Cannabinoid receptors are widely expressed in goldfish: molecular cloning of a CB2-like receptor and evaluation of CB1 and CB2 mRNA expression profiles in different organs.

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 puffer fish 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 and 40 kDa and other faint bands with apparent molecular masses around 70, 57 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 qReal-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 nonmammalian vertebrates.

Interplay of the endocannabinoid system with neuropeptide Y and corticotropin-releasing factor in the goldfish forebrain.

Based on pharmacological, behavioral, and neuroanatomical studies, the endocannabinoid system appears to be pivotal in some neuroendocrine mechanisms, such as modulation of vertebrate reproduction, stress, and food intake. The present study aimed to investigate the involvement of the endocannabinoid system in the control of the feeding response in the goldfish. By means of immunohistochemistry techniques, using anti-CB1 cannabinoid receptor, anti-corticotropin-releasing factor (CRF), and anti-neuropeptide Y (NPY) antisera on brain sections of Carassius auratus, we found a topographical co-distribution of the three signaling molecules through the preoptic area and posterior lobes of the hypothalamus and even a co-localization of CB1 and NPY in the telencephalon. Previous results have shown that food deprivation in goldfish is accompanied by a significant increase of anandamide (AEA) levels in the telencephalon and AEA causes a dose-dependent effect on food intake. We have thus investigated the possible influence of intraperitoneal AEA injections on NPY expression. Our results indicate an interplay between the endocannabinoid system and orexigenic and anorexigenic molecules, such as NPY and, possibly, CRF.

Goldfish CB1 mRNA expression is affected by fasting and anandamide administration.

The endocannabinoid system has a well-documented pivotal role in the control of mammalian feeding response; nevertheless, some evidence is available regarding a similar role in nonmammalian vertebrates and invertebrates. As in the bonyfish Carassius auratus, CB1 cannabinoid receptors are abundant in brain regions involved in the control of food intake, and fasting affects endocannabinoid levels, in this study the effects of food deprivation and anandamide administration on CB1 expression were evaluated. Fasting led to a time-dependent increase of CB1 mRNA levels in the forebrain, an effect reversed by refeeding. Furthermore, the administration of exogenous anandamide reduced CB1 expression in food-deprived goldfish. Our results support the involvement of CB1 receptors in the control of energy intake in nonmammalian vertebrates.

Interplay between the endocannabinoid system and GnRH-I in the forebrain of the anuran amphibian Rana esculenta.

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.

Are the cannabinoids involved in bony fish reproduction?

Following the discovery of two CB1 genes in the fish Fugu rubripes, investigations on the phylogeny of endocannabinoids have indicated that this system is highly conserved. Our study demonstrated that CB1 receptors are expressed in the CNS and gonads of two teleosts, Carassius auratus and Pelvicachromis pulcher, and they show a high percentage of sequence identity with Fugu rubripes CB(1A) and Danio rerio CB1. By means of immunohistochemistry for CB1, sGnRH, and TH, we found a codistribution of these signaling molecules in the basal telencephalon/preoptic area, which are key centers for gonadotropic regulation. We therefore suggest that endocannabinoids are possibly involved in modulating fish reproduction at both the central and peripheral levels.

Expression and distribution of CB1 cannabinoid receptors in the central nervous system of the African cichlid fish Pelvicachromis pulcher.

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.