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Xenopus laevis CB1 cannabinoid receptor: molecular cloning and mRNA distribution in the central nervous system.
Cottone E
,
Salio C
,
Conrath M
,
Franzoni MF
.
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In the present research we isolated and characterized Xenopus laevis CB1 cannabinoid receptor mRNA. The CB1 coding sequence shows a high degree of identity with those of other vertebrates, mammals included, confirming that CB1 receptor is conserved over the course of vertebrate evolution. Notably, the similarity between the X. laevis CB1 sequence and that of the urodele amphibian Taricha granulosa is not higher than the similarity existing between Xenopus and mammals, thus supporting phylogenetic distance between anurans and urodeles. By means of in situ hybridization histochemistry, CB1 mRNA expression and distribution was investigated in the X. laevis central nervous system. As revealed, CB1 mRNA-containing neurons are numerous in the prosencephalon, especially in the olfactory bulbs, telencephalic pallium, and hypothalamus. In the midbrain and hindbrain, labeled cells were observed in the mesencephalic tegmentum and dorsolateral romboencephalon. Abundant CB1 mRNA positive neurons are localized throughout the gray matter of the spinal cord, in particular in the dorsal and ventral fields, where labeled motor neurons are also observed. The distribution of CB1 mRNA in the Xenopus CNS is generally consistent with the CB1-like-immunohistochemistry results we have previously obtained, showing in amphibians a well developed cannabinergic system almost comparable to that described in mammals. However, some differences, such as the abundance of CB1 mRNA-containing neurons in the olfactory system and the rich CB1 spinal innervation, are found.
Figure 1. Northern blotting analysis of Xenopus laevis brain total RNA. Hybridization with a CB1 antisense RNA probe allowed to identify a single band of â¼4.3 kb (A). Hybridization with a negative control RNA probe failed to give any signal (B). Numbers on the left indicate RNA molecular weight standard migration.
Figure 4. Distribution of CB1 mRNA labeling in the different layers of the olfactory bulb and anterior olfactory nucleus (A,B, longitudinal parasagittal sections). Some lightly stained periglomerular cells are shown in the glomerular layer of the olfactory bulb; some positive cell bodies are seen in the anterior olfactory nucleus. Intensely labeled cell bodies occur in the mitral and tufted cell layer as well in the granule cell layer (A). Positive cell bodies of different sizes are shown in more detail within the layer containing both mitral (arrows) and tufted cells (arrowhead) (B). A transverse section of the prosencephalon (C) shows abundant positive cell bodies distributed throughout the pallium and subpallial sites of the telencephalon such as amygdaloid nucleus and striatum. An intense staining is also observed in the anterior preoptic nucleus. Higher magnifications of CB1 mRNA-positive neurons in the medialpallium (D), in the pars medialis of the amygdaloid nucleus (E), and in the dorsal pallium (F). A: anterior; AON: anterior olfactory nucleus; Apl: amygdala pars lateralis; Apm: amygdala pars medialis; Dp: dorsal pallium; gcl: granule cell layer; gl: glomerular layer; ml: mitral cell layer; Mp: medialpallium; P: posterior; Poa: anterior preoptic nucleus; Str: striatum. Scale bars = 80 μm in A, 40 μm in B, 450 μm in C, 140 μm in D, 80 μm in E and F.
Figure 5. CB1 mRNA containing neurons in the eminentia thalami (A) and periventricular gray of the retrochiasmatic hypothalamus (B; the ventricular lumen, at the right, is not visible in the micrograph) (longitudinal parasagittal sections). In the ventral mesencephalic tegmentum positive cell bodies of different sizes are seen; two bipolar neurons with dorsoventrally oriented processes are indicated by arrows (C, longitudinal parasagittal section). The distribution of the CB1 mRNA-positive cell bodies in the spinal cord is shown in a transverse hemisection, cervical level (D). A transverse hemisection of the spinal cord, adjacent to D, processed for negative control, does not show any labeling (E). Numerous small (arrowheads) and medium-sized (arrows) neurons express CB1 receptor mRNA in the dorsal field (F). Labeled motor neurons (arrowheads) and smaller cells (arrows) are observed in the ventral field of the spinal cord (G). A: anterior; cf: central field; df: dorsal field; Lt: Lissauer's tract; ot: optic tract; P: posterior; v: ventricle; vf: ventral field. Scale bars = 100 μm in A, 120 μm in B, 40 μm in C, 220 μm in D and E, 50 μm in F and G.
