Claudia Pinelli

Dehydration-regulated processing of late embryogenesis abundant protein in a desiccation-tolerant nematode

Late embryogenesis abundant (LEA) proteins occur in desiccation‐tolerant organisms, including the nematode Aphelenchus avenae, and are thought to protect other proteins from aggregation. Surprisingly, expression of the LEA protein AavLEA1 in A. avenae is partially discordant with that of its gene: protein is present in hydrated animals despite low cognate mRNA levels. Moreover, on desiccation, when its gene is upregulated, AavLEA1 is specifically cleaved to discrete, smaller polypeptides. A processing activity was found in protein extracts of dehydrated, but not hydrated, nematodes, and main cleavage sites were mapped to 11‐mer repeated motifs in the AavLEA1 sequence. Processed polypeptides retain function as protein anti‐aggregants and we hypothesise that the expression pattern and cleavage of LEA protein allow rapid, maximal availability of active molecules to the dehydrating animal.

Comparative immunocytochemical study of FMRFamide neuronal system in the brain of Danio rerio and Acipenser ruthenus during development.

The distribution of FMRFamide-like immunoreactive (ir) neurons and fibers was investigated in the central nervous system of developing zebrafish and juvenile sturgeon (sterlet). Adult zebrafish was also studied. In zebrafish embryos FMRFamide-ir elements first appeared 30 h post-fertilization (PF). Ir somata were located in the olfactory placode and in the ventral diencephalon. FMRFamide-ir fibers originating from diencephalic neurons were found in the ventral telencephalon and in ventral portions of the brainstem. At 48 h PF, the ir perikarya in the olfactory placode displayed increased immunoreactivity and stained fibers emerged from the somata. At 60 h PF, bilaterally, clusters of FMRFamide-ir neurons were found along the rostro-caudal axis of the brain, from the olfactory placode to rostral regions of the ventro-lateral telencephalon. At 60 h PF, numerous ir fibers appeared in the dorsal telencephalon, optic lobes, optic nerves, and retina. Except for ir fibers in the hypophysis at the age of 72 h PF, and a few ir cells in the nucleus olfacto-retinalis (NOR) at the age of 2 months PF, no major re-organization was noted in subsequent ontogenetic stages. The number of stained NOR neurons increased markedly in sexually mature zebrafish. In adult zebrafish, other ir neurons were located in the dorsal zones of the periventricular hypothalamus and in components of the nervus terminalis. We are inclined to believe that neurons expressing FMRFamide originate in the olfactory placode and in the ventricular ependyma in the hypothalamus. On the same grounds, a dual origin of FMRFamide-ir neurons is inferred in the sturgeon, an ancestral bony fish: prior to the observation of ir cells in the nasal area and in the telencephalon stained neurons were noted in circumventricular hypothalamic regions.

Proliferative activity in the frog brain: A PCNA-immunohistochemistry analysis

By means proliferating cell nuclear antigen (PCNA) immunohistochemistry, we have provided a detailed neuroanatomical mapping of proliferative activity during development and adulthood in the frog (Rana esculenta) brain. Western blot analysis confirmed the presence of this protein in brain extracts from adults and tadpoles. Proliferative activity was observed in the ventricular and subventricular zones throughout the brain. The present study provides details as to which of the morphologically distinguishable brain region(s) has a long-lasting proliferative activity and in which region this activity undergoes a progressive decrease during development. In the subventricular zones of the third ventricle, PCNA-labeled cells were particularly abundant in the magnocellular preoptic nucleus and the ventromedial thalamic nucleus. It was observed that proliferation zones are present practically in all major subdivisions of the forebrain, midbrain and hindbrain, including the cerebellum in which PCNA-labeled cells were located in the outer granular layer and the inner molecular layer. The habenulae, epiphysis and isthmic nuclei never showed the presence of PCNA-immunoreactive nuclei. The widespread proliferative activity implies that the frog brain has a great potential for neurogenesis/gliogenesis not only during larval development but also in the adulthood.

Neuropeptide Y: localization in the brain and pituitary of the developing frog (Rana esculenta)

