RESUMEN
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.
Asunto(s)
Carpa Dorada/metabolismo , ARN Mensajero/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/aislamiento & purificación , Receptor Cannabinoide CB2/metabolismo , Animales , Secuencia de Bases , Western Blotting/veterinaria , Clonación Molecular , Análisis por Conglomerados , Cartilla de ADN/genética , Perfilación de la Expresión Génica/veterinaria , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB2/genética , Análisis de Secuencia de ADN/veterinaria , Homología de Secuencia , Especificidad de la EspecieRESUMEN
Based on pharmacological, behavioral and neuroanatomical studies, the endocannabinoids appear to be pivotal in some important neuroendocrine regulations of both vertebrates and invertebrates. Interestingly, a well developed endocannabinoid system was recently demonstrated by us in different bonyfish brain areas which control reproduction, energy balance and stress. Fish in particular are very sensitive to different types of stressors which can heavily affect their reproductive activity and negatively reverberate on aquaculture. Since recent new data have been reported on endocrine disruptors (EDs) impact on zebrafish receptor CB1 expression, in the present research we have investigated the response of the endocannabinoid system to acute treatment with an environmental stressor such as the xenoestrogen nonylphenol (4NP) in the brain and peripheral tissues of the goldfish Carassius auratus. First of all the estrogenic effects induced by 4NP were demonstrated by a dose-dependent increase of plasma levels and gene expression of the biomarker vitellogenin, then changes in cannabinoid receptors and anandamide degradative enzyme, the fatty acid amide hydrolase (FAAH), were analysed by means of Real Time PCR. As the exposure to EDs may lead to an activation of estrogen receptors and affects the Aromatase (AROB) transcription, changes in mRNA levels for ER subtypes and AROB were also evaluated. Our results confirm in goldfish the effect of 4NP on ERα and ERß1 receptors and point out a different sensitivity of CB1 and CB2 for this compound, suggesting distinct roles of these cannabinoid receptors in some adaptive processes to contrast stress induced by xenoestrogen exposure.
Asunto(s)
Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Carpa Dorada/metabolismo , Fenoles/toxicidad , Receptores de Estrógenos/metabolismo , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Animales , Aromatasa/genética , Aromatasa/metabolismo , Ensayo de Inmunoadsorción Enzimática , Carpa Dorada/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Cannabinoides/genética , Receptores de Cannabinoides/metabolismo , Receptores de Estrógenos/genética , Vitelogeninas/genética , Vitelogeninas/metabolismoRESUMEN
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.
Asunto(s)
Moduladores de Receptores de Cannabinoides/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Endocannabinoides , Carpa Dorada/metabolismo , Neuropéptido Y/metabolismo , Animales , Regulación de la Expresión Génica , Neuropéptido Y/genéticaRESUMEN
The endocannabinoid system, through the cannabinoid receptor CB1, is involved in the modulation of adaptive responses to environmental conditions. However, little is known about the role of the cannabinergic system, particularly CB1 receptor expression, in relation to the effects induced by xenoestrogens concerning the reproductive axis. Our results demonstrate that only 10(-8) mol/L of 17beta-estradiol was able to induce significantly higher levels of CB1A mRNA, while no effects were found after treatment with 4-nonylphenol (10(-8) or 10(-6) mol/L); moreover, mRNA expression titers of CB1B did not show any significant change. The estrogenic effects of treatments were evidenced by a dose-dependent induction of plasma hepatic vitellogenin titers. It can be concluded that low doses of estrogens, and possibly of xenoestrogens, may increase endocannabinoid signaling pathways.
Asunto(s)
Estrógenos/química , Estrógenos/farmacología , Receptor Cannabinoide CB1/genética , Estrés Fisiológico/efectos de los fármacos , Animales , Peces Planos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genéticaRESUMEN
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.
