Elsevier

Neuroscience

Volume 83, Issue 2, 1998, Pages 393-411
Neuroscience

Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system

https://doi.org/10.1016/S0306-4522(97)00436-3Get rights and content

Abstract

Immunohistochemical distribution of cannabinoid receptors in the adult rat brain was studied using specific purified antibodies against the amino-terminus of the CB1 receptor. Our results generally agree well with the previous studies using CB1 receptor autoradiography and messenger RNA in situ hybridization. However, because of its greater resolution, immunohistochemistry allowed identification of particular neuronal cells and fibers that possess cannabinoid receptors. CB1-like immunoreactivity was found in axons, cell bodies and dendrites, where it appeared as puncta in somata and processes. Both intensely and moderately or lightly stained neurons were observed. The intensely stained neurons were dispersed and only occur in cortical structures including hippocampal formation and olfactory bulb. Moderately or lightly stained neurons were found in caudate–putamen and amygdala. In the hippocampal formation only intensely stained neurons were observed. The cell bodies of pyramidal neurons in CA1 and CA3 fields appeared to be unstained but surrounded by a dense plexus of immunoreactive fibers. The granule cells in the dentate area were also immunonegative. Many intensely stained neurons were located at the base of the granule cell layer. CB1-like immunoreactive neurons and fibers were also found in the somatosensory, cingulate, perirhinal, entorhinal and piriform cortices, in claustrum, amygdaloid nuclei, nucleus accumbens and septum. Beaded immunoreactive fibers were detected in periaqueductal gray, nucleus tractus solitarius, spinal trigeminal tract and nucleus, dorsal horn and lamina X of the spinal cord. A triangular cap-like mass of immunoreactivity was found to surround the basal part of the Purkinje cell body in the cerebellum. Only small, lightly stained cells were found in the molecular layer in the cerebellum close to the Purkinje cell layer.

The CB1 receptor is widely distributed in the forebrain and has a more restricted distribution in the hindbrain and the spinal cord. It appears to be expressed on cell bodies, dendrites and axons. According to the location and morphology, many, but not all, CB1-like immunoreactive neurons appear to be GABAergic. Therefore, cannabinoids and cannabinoid receptors may play a role in modulating GABAergic neurons.

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

References (63)

  • A Mansour et al.

    Immunohistochemical localization of the cloned k1 receptor in the rat CNS and pituitary

    Neuroscience

    (1996)
  • A.S Miller et al.

    Effects of a cannabinoid on spontaneous and evoked neuronal activity in substantia nigra pars reticulata

    Eur. J. Pharmac.

    (1995)
  • A.S Miller et al.

    Electrophysiological effects of a cannabinoid on neural activity in the globus pallidus

    Eur. J. Pharmac.

    (1996)
  • R.G Pertwee et al.

    Enhancement by chlordiazepoxide of catalepsy induced in rats by intravenous or intrapallidal injections of enantiomeric cannabinoids

    Neuropharmacology

    (1991)
  • A.V Revuelta et al.

    The dimethylheptyl derivative of (-)-Δ9-tetrahydrocannabinol reduces the turnover of gamma-aminobutyric acid in the septum and nucleus accumbens

    Life Sci.

    (1982)
  • M.C Sañudo-Peña et al.

    Effects of intranigral cannabinoids on rotational behavior in rats: interactions with the dopaminergic system

    Neurosci. Lett.

    (1996)
  • C Song et al.

    Rat brain cannabinoid receptors are N-linked glycosylated proteins

    Life Sci.

    (1995)
  • K Tsou et al.

    Immunocytochemical localization of opiomelanocortin-derived peptides in the adult rat spinal cord

    Brain Res.

    (1986)
  • K Tsou et al.

    Physical withdrawal in rats tolerant to Δ9-tetrahydrocannabinol precipitated by a cannabinoid receptor antagonist

    Eur. J. Pharmac.

    (1995)
  • K Tsou et al.

    Suppression of noxious stimulus-evoked expression of fos-like immunoreactivity in rat spinal cord by a selective cannabinoid agonist

    Neuroscience

    (1996)
  • T.M Westlake et al.

    Cannabinoid receptor binding and messenger RNA expression in human brain: an in vitro receptor autoradiography and in situ hybridization histochemistry study of normal aged and Alzheimer's brains

    Neuroscience

    (1994)
  • Amaral D. G. and Witter M. P. (1995) Hippocampal formation. In The Rat Nervous System, 2nd Edn (ed. Paxinos G.), pp....
  • Anderson P. (1975) Organization of hippocampal neurons and their interconnections. In The Hippocampus. Vol. 1:...
  • K.A Campbell et al.

    Δ9-THC differentially affects sensory-evoked potentials in the rat dentate gyrus

    J. Pharmac. exp. Ther.

    (1986)
  • D.R Collins et al.

    Prevention by cannabinoid antagonist, SR 141716A, of cannabinoid-mediated blockade of long-term potentiation in the rat hippocampal slice

    Br. J. Pharmac.

    (1995)
  • S.A Deadwyler et al.

    Cannabinoids modulate voltage-sensitive potassium A current in hippocampal neurons via a cAMP-dependent process

    J. Pharmac. exp. Ther.

    (1995)
  • S.A Deadwyler et al.

    The effects of delta-9-THC on mechanisms of learning and memory

    NIDA Res. Monogr.

    (1990)
  • Derkinderen P., Toutant M., Burgaya F., Le Bert M., Siciliano J. C., de Franciscis V., Gelman M. and Girault J.-A....
  • W.A Devane et al.

    Isolation and structure of a brain constituent that binds to the cannabinoid receptor

    Science

    (1992)
  • F.R de Fonseca et al.

    Activation of corticotropin-releasing factor in the limbic system during cannabinoid withdrawal

    Science

    (1997)
  • N Gifford et al.

    Electrically evoked acetylcholine release from hippocampal slices is inhibited by the cannabinoid receptor agonist, WIN 55212-2, and is potentiated by the cannabinoid antagonist SR 141716A

    J. Pharmac. exp. Ther.

    (1996)
  • Cited by (0)

    View full text