Abstract.The immunohistochemical localization of neuropeptide Y (NPY)-like peptide has been investigated in the peripheral terminal nerve, brain and pituitary of the frog, Rana esculenta, during development. Soon after hatching, a rather simple NPY-immunoreactive (-ir) neuronal system is present, with elements located mainly in the diencephalon. When hind limbs appear and develop, the NPY-neuronal system undergoes considerable elaboration and NPY-ir perikarya appear in several regions of the telencephalon (dorsal, medial, and lateral pallium; medial septum; medioventral telencephalon; anterior preoptic area), diencephalon (ventromedial, central and posterior thalamic nuclei; suprachiasmatic nucleus; infundibulum), mesencephalon (anteroventral mesencephalic tegmentum), and rhombencephalon (central grey; area of the cerebellar and vestibular nuclei). The frequency of NPY-ir neurons increases during larval development, and then decreases in the anterior preoptic area during the metamorphic climax. Dense plexuses of NPY-ir fibers are formed in several brain areas. NPY-ir fibers are found in the peripheral terminal nerve, and ir-neurons through its course along the ventromedial surface of the olfactory bulbs. NPY-ir fiber projections to the median eminence and pars intermedia derive mainly from the ventral infundibular group of NPY-ir neurons, with a contribution from the suprachiasmatic group of NPY neurons. NPY and carboxyl terminal flanking peptide of proneuropeptide Y coexist in the same neurons throughout the brain. The ontogenetic pattern of NPY-ir neuronal system in the brain of Rana esculenta is remarkably different than that reported for Xenopus laevis.

Distribution of FMRFamide-like Immunoreactivity in the Amphibian Brain: Comparative Analysis

FMRFamide is a small neuropeptide present in particular neurons of the basal forebrain and midbrain of the vertebrate groups studied, especially fishes and mammals. In order to assess interspecies variation, the distribution of FMRFamide‐like immunoreactivity was studied in the brains of 13 species of amphibian. Although FMRFamide‐immunoreactive (IR) terminals occurred throughout much of the brain, IR cell groups were noted in circumscribed regions of the CNS. In the eight anuran species studied, two major populations of labeled perikarya were observed: one in the septopreoptic area and another one in the caudal portion of the diencephalon. The rostrocaudal extent of both and the number of labeled somata in each neuronal group displayed species‐specific differences. In urodeles and gymnophiones, labeled perikarya were located in the diencephalon, but there were remarkable species differences in the number of such cells. It is discussed whether sex or season of collection may account for some of the differences observed. The distribution of FMRFamide‐IR perikarya, fibers, and pathways in the brain of anurans, urodeles, and gymnophiones was compared. The existence of FMRFamide perikarya in the anterior preoptic neuropil and medial septum appeared to be a feature common to all anurans; labeled neurons in the dorsal thalamus, however, may be present only in the (viviparous) gymnophione Typhlonectes compressicauda. Cerebrospinal fluid contacting FMRFamide neuronal cell bodies and fibers were observed in each of the three taxonomic orders. The data are compared with those previously obtained for other groups of vertebrates. J. Comp. Neurol. 414:275–305, 1999.

Mast cells in nonmammalian vertebrates: an overview.

Mast cells are best known as multifunctional entities that may confer a benefit on immune system. This review presents the known facts on mast-cell system and breakthroughs in mast-cell biology in fish, amphibians, reptiles, and birds. As compared to mammals, there are relatively few data available on mast cells in many nonmammalian vertebrates. Nevertheless, like in mammals, mast cells in nonmammalian vertebrates contain a wide range of bioactive compounds including histamine, heparin, neuropeptides, and neutral proteases. In bony fishes, these cells secrete antimicrobial peptides as well. Mast cells have a widespread distribution in the brain, endocrine glands, intestine, liver, kidney, skin, tongue, and lungs, the highest concentration occurring in different tissues in the different taxa. Currently, researchers are grappling with the nature of scientific support to substantiate the functional importance of mast cells in nonmammalian vertebrates. Ultimately, the origin and evolution of vertebrate mast cell is of great interest to comparative immunologists seeking an underlying trend in the phylogenetic development of immunity.

Distribution of FMRFamide-like immunoreactivity in the brain and pituitary of Rana esculenta during development

Developmental aspects of the distribution of FMRFamide (Phe-Met-Arg-Phe-NH2) immunoreactivity (ir) were investigated by indirect immunofluorescence in the brain, pituitary and terminal nerve of the frog, Rana esculenta. Soon after hatching. FMRFamide neurons were found in the proximal terminal nerve, mediobasal olfactory bulb, caudal dorsolateral pallium, diagonal band of Broca, anterior preoptic area, suprachiasmatic area, thalamus, infundibulum, and developing pituitary. FMRFamide fibers were present in the olfactory epithelium, terminal nerve, olfactory bulbs, dorsal and midventral telencephalon, epiphysis, mediolateral thalamus, pretectal gray, optic tectum, infundibulum, posterior interpeduncular nucleus-tegmentum area, and rostral rhombencephalon. During successive developmental stages, ir neurons were no longer observed in the dorsal telencephalon and pituitary. In late larval stages, ir neurons appeared in the medial septal area, and ir fibers in the cerebellum and torus semicircularis. At the same time, the frequency of ir neurons increased progressively in the anterior preoptic area, suprachiasmatic area and infundibulum. FMRFamide-ir neurons were never revealed in mesencephalon and rhombencephalon. Numerous ir fibers terminated in the median eminence and intermediate lobe of the pituitary. The adult pattern of distribution of FMRFamide-ir elements in the brain was achieved during the postmetamorphic development. In light of the existing literature, the possible placodal origin of forebrain-located FMRFamide neurons is briefly discussed.