Asunto(s)
Moduladores de Receptores de Cannabinoides/fisiología , Endocannabinoides , Hormona Liberadora de Gonadotropina/fisiología , Prosencéfalo/metabolismo , Rana esculenta/fisiología , Receptor Cannabinoide CB1/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Buserelina/farmacología , Moduladores de Receptores de Cannabinoides/metabolismo , Clonación Molecular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hormona Liberadora de Gonadotropina/genética , Hormona Liberadora de Gonadotropina/metabolismo , Masculino , Modelos Biológicos , Datos de Secuencia Molecular , Neuronas/metabolismo , Prosencéfalo/efectos de los fármacos , ARN Mensajero/metabolismo , Rana esculenta/metabolismo , Receptor Cannabinoide CB1/metabolismo , Reproducción/genética , Homología de Secuencia de AminoácidoRESUMEN
N-arachidonoylethanolamide (anandamide [AEA]) is the main endocannabinoid described to date in the testis. It exerts its effects through the activation of G-protein coupled cannabinoid receptors (CNR). However, the activity of AEA in controlling male reproduction is still poorly known. Here we provide direct evidence on the presence of the "endocannabinoid system," constituted by type-1 cannabinoid receptor (CNR1) and fatty acid amide hydrolase (FAAH), in the frog Rana esculenta testis demonstrating its expression in tubular compartment. In fact, during the annual reproductive cycle, both proteins increase in September, when the appearance of spermatids (SPT) occurs. Immunocytochemistry confirms their localization in germ cells and, in particular, in elongated SPT. Signals are still present in spermatozoa (SPZ), as demonstrated by Western blot analysis. Furthermore, the activation of CNR1 reduces sperm motility. Comparative research, carried out using mouse and rat SPZ, definitely indicates that the endocannabinoid system operates in SPZ of phylogenetically distant species. A conserved physiological role of endocannabinoid system in controlling the inhibition of sperm motility is suggested.
Asunto(s)
Amidohidrolasas/metabolismo , Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Receptores de Cannabinoides/metabolismo , Espermatozoides/metabolismo , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/genética , Animales , Ácidos Araquidónicos/farmacología , Evolución Biológica , Inhibidores Enzimáticos/farmacología , Masculino , Ratones , Alcamidas Poliinsaturadas , Rana esculenta , Ratas , Ratas Sprague-Dawley , Receptores de Cannabinoides/genética , Espermatozoides/efectos de los fármacos , Espermatozoides/enzimología , Testículo/metabolismoRESUMEN
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.
Asunto(s)
Sistema Nervioso Central/metabolismo , Cíclidos/metabolismo , Regulación de la Expresión Génica/fisiología , Receptor Cannabinoide CB1/biosíntesis , Secuencia de Aminoácidos/fisiología , Animales , Sistema Nervioso Central/química , Cíclidos/genética , Femenino , Masculino , Datos de Secuencia Molecular , Receptor Cannabinoide CB1/análisis , Receptor Cannabinoide CB1/genéticaRESUMEN
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.
Asunto(s)
Sistema Nervioso Central/metabolismo , Clonación Molecular , ARN Mensajero/metabolismo , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Xenopus laevis/metabolismo , Secuencia de Aminoácidos/genética , Animales , Encéfalo/metabolismo , Femenino , Datos de Secuencia Molecular , Médula Espinal/metabolismo , Distribución Tisular , Vertebrados/metabolismoRESUMEN
Cannabinoids are involved in the control of pain at the spinal level through the cannabinoid receptor-1 (CB1) localized pre- and postsynaptically on primary afferent fibres and dorsal horn interneurones, respectively. Using immunocytochemistry, we show that in addition to its neuronal localization, CB1 is also expressed in numerous astrocytes in laminae I and II of the rat dorsal horn. This ubiquitous localization may account for the complex role played by cannabinoids in antinociception. CB1 receptors in astrocytes may be involved in the anti-hyperalgesic action of exogenous cannabinoids.
Asunto(s)
Astrocitos/química , Células del Asta Posterior/química , Receptores de Droga/análisis , Animales , Astrocitos/ultraestructura , Interneuronas/química , Interneuronas/ultraestructura , Masculino , Microscopía Inmunoelectrónica , Nociceptores/fisiología , Dolor/metabolismo , Células del Asta Posterior/ultraestructura , Ratas , Ratas Wistar , Receptores de CannabinoidesRESUMEN
In an effort to establish distinct phylogenetic values for the major GABA(A) receptor subunit mRNAs (alpha(1), beta(2) and gamma(2)) in the telencephalon, the cellular expression capacity of these mRNAs was evaluated in this brain region of two distantly related mammals (hedgehog and rat). Application of emulsion hybridization histochemistry and quantitative film autoradiography approaches allowed us to detect elevated ( P<0.01) beta(2) mRNA levels in primitive telencephalic neuronal populations such as those of the glomerular olfactory layer of the hedgehog. Conversely, notable levels of the alpha(1)-subunit mRNA were also registered for the higher telencephalic regions such as the oriens-pyramidalis layer of the hippocampus although elevated levels were mostly obtained for the rat. Interestingly, it was the high number of giant pyramidal neurons that were actively involved in the expression of this subunit. A similar pattern was also detected for pyramidal-like cells of the rat lateral amygdala region expressing the gamma(2)-subunit mRNA. Taken together, these results suggest an early phylogenetic success of GABAergic neuronal mechanisms relying mainly on elevated beta(2)-subunit levels. On the other hand, elevated quantities of gamma(2)- and, above all, of alpha(1)-subunit mRNAs in the rat seem instead to be related to the highly advanced behavioral and mnemonic strategies that are under the control of these telencephalic regions.