Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system
Section snippets
Western blotting
The brain from a single 150-g Sprague–Dawley rat was rapidly dissected isolating the cortex, cerebellum, striatum, and hippocampus. Brain regions were disrupted using a glass-teflon homogenizer in five-fold (volume/weight) excess buffer A [25 mM HEPES, 1 mM EDTA, 6 mM MgCl2, 100 μM phenylmethylsulfonyl fluoride (PMSF), 100 μg/ml soybean trypsin inhibitor, and 10 μM/ml leupeptin, pH 7.4 with NaOH]. Homogenates were spun for 5 min at 1000 g, the supernatants were saved, and the pellets were
CB1 receptor distribution by western blotting
Antibodies to the amino terminus (residues 1–77) of the CB1 receptor recognized a major band of ∼63,000 mol. wt in cortex, hippocampus, striatum, and cerebellum (Fig. 1A). A minor band of ∼180,000 mol. wt was also seen. No immunoreactivity was detected in any of these regions when the primary antiserum was preincubated with the immunizing protein (Fig. 1B). The ∼63,000 mol. wt band of the major immunoreactive protein agrees well with the expected molecular weight of the CB1 receptor,[38]as it is
Discussion
The antibodies used in this study were raised against the first 77 residues of the rat CB1 receptor fused to glutathione S-transferase. Tissue sections, cultured neurons, and transfected cell lines[62]were stained with the antibodies, and the staining was blocked when the antibodies were preincubated with the immunizing protein. In the western blots of brain regions, the immunoreactive bands were also abolished by similar preincubation. Further support for the specificity of the antibodies may
Conclusions
In summary, we have used an antibody to the CB1 receptor to determine its distribution in rat CNS. The CB1 receptor is widely distributed in the forebrain and has a more restricted distribution in the midbrain, hindbrain and spinal cord. It appears to be expressed on cell bodies, dendrites, and axons. Many neurons that are identifiable as GABAergic express CB1 receptors suggesting that cannabinoids have an important role in regulating GABAergic neurotransmission. Co-localization studies of CB1
Acknowledgements
The authors express their gratitude to Saundra L. Patrick for technical assistance, Dr Alfred Mansour for sharing his immunohistochemical protocol, Dr Rebecca Burwell for loaning microscope equipment, Dr Charles Ouimet, Dr Ruth Westenbroek, Dr Michael S. Beattie, and Dr Jacqueline Bresnahan for critical reading of the manuscript. We are grateful for the financial support provided by the NIH(NS33247, DA10536 to J.M.W. and K.T., DA10043, MH01083 to J.M.W.; NS01588, NS08174, DA00286, DA08934 to
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