Maria Fosca Franzoni

CB1-cannabinoid and mu-opioid receptor co-localization on postsynaptic target in the rat dorsal horn.

Cannabinoids and opioids interact in the control of nociception at the spinal level. Likely, several mechanisms are involved, with one of them being co-localization of cannabinoid and opioid receptors. In order to validate this hypothesis, a double labeling study of CB1 cannabinoid receptors and μ-opioid receptors in the dorsal horn of the rat spinal cord was performed. A strong co-localization of CB1 and μ-opioid receptors was observed in lamina II interneurons at the ultrastructural level. The physiological consequences of the co-localization are discussed.

Endocannabinoid System in Frog and Rodent Testis: Type-1 Cannabinoid Receptor and Fatty Acid Amide Hydrolase Activity in Male Germ Cells

Abstract 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.

Neuronal and astrocytic localization of the cannabinoid receptor-1 in the dorsal horn of the rat spinal cord.

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.

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.

Xenopus laevis CB1 cannabinoid receptor: molecular cloning and mRNA distribution in the central nervous system.

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. J. Comp. Neurol. 464:487–496, 2003.

Cannabinoid receptor CB1-like and glutamic acid decarboxylase-like immunoreactivities in the brain of Xenopus laevis.

Investigation of the cannabinoid system in a vertebrate group phylogenetically distant from mammals might improve understanding of its physiological role. Thus, in the present study, the distribution of the cannabinoid CB1 receptor has been investigated in the brain of Xenopus laevis (anuran amphibians) by immunohistochemistry, using both light and confocal laser-scanning microscopy. Immunostained neuronal perikarya and terminals were found in the olfactory bulb, dorsal and medial pallium, striatum, and amygdala. Varicosities and nerve terminals containing CB1-like immunoreactivity were also seen in the thalamus and hypothalamus. A number of stained cells were observed in the pars distalis of the pituitary gland. Positive nerve fibers were distributed throughout mesencephalic tegmentum, and in the cerebellum immunolabeling was observed in some Purkinje and possibly Golgi cells. The confocal microscopic analysis of CB1-like and glutamic acid decarboxylase-like immunoreactivities in both the medial pallium of the telencephalon and the olfactory bulbs showed a wide codistribution of the two markers. The present results indicate that distribution of CB1 is conserved in the course of phylogeny. Furthermore, the close relationship between CB1-like and glutamic acid decarboxylase-like immunolabelings point toward the existence of a functional link between cannabinergic and GABAergic innervations also in amphibian brain.

A golgi study on tanycytes and liquor-contacting cells in the posterior hypothalamus of the newt

SummaryGolgi methods were employed to study neurons and ependymal tanycytes in the posterior hypothalamus of the newt. The tanycytes send a few coarse, spiny or barbed processes towards the pia mater. In the periventricular grey, the neurohistological methods show common neurons, ranging from a multipolar to a plumed organization, and abundant liquor-contacting cells. These cells, possibly neurons, give rise to a process that reaches the cerebro-spinal fluid, and terminates in a spindle-shaped swelling, with a thin thread at its tip. In other cells, the intraventricular endings are bulbous or finger-like. The occurrence of: (1) branches of the liquor-contacting process, running parallel to the infundibular surface; (2) infundibular processes which end at the base or between the ependymal cell bodies; and (3) axons coursing in the same position, all indicates that the subependymal layer is a site for complex intercellular relationships. The significance of liquor-contacting cells and tanycytes is discussed, in view of the possibility that they may represent part of a system for hypothalamic regulation in response to changes in the CSF.

A quantitative autoradiographic study of GABAA and benzodiazepine receptors in the brain of the frog, Rana esculenta

Specific binding sites for GABAA and benzodiazepines were detected in the brain of the frog Rana esculenta after the in vitro incubation of tissue sections with their respective specific agonists [3H] muscimol and [3H] flunitrazepam. Conditions for the binding assay were optimized and as a result binding was saturable and specific. Quantitative autoradiographic receptor measurements in the different brain sections showed that elevated levels of [3H] muscimol binding sites were found in the two layers of the cerebellum (periventricular and external) with the highest binding densities being detected in the periventricular layer. Relatively high densities of [3H] muscimol binding sites were also observed in the torus semicircularis of the mesencephalon and in the thalamic nucleus rotundus and posterolateral nucleus, plus the mitral cell layer of the olfactory bulb, the amygdala pars lateralis and the striatum of the telencephalon. Intermediate to low binding levels were obtained in the remaining brain areas such as the external layer of the optic tectum, the dorsomedial and dorsolateral anterior thalamic nuclei, the medial and lateral pallium, the medial septal nucleus, the preoptic nucleus, the dorsal and ventral infundibular nuclei of the hypothalamus and the interpeduncular nucleus. Autoradiographic evaluation of benzodiazepine receptors revealed that binding levels of [3H] flunitrazepam were overall lower than those of the GABAA sites. In fact the highest [3H] flunitrazepam binding levels were observed in the striatum, external layer of the optic tectum and the torus semicircularis.(ABSTRACT TRUNCATED AT 250 WORDS)

Xenoestrogens elicit a modulation of endocannabinoid system and estrogen receptors in 4NP treated goldfish, Carassius auratus.

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 ERβ1 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.