The morphofunctional relationship between the endocannabinoid system and GnRH activity in the regulation of reproduction has poorly been investigated in vertebrates. Due to the anatomical features of lower vertebrate brain, in the present paper, we chose the frog Rana esculenta (anuran amphibian) as a suitable model to better investigate such aspects of the reproductive physiology. By using double-labeling immunofluorescence aided with a laser-scanning confocal microscope, we found a subpopulation of the frog hypothalamic GnRH neurons endowed with CB1 cannabinoid receptors. By means of semiquantitative RT-PCR assay, we have shown that, during the annual sexual cycle, GnRH-I mRNA (formerly known as mammalian GnRH) and CB1 mRNA have opposite expression profiles in the brain. In particular, this occurs in telencephalon and diencephalon, the areas mainly involved in GnRH release and control of the reproduction. Furthermore, we found that the endocannabinoid anandamide is able to inhibit GnRH-I mRNA synthesis; buserelin (a GnRH agonist), in turn, inhibits the synthesis of GnRH-I mRNA and induces an increase of CB1 transcription. Our observations point out the occurrence of a morphofunctional anatomical basis to explain a reciprocal relationship between the endocannabinoid system and GnRH neuronal activity.
Neuroanatomical investigation of the cannabinoid system in a lower vertebrate group such as teleost fishes might improve our understanding of the physiological role of such a signaling system. In the present study, the expression of a CB1 cannabinoid receptor has been demonstrated in the CNS of a teleost fish, the cichlid Pelvicachromis pulcher. Moreover, CB1-like immunoreactivity has been analyzed by using a purified antibody against the CB1 receptor amino-terminus. Immunostained neurons and varicosities were found through the telencephalon as well as in the preoptic area and lateral infundibular lobes of the hypothalamus. Stained cells were observed in the pituitary gland. Several cell bodies and nerve terminals containing an intense CB1-like immunoreactivity were found in the pretectal central nucleus and posterior tuberculum, both lying in a transitional region between diencephalon and mesencephalon. In the brainstem, the CB1 immunopositivity was more restricted than in the prosencephalon, with the exception of some large, intensely immunopositive nerve cells within the dorsal mesencephalic tegmentum, possibly motor neurons of the third cranial nerve. In the cerebellum, among a majority of immunonegative granule cells, a subset of them was immunostained. Some positive Purkinje cells were also observed. In the spinal cord, ventral gray matter, several alpha-motoneurons were stained. Similarities to and discrepancies from the CB1 receptor distributions in other vertebrate CNS are discussed, paying particular attention to the abundant CB1 immunoreactivity observed in the area encompassing the pretectum and glomerular nucleus, which is characterized by a peculiar differentiation in bony fishes.
Cannabinoids, the bioactive constituents of Cannabis sativa, and endocannabinoids, among which the most important are anandamide and 2-arachidonoylglycerol, control various biological processes by binding to specific G protein-coupled receptors, namely CB1 and CB2 cannabinoid receptors. While a vast amount of information on the mammalian endocannabinoid system does exist, few data have been reported on bony fish. In the goldfish, Carassius auratus, the CB1 receptor has been cloned and its distribution has been analyzed in the retina, brain and gonads, while CB2 had not yet been isolated. In the present paper we cloned the goldfish CB2 receptor and show that it presents a quite high degree of amino acid identity with zebrafish Danio rerio CB2A and CB2B receptors, while the percentage of identity is lower with the pufferfish Fugu rubripes CB2, as also confirmed by the phylogenetic analysis. The sequence identity becomes much lower when comparing the goldfish and the mammalian CB2 sequences; as for other species, goldfish CB2 and CB1 amino acid sequences share moderate levels of identity. Western-blotting analysis shows the CB2 receptor as two major bands of about 53 kDa and 40 kDa, and other faint bands with apparent molecular masses around 70 kDa, 57 kDa and 55 kDa. Since the distribution of a receptor could give information on its physiological role, we evaluated and compared CB1 and CB2 mRNA expression in different goldfish organs by means of quantitative Real-Time PCR. Our results show that both CB1 and CB2 receptors are widely expressed in the goldfish, displaying some tissue specificities, thus opening the way for further functional studies on bony fish and other non-mammalian vertebrates.
Following the discovery in the brain of the bonyfish Fugu rubripes of two genes encoding for type 1 cannabinoid receptors (CB1A and CB1B), investigations on the phylogeny of these receptors have indicated that the cannabinergic system is highly conserved. Among the multiple functions modulated by cannabinoids/endocannabinoids through the CB1 receptors one of the more investigated is the mammalian reproduction. Therefore, since studies performed in animal models other than mammals might provide further insight into the biology of these signalling molecules, the major aim of the present paper was to review the comparative data pointing toward the endocannabinoid involvement in the reproductive control of non mammalian vertebrates, namely bonyfish and amphibians.The expression and distribution of CB1 receptors were investigated in the CNS and gonads of two teleosts, Pelvicachromis pulcher and Carassius auratus as well as in the anuran amphibians Xenopus laevis and Rana esculenta. In general the large diffusion of neurons targeted by cannabinoids in both fish and amphibian forebrain indicate endocannabinoids as pivotal local messengers in several neural circuits involved in either sensory integrative activities, like the olfactory processes (in amphibians) and food response (in bonyfish), or neuroendocrine machinery (in both). By using immunohistochemistry for CB1 and GnRH, the codistribution of the two signalling molecules was found in the fish basal telencephalon and preoptic area, which are key centers for gonadotropic regulation in all vertebrates. A similar topographical codistribution was observed also in the septum of the telencephalon in Rana esculenta and Xenopus laevis.Interestingly, the double standard immunofluorescence on the same brain section, aided with a laser confocal microscope, showed that in anurans a subset of GnRH neurons exhibited also the CB1 immunostaining. The fact that CB1-LI-IR was found indeed in the FSH gonadotrophs of the Xenopus pituitary gland and CB1 receptors together with the fatty acid amide hydrolase, the degradative enzyme of the endocannabinoid anandamide, were demonstrated in both bonyfish and frog gonads, strongly suggests that endocannabinoids are involved in central and peripheral gonadotropic functions of teleosts and amphibians.
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