Alda Guastalla

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 α‐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. J. Comp. Neurol. 485:293–303, 2005.

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

Abstract: 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 CB1A 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.

Identification by immunofluorescence of prolactin- and somatotrophin-producing cells in the pituitary gland of the tree frog Hyla arborea

The indirect immunofluorescence procedure was used to localize prolactin (PRL)- and somatotrophin (STH)- producing cells in the pituitary distal lobe from Hyla arborea adult specimens. The following mammalian antisera were employed: rabbit anti-ovine PRL, antibovine PRL, anti-human PRL, anti-rat PRL, anti-ovine STH, anti-bovine STH, anti-human STH; monkey anti-rat STH. Immunocytochemical staining was suppressed by solid phase absorption of both anti-PRL and anti-STH with the specific antigen. Absorption of anti-PRL with STH and of anti-STH with PRL did not appreciably affect immunocytochemical staining. Treatment with the two antisera revealed two different reactive cell types, both acidophils. Using PRL antisera a strong fluorescence was found in the large acidophils located chiefly in the rostro-central and ventral areas of the distal lobe sagittal sections. A somewhat weaker fluorescence was observed using STH antisera in the fewer, small acidophils mostly concentrated in the dorso-caudal region and only sparsely scattered in the other areas of the pars distalis. Strikingly, the overall pattern of localization shown by the two cell types is similar to their already known distribution in the pituitary distal lobe of some other species of urodele and anuran amphibians.

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.

Immunocytochemical identification of growth hormone- and prolactin-producing cells in the hypophysis of the newt Triturus cristatus carnifex Laur

Single indirect immunocytochemical methods (immunofluorescence, PAP, and ABC) and double sequential staining (ABC followed by immunofluorescence) were used to localize GH- and PRL-producing cells in the pituitary distal lobe from Triturus cristatus. The following antisera were employed: rabbit anti-ovine PRL, anti-Rana catesbeiana PRL, anti-ovine GH, anti-bovine GH, and monkey anti-rat GH. A cell population corresponding to type-2 acidophils localized in the dorsal and central region, under the intermediate lobe, immunoreacted with GH antisera. Both ovine and bullfrog PRL antisera labeled a large cell population, corresponding to type-1 acidophils, predominantly localized in the ventral anterior two-thirds of the gland. The pattern of localization shown by the two cell types, although consistent with the majority of data on adult amphibians, disproves the findings obtained on the same species by other authors.

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.

Pituitary immunocytochemistry and prolactin plasma levels in hypophysectomized female newts, Triturus camifex, bearing a long‐term pituitary autograft

Abstract The use of specific immunocytochemical techniques to identify prolactin (PRL)‐producing cells allowed us to define more precisely hypophyseal cytology in summer and winter animals and to study its modifications in the ectopic pituitary autograft, namely in a situation in which hypothalamic control is abolished. In the normal adenohypophysis, PRL‐cells are preferentially localized in the an‐teroventral region; in the autograft, these cells are well preserved and a radioimmunoassay (RIA) for amphibian PRL shows that PRL secretion, compared with that of the adenohypophysis in situ, does not vary in winter, while it increases in summer. These results are in agreement with previous data (increase in fin caudal height, a PRL‐dependent character, and in body weight) and demonstrate that, PRL hypophyseal secretion in Triturus camifex comes under a temperature‐dependent hypothalamic inhibitory control, active only in summer.

Identification by immunofluorescence of ACTH-producing cells in the pituitary gland of the tree frog Hyla arborea

The indirect immunofluorescence procedure was used to localize ACTH-producing cells in the pituitary distal lobe from Hyla arborea adult specimens; the antiserum employed was rabbit anti-synthetic ACTH (1-24) conjugated with BSA. Immunohistochemical staining was suppressed by solid-phase absorption of the anti-ACTH with the specific antigen. In the distal lobe one cell population is immunoreactive; these cells are predominantly localized in the anterior half, both ventrally and dorsally; they exclusively correspond to type-3 basophils. In the intermediate lobe the total cell population is immunoreactive. Parallel inspections carried out on the pituitary of Xenopus laevis specimens gave results fully consistent with those reported by other authors.

PROLACTIN BINDING SITES IN XENOPUS LAEVIS (DAUDIN) (ANURA PIPIDAE) KIDNEY: AN AUTORADIOGRAPHIC AND IMMUNOCYTOCHEMICAL STUDY

SUMMARY We have studied prolactin binding sites in Xenopus laevis (Daudin) (Anura Pipidae) kidney by autoradiography and immunocytochemistry. By the autoradiographic technique after intracardiac injection of 125I- oPRL labelling is appreciable in Xenopus laevis kidney at the level of the large vessels and capillaries, the renal corpuscle and, to a variable extent, within the lumina and epithelial cells of the proximal convoluted tubules. Compared to the autoradiographic reaction, the immunocytochemical method failed to reveal the hormone when extremely low doses of it were injected but allowed a much more exact localization of prolactin uptake by the epithelial cells of the proximal convoluted tubules. On the basis of the autoradiographs we could not ascertain wheter, or to what extent, the hormone was bound to specific renal cell PRL receptors: in fact, not only insulin and luteinizing hormone, but also unlabelled prolactin, injected as a specific competitor, failed to bring about the recognizable displa...