Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study

Abstract

A potent, synthetic cannabinoid was radiolabeled and used to characterize and precisely localize cannabinoid receptors in slide-mounted sections of rat brain and pituitary. Assay conditions for 3H-CP55,940 binding in Tris-HCl buffer with 5% BSA were optimized, association and dissociation rate constants determined, and the equilibrium dissociation constant (Kd) calculated (21 nM by liquid scintillation counting, 5.2 nM by quantitative autoradiography). The results of competition studies, using several synthetic cannabinoids, add to prior data showing enantioselectivity of binding and correlation of in vitro potencies with potencies in biological assays of cannabinoid actions. Inhibition of binding by guanine nucleotides was selective and profound: Nonhydrolyzable analogs of GTP and GDP inhibited binding by greater than 90%, and GMP and the nonhydrolyzable ATP analog showed no inhibition. Autoradiography showed great heterogeneity of binding in patterns of labeling that closely conform to cytoarchitectural and functional domains. Very dense 3H-CP55,940 binding is localized to the basal ganglia (lateral caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), cerebellar molecular layer, innermost layers of the olfactory bulb, and portions of the hippocampal formation (CA3 and dentate gyrus molecular layer). Moderately dense binding is found throughout the remaining forebrain. Sparse binding characterizes the brain stem and spinal cord. Densitometry confirmed the quantitative heterogeneity of cannabinoid receptors (10 nM 3H-CP55,940 binding ranged in density from 6.3 pmol/mg protein in the substantia nigra pars reticulata to 0.15 pmol/mg protein in the anterior lobe of the pituitary). The results suggest that the presently characterized cannabinoid receptor mediates physiological and behavioral effects of natural and synthetic cannabinoids, because it is strongly coupled to guanine nucleotide regulatory proteins and is discretely localized to cortical, basal ganglia, and cerebellar structures involved with cognition and movement.

Comparative analysis of fatty acid amide hydrolase and cb1 cannabinoid receptor expression in the mouse brain: evidence of a widespread role for fatty acid amide hydrolase in regulation of endocannabinoid signaling

“…1H, J). This is consistent with the findings of Herkenham et al (1991) who detected only low concentrations of cannabinoid-binding sites in thalamic nuclei of the rat brain. Thus, the thalamus represents a region of the brain where FAAH-immunoreactive neurons are particularly abundant but with few or no associated CB 1 -expressing fibers.…”

“…The cerebellar nuclei are, however, void of CB 1 -immunoreactivity (Fig. 1P), consistent with the absence of cannabinoid-binding sites in these nuclei in the rat brain (Herkenham et al, 1991).…”

“…In some brain regions (e.g. hypoglossal nucleus, median raphe nucleus), the absence of CB 1 -immunoreactivity may simply reflect insensitivity of the immunocytochemical methods used here because there is evidence for the presence of low-level cannabinoid receptor expression from radioligand binding studies (Herkenham et al, 1991). However, there clearly are some regions of the brain (e.g.…”

Acute Δ9-Tetrahydrocannabinol-Induced Deficits in Reversal Learning: Neural Correlates of Affective Inflexibility

“…This regional profile of effects is largely in accordance with the distribution of CB1 cannabinoid receptors at which THC acts (Devane et al, 1988;Mechoulam et al, 1970;Glass et al, 1997;Herkenham et al, 1990Herkenham et al, , 1991a, and THC-induced alterations in activity in these areas have also been demonstrated in previous IEG and metabolic mapping studies performed in rodents (Bloom et al, 1997;ErdtmannVourliotis et al, 1999;Mailleux et al, 1994;Margulies and Hammer, 1991;McGregor et al, 1998;Whitlow et al, 2002). In addition, human imaging studies have consistently demonstrated marked alterations in activity in frontal brain regions following acute marijuana/THC intake or chronic marijuana use (Lundqvist et al, 2001;Mathew and Wilson, 1993;Mathew et al, 1997Mathew et al, , 2002O'Leary et al, 2000O'Leary et al, , 2002Volkow et al, 1996).…”

The Psychotomimetic Effects of Intravenous Delta-9-Tetrahydrocannabinol in Healthy Individuals: Implications for Psychosis

“…The primary effect of cannabinoids is the modulation of neurotransmitter release via activation of presynaptic CB-1Rs (reviewed in Belue et al, 1995;Freund et al, 2003;Pertwee, 1999a). CB1Rs are distributed with high density in the cerebral cortex, particularly frontal regions, basal ganglia, hippocampus, anterior cingulate cortex, and cerebellum (Egertova and Elphick, 2000;Egertova et al, 1998;Elphick and Egertova, 2001;Glass et al, 1997;Herkenham et al, 1991Herkenham et al, , 1990), brain regions that are relevant to both the known effects of cannabinoids and also regions that have been implicated in the putative neural circuitry of psychosis.…”