunoblots from mouse brain and spleen lysates revealed a major band of approximately 53 kDa, with other visible bands around 37 kDa and 75 kDa, similar to those observed recently (Van Sickle et al., 2005). The use of three anti-CB2 affinity purified polyclonal antibodies with peptide conjugates corresponding to different amino acid terminals and yielding similar patterns of staining indicated further specificity of CB2 receptor localization. The first two different CB2 antibodies (one from
Discussion
As accumulating evidence from our studies and those of others indicate that CB2 cannabinoid receptors may be present in the brain but has not been systematically characterized, our main goal was to localize CB2 cannabinoid receptors and their distribution patterns in the rat brain. An initial publication of data from this work has appeared in abstract form (Gong et al., 2005, Onaivi et al., in press). Numerous previous studies for over a decade since the cloning of CB2 cannabinoid receptors
Animals and preparation of tissue
Sprague–Dawley rats were anesthetized with pentobarbital (97.2 mg/kg i.p.), briefly perfused transcardially with saline and then with 4% paraformaldehyde in phosphate buffer (PB; 0.1 M, pH 7.4) for 5 min. Brains and spleens were dissected, post-fixed in buffered paraformaldehyde for 2 h at room temperature, equilibrated with 30% sucrose in phosphate buffer at 4 °C, frozen, and cut into coronal or sagittal 20–40 μm sections using a sliding microtome. Experiments confirmed to National Institutes
Acknowledgments
This work was supported financially by NIDA/IRP, NIH, and DHSS, and ESO acknowledges financial support from William Paterson University center for research. The CB2 knockout and their wild type control mice used in this were developed by Buckley et al., 2000 and obtained from the National Institutes of Health.
References (41)
- et al.
An entourage effect: inactive endogenous fatty acid glycerol esters enhance 2-arachidonyl-glycerol cannabinoid activity
Eur. J. Pharmacol.
(1998) Cannabinoid receptors and the regulation of immune response
Chem. Phys. Lipids
(2000)- et al.
Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB(2) receptor
Eur. J. Pharmacol.
(2000 (May 19)) - et al.
Differential expression of the CB2 cannabinoid receptor by rodent macrophages and macrophage-like cells in relation to cell activation
Int. J. Immunopharmacol.
(2002) - et al.
Cannabinoids enhance human B-cell growth at low nanomolar concentrations
FEBS Lett.
(1995) - et al.
Human brain endothelium: coexpression and function of vanilloid and endocannabinoid receptors
Mol. Brain Res.
(2004) - et al.
Evaluation of the cannabinoid CB2 receptor-selective antagonist, SR144528, further evidence for CB2 receptor absence in the rat central nervous system
Eur. J. Pharmacol.
(1999) - et al.
Cannabis and alcohol—A close friendship
Trends Pharmacol. Sci.
(2003) - et al.
Endocannabinoids and cannabinoid receptor genetics
Prog. Neurobiol.
(2002) - et al.
Role of the endocannabinoid system in Alzheimer's disease: new perspectives
Life Sci.
(2004)