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.

ABSTRACT[3H]CP 55,940, a radiolabeled synthetic cannabinoid, which is 10-100. times more potent in vivo than 49-tetrahydrocannabinol, was used to characterize and localize a specific cannabinoid receptor in brain sections. The potencies of a series of natural and synthetic cannabinoids as competitors of [3HJCP 55,940 binding~correlated closely with their relative potencies in several biological assays, suggesting that the receptor characterized in our in vitro assay is the same receptor that mediates behavioral and pharmacological effects of cannabinoids, including human subjective experience. Autoradiography of cannabinoid receptors in brain sections from several mammalian species, including human, reveals a unique and conserved distribution; bing is most dense in outflow nuclei ofthe basal ganglia-the substantia nigra pars reticulata and globus pallidus-and in the hippocampus and cerebellum. Generally high densities in forebrain and cerebellum implicate roles, for cannabinoids in cognition and movement. Sparse densities in lower brainstem areas controlling cardiovascular and respiratory functions may explain why high doses of A9-tetrahydrocannabinol are not lethal.Marihuana (Cannabis sativa) is one of the oldest and most widely used drugs in the world (1, 2). The major psychoactive ingredient of the marihuana plant is A9-tetrahydrocannabinol (A9-THC) (3). A9-THC and other natural and synthetic cannabinoids produce characteristic motor, cognitive, and analgesic effects (4, 5). Early reports showing cannabinoid-like activity of 9f3-hydroxyhexahydrocannabinol (13-HHC) (6)(7)(8) inspired the synthesis of several distinct cannabinoids for studies of their potential use as analgesics (9) 55,940 was extracted, giving a radiochemical yield of 15% and a specific activity of 79 Ci/mmol (1 Ci = 37 GBq). Optimization and competition studies were carried out with slidemounted sections cut from unfixed frozen rat brains. Incubations were in plastic cytomailers (CMS), each containing eight 30-gm-thick "sausage" sections on four gelatin-coated slides in 5 ml of solution (13). The sausage sections were prepared by combining and mincing three whole rat brains to achieve relative homogeneity of receptor and protein conAbbreviations: A8-and A9-THC, A8_ and A9-tetrahydrocannabinol, respectively; BSA, bovine serum albumin; HHC, hydroxyhexahydrocannabinol; SNr, substantia nigra pars reticulata. tTo whom reprint requests should be addressed. 1932The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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This study was designed to examine the mechanisms by which systemic interleukin-1 affects neuroendocrine systems in the brain. Intraperitoneal injections of interleukin-1 beta (1.25 micrograms/rat) were administered to rats. One or three hours after injection, the expression levels of the immediate-early gene c-fos and of genes for several neuropeptides, receptors, and enzymes were examined by in situ hybridization histochemistry. In the brainstem at 1 hr, c-fos mRNA was elevated in the area postrema and nucleus of the solitary tract, but not in the locus coeruleus. At 3 hr, the c-fos mRNA levels had increased further in the nucleus of the solitary tract. Rostrally, elevations in c-fos mRNA levels were found in the hypothalamic and thalamic paraventricular nuclei, central nucleus of amygdala, bed nucleus of the stria terminalis, and medial preoptic area, peaking at 1 hr and diminishing at 3 hr. In addition, at 3 hr a new pattern of c-fos activity emerged--the arcuate nucleus and cells at the external margins throughout the brain now expressed c-fos mRNA. Corticotropin-releasing hormone mRNA levels were doubled in the paraventricular nucleus at 1 and 3 hr, concomitant with elevations in plasma adrenocorticotrophic hormone (ACTH) and corticosterone. Tyrosine hydroxylase mRNA levels in the brainstem did not change. The c-fos mRNA induction patterns reveal a temporally dynamic response to interleukin-1 administration. We propose that the early set of structures responding to interleukin-1 initiates the neuroendocrine response to cytokines. Coactivation of the area postrema and nucleus of the solitary tract may reflect entry into the brain and neural transduction of the peripheral signal. The late set--including the nucleus of the solitary tract, arcuate nucleus, and the brain's edge--may reflect cellular activation along the diffusion routes traveled by interleukin-1 or a bioactive transduction product, because the pattern of edge labeling is similar to the autoradiographic pattern of flow lf radiolabeled tracer substances in the cerebrospinal fluid. The late c-fos mRNA response to interleukin-1, therefore, may represent a demonstration of information transfer in the parasynaptic mode, also known as volume transmission.