Distribution of Gonadotropin-Releasing Hormone Immunoreactivity in the Brain of Ichthyophis beddomei (Amphibia: Gymnophiona)

From a comparative viewpoint, we have investigated the presence and neuroanatomical distribution of gonadotropin‐releasing hormone (GnRH)‐immunoreactive material in the brain of a gymnophione amphibian, Ichthyophis beddomei. Immunocytochemical analysis of the adult brain and terminal nerves in both sexes shows the presence of neurons and fibers containing mammalian GnRH (mGnRH)‐ and chicken GnRH‐II (cGnRH‐II)‐like peptides. With respect to GnRH‐immunoreactive material, there are two distinct neuronal systems in the brain: one containing mGnRH, which is located in the forebrain and terminal nerve, and the other containing cGnRH‐II, which is restricted to the midbrain tegmentum. Basically, this distribution pattern parallels that of many species of anurans and a urodele. Whereas the presence of cGnRH‐II‐immunoreactive fibers in the dorsal pallium of I. beddomei is a feature in common with a urodele amphibian, the total absence of cGnRH‐II‐like material in the median eminence is unique to this species. It is suggested here that the distribution profile of GnRH‐like material within the brain and terminal nerve of I. beddomei represents a primitive pattern. J. Comp. Neurol. 384:283‐292, 1997.

Extrabulbar olfactory system and nervus terminalis FMRFamide immunoreactive components in Xenopus laevis ontogenesis

The extrabulbar olfactory system (EBOS) is a collection of nerve fibers which originate from primary olfactory receptor-like neurons and penetrate into the brain bypassing the olfactory bulbs. Our description is based upon the application of two neuronal tracers (biocytin, carbocyanine DiI) in the olfactory sac, at the cut end of the olfactory nerve and in the telencephalon of the developing clawed frog. The extrabulbar olfactory system was observed already at stage 45, which is the first developmental stage compatible with our techniques; at this stage, the extrabulbar olfactory system fibers terminated diffusely in the preoptic area. A little later in development, i.e. at stage 50, the extrabulbar olfactory system was maximally developed, extending as far caudally as the rhombencephalon. In the metamorphosing specimens, the extrabulbar olfactory system appeared reduced in extension; caudally, the fiber terminals did not extend beyond the diencephalon. While a substantial overlapping of biocytin/FMRFamide immunoreactivity was observed along the olfactory pathways as well as in the telencephalon, FMRFamide immunoreactivity was never observed to be colocalized in the same cellular or fiber components visualized by tracer molecules. The question whether the extrabulbar olfactory system and the nervus terminalis (NT) are separate anatomical entities or represent an integrated system is discussed.

Comparative analysis of FMRFamide-like immunoreactivity in caiman (Caiman crocodilus) and turtle (Trachemys scripta elegans) brains.

Abstract. The distribution of FMRFamide (FMRFa)-like peptides in caiman (Caiman crocodilus) and turtle (Trachemys scripta elegans) brains was studied by immunohistochemistry. In both species, distinct groups of FMRFa-like immunoreactive (ir) perikarya were present in the medial septal nucleus, accumbens nucleus, nucleus of the diagonal band of Broca, suprachiasmatic area, lateral hypothalamic area, and periventricular hypothalamic nucleus. A few FMRFa-ir neurons in the hypothalamic area were located in the neuroepithelial cell lining of the third ventricle. FMRFa-ir fibers were scattered in all major areas of the brain, from the olfactory bulbs to the rhombencephalon. They formed dense aggregates in the medial septal area, basal telencephalon, median eminence, and infundibulum, and adjacent to the fourth ventricle. The most obvious difference between the FMRFa-ir systems in caimans and turtles concerned the number of nuclei that contained neurons with this immunoreactivity. Eight such clusters were present in the caiman brain, whereas thirteen clusters were found in the turtle brain. The turtle also displayed scattered FMRFa-ir somata in the anterior olfactory nucleus, striatum, lateral septal nucleus, medial and lateral cortex, medial forebrain bundle, lateral preoptic area, and lateral geniculate nucleus. In the caiman brain, a few FMRFa-ir neurons were noted in the ventrolateral area of the pallial commissure and an even smaller number of ir neurons was found dispersed in the optic tracts. Neither formed nuclear aggregates. The results are compared with those described for other vertebrates.