| Background:Cannabinoid now is mainly used for refractory diseases, such as some nervous system diseases, including multiple sclerosis (MS), motor neuron disease, chronic intractable pain and drug-induced vomiting. In addition, cannabinoid exerts certain therapeutic effects on glaucoma, asthma and cardiovascular diseases. Although cannabinoid has not been extensively used in clinical applications yet, it has aroused wide interest of researchers.Cannabinoid is commonly divided into three categories: extracted from natural plant, synthetic and endogenous. In 1990s, its receptors, CB1 and CB2, were cloned in vitro. CB1 receptor is constituted of 473 amino acids and 7 transmembrane domains, and CB2 receptor of 360 amino acids and 7 transmembrane domains. They are G protein-coupled receptors. Research on gene cloning found that these two receptors are homologous in 44% amino acid sequence. Human and rat cannabinoid receptors have a high homology in amino acid sequence. Therefore, adult rats are used as ideal experimental animals to study the distribution of cannabinoid receptors and functions.CB1 receptors are thought to be the most widely expressed G-protein coupled receptor in the brain, mainly distributed in presynaptic membrane of nerve terminals and regulating the release of neurotransmitters. CB2 receptors are mainly located in immune tissues and cells, such as the marginal zone of the spleen, tonsils, T cells, B cells and macrophages and so on, playing roles in immune regulation and inhibiting the release of cytokines. CB2 receptors also have a higher expression in the nervous system. Evidence shows that a number of glial cells express both cannabinoid receptors CB1and CB2. Up to now, the studies on the distribution of CB1 and CB2 receptors in the brain were limited in some parts of brain regions. And the receptors were found to be expressed in more brain regions. Thus, further studies need to be done on their distribution in the brain. The latest studies showed that activation of CB1and CB2 receptors has a protective effect on demyelinating disease. On the one hand, they can inhibit the immune response, reducing the destruction of myelin. On the other hand, more importantly, they promote the survival of oligodendrocyte and its precursor and thus impact the regeneration of myelin directly. Myelin regeneration needs a variety of cells and cytokines, including oligodendrocytes, astrocytes, Id2, olig2 and so on. Further study on the positive cell types of cannabinoid receptors in the brain will be helpful in profound understanding on through which cells cannabinoid functions, and will provide the targets for the treatment of demyelinating disease at the same time.Objective:The aim of this study was to investigate the distribution of two types of cannabinoid receptors CB1 and CB2 in the whole brain, clarify the features, and similarities and differences between the two types of receptors in different brain regions in order to found a basis for further study on the functions of cannabinoid and the two receptors. We studies the expression of cannabinoid receptor CB1, CB2 in the brain of SD rats, determined the major cell types which expressed the receptors, especially in oligodendrocyte cells, observed whether the two receptors co-expressed in one cell, so as to provide the basis for cannabinoids function in the demyelinating disease.Methods:Totally 10 adult SD rats, weighting 240 to 260 g, with both sexes were employed in this study. Immunohistochemical method was used to demonstrate the expression of CB1 and CB2 receptors in different regions of adult rat brain. Immunofluorescence staining was used to examine the cell types. Adjacent sections was used to study the co-expression of two types of receptor in the one cell.Results:1 Distribution of cannabinoid receptors CB1 and CB2Cannabinoid receptors were distributed extensively in adult rat brain, but the number of positive cells was various among the different brain regions. In comparison of two types of receptors, CB2 receptor had a similar distribution pattern as CB1 receptor in most region of the brain. But there were significant differences of their expressions in the callosum, white matter of cerebellum, pons and medulla oblongata. The CB2 positive cells were much more in the callosum and white matter of cerebellum than CB1 positive cells, but the condition was opposite in the medulla. CB2 positive cells showed long cell processes in the regions of pons, medulla oblongata, cerebellum white matter and callosum, the same case of CB1 receptors only existed in the callosum and medulla oblongata.Cannabinoid receptor CB1 was strongly positive in the cerebral cortex, hippocampus, basal ganglia, olfactory brain granule cell layer, the Purkinje cell layer of cerebellar; moderate positive in the olfactory brain globule layer, mitral cell layer, pons, flocculus and medulla oblongata; and mildly positive in the callosum, cerebellum molecular layer, granular layer and white matter of cerebellum. Cannabinoid receptor CB2 was highly expressed in the cerebral cortex, callosum, hippocampus, basal ganglia, olfactory brain granule cell layer, the Purkinje cell layer and white matter of cerebellum; moderately in the olfactory brain globule layer, mitral cell layer, pons and flocculus; and mildly in the cerebellum molecular layer, granular layer and medulla oblongata. The immunoreactive stain of cannabinoid receptors was mostly observed in the cell body. But that of CB2 receptor was distributed in the cell body and long processes in the callosum, pons and medulla oblongata. CB1 receptor had the same pattern in the callosum and medulla oblongata.2. Positive cell types of cannabinoid receptors in the brain and co-expressionDouble-labeled immunofluorescence staining showed that CB1 receptor was mainly expressed in the neurons. A great deal of CB1 positive neurons were observed in the cerebral cortex, hippocampus and cerebellum Purkinje cell layer. At the same time, there were a few oligodendrocytes expressing CB1 receptor in the cerebral cortex. Only few astrocytes expressed CB1 receptors which were located mainly in cerebellar white matter. The major cell types positive to CB2 receptor were the neurons, oligodendrocytes and astrocytes. In the area of cerebellar white matter, a large number of oligodendrocytes expressed CB2 receptor, and astrocytes expressed more CB2 receptors than CB1 receptor. There were three distribution forms of CB2 receptors in the oligodendrocytes, that is, in the nucleus, in the nucleus and perinuclear area, and in the perinuclear area.Adjacent sections were used to study the co-expression of two types of receptors in the one cell. The co-expression of the two receptors was found in the cerebral cortex and flocculus cerebelli. In the cerebral cortex, there were very little cells double-positive to the two types receptor at the same. The morphology of these co-expression cells had no significant difference with other cells. In the flocculus cerebelli, the number of Purkinje cell which co-expressed two types receptor was small, and there was no significant difference with other Purkinje cells in the morphology.Conclusion:1. Cannabinoid receptors CB1and CB2 are widely expressed in the brain. The two types of receptors are similarly distributed in most brain regions, but there is certain difference of the same receptor in different brain regions. The similar distribution may suggest that they are involved in some pathophysiological processes. Differences in the number and staining parts of cannabinoid receptor-positive cells in different brains suggest that the two receptors in the brain may play different roles. These results will lay a foundation for the further study of cannabinoid receptors.2. The major cell type positive to CB1 cannabinoid receptors are neurons in the brain, and oligodendrocytes and astrocytes are more lesser. The major cell types of CB2 are neurons and oligodendrocytes, and astrocytes are more less. The co-expression of two types of receptor is observed in the same cell. The results indicate that cannabinoids in the brain can act on various types of cells, especially on oligodendrocyte, which maybe play an important role in the brain. |
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