Elsevier

Brain Research

Volume 1071, Issue 1, 3 February 2006, Pages 10-23
Brain Research

Research Report
Cannabinoid CB2 receptors: Immunohistochemical localization in rat brain

https://doi.org/10.1016/j.brainres.2005.11.035Get rights and content

Abstract

Brain expression of CB2 cannabinoid receptors has been much less well established and characterized in comparison to the expression of brain CB1 receptors. Since CB2 receptors are intensely expressed in peripheral and immune tissues, expression in brain microglia has been anticipated. Nevertheless, we now describe expression of CB2-receptor-like immunoreactivity in brain in neuronal patterns that support broader CNS roles for this receptor. Two anti-CB2 affinity purified polyclonal antibodies were raised in rabbits immunized with peptide conjugates that corresponded to amino acids 1–33 and 20–33. Western blot analyses revealed specific bands that were identified using these sera and were absent when the sera were preadsorbed with 8.3 μg/ml of the immunizing peptides. These studies, and initial RT-PCR analyses of brain CB1 and CB2 mRNAs, also support the expression of brain CB2 receptor transcripts at levels much lower than those of CB1 receptors. CB2 cannabinoid receptor mRNA was clearly expressed in the cerebellum of wild type but not in CB2 knockout mice. CB2 immunostaining was detected in the interpolar part of spinal 5th nucleus of wild type but not in CB2 knockout mice, using a mouse C-terminal CB2 receptor antibody. Immunohistochemical analyses revealed abundant immunostaining for CB2 receptors in apparent neuronal and glial processes in a number of rat brain areas. Cerebellar Purkinje cells and hippocampal pyramidal cells revealed substantial immunoreactivity that was absent when sections were stained with preadsorbed sera. CB2 immunoreactivity was also observed in olfactory tubercle, islands of Calleja, cerebral cortex, striatum, thalamic nuclei, hippocampus, amygdala, substantia nigra, periaqueductal gray, paratrochlear nucleus, paralemniscal nucleus, red nucleus, pontine nuclei, inferior colliculus and the parvocellular portion of the medial vestibular nucleus. In-vitro, CB2 immunoreactivity was also present in hippocampal cell cultures. The multifocal expression of CB2 immunoreactivity in glial and neuronal patterns in a number of brain regions suggests reevaluation of the possible roles that CB2 receptors may play in the 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.

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