Gianluca Polese

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.

Frogs, sentinels of DNA damage induced by pollution in Naples and the neighbouring provinces.

Many DNA mutation-based diseases recognised in Campania have recently been related to toxic substances in illegal dumping areas. We performed a comet assay on edible frog erythrocytes to evaluate DNA damage. Differences in genotoxic parameters were observed among populations. We show that severe DNA damage occurred in the north Campania where the emergence of environmental waste exploded recently. Although a similar magnitude of genotoxic damage was observed in some southern populations, it is attributable to a massive pesticide pollution related to intensive farming. The frog species analysed seems to be a good bioindicator for detecting genotoxic effects of chemical environmental hazards.

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.

Sensing marine biomolecules: smell, taste, and the evolutionary transition from aquatic to terrestrial life.

The usual definition of smell and taste as distance and contact forms of chemoreception, respectively, has resulted in the belief that, during the shift from aquatic to terrestrial life, odorant receptors (ORs) were selected mainly to recognize airborne hydrophobic ligands, instead of the hydrophilic molecules involved in marine remote-sensing. This post-adaptive evolutionary scenario, however, neglects the fact that marine organisms 1) produce and detect a wide range of small hydrophobic and volatile molecules, especially terpenoids, and 2) contain genes coding for ORs that are able to bind those compounds. These apparent anomalies can be resolved by adopting an alternative, pre-adaptive scenario. Before becoming airborne on land, small molecules, almost insoluble in water, already played a key role in aquatic communication, but acting in “contact” forms of olfaction that did not require major molecular innovations to become effective at a distance in air. Rather, when air was “invaded” by volatile marine terpenoids, an expansion of the spatial range of olfaction was an incidental consequence rather than an adaptation.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Ontogenetic organization of the FMRFamide immunoreactivity in the nervus terminalis of the lungfish, Neoceratodus forsteri

The development of the nervus terminalis system in the lungfish, Neoceratodus forsteri, was investigated by using FMRFamide as a marker. FMRFamide immunoreactivity appears first within the brain, in the dorsal hypothalamus at a stage around hatching. At a slightly later stage, immunoreactivity appears in the olfactory mucosa. These immunoreactive cells move outside the olfactory organ to form the ganglion of the nervus terminalis. Immunoreactive processes emerge from the ganglion of the nervus terminalis in two directions, one which joins the olfactory nerve to travel to the brain and the other which courses below the brain to enter at the level of the preoptic nucleus. Neither the ganglion of the nervus terminalis nor the two branches of the nervus terminalis form after surgical removal of the olfactory placode at a stage before the development of FMRFamide immunoreactivity external to the brain. Because this study has confirmed that the nervus terminalis in lungfish comprises both an anterior and a posterior branch, it forms the basis for discussion of homology between these branches and the nervus terminalis of other anamniote vertebrates. J. Comp. Neurol. 450:115–121, 2002.

Morphofunctional characterization and antibacterial activity of haemocytes from Octopus vulgaris

This study focused on the morphological and functional characterization of the haemocytes from Octopus vulgaris as the first agents responsible for innate immunity. Three major haemocytes types were identified by light microscopy based on nucleus/cytoplasm ratio and the presence or absence of cytoplasm granules: haemoblast-like cells, hyalinocytes and granulocytes. The presence of three haemocyte populations was also confirmed by flow cytometry. Cytochemical characterization suggests that they perform different activities during humoral responses. Using the plate radial diffusion method we demonstrated the bactericidal activity of haemocytes in the presence of different bacteria strains. To investigate the presence of soluble biotic compounds responsible for antibacterial activity, using a disc diffusion method and the minimum inhibitory concentration, we have also tested the methanolic acid extract from these cells. Our results pave the way for the development of potent antibacterial drugs that could lead to several applications.

Olfactory organ of Octopus vulgaris: morphology, plasticity, turnover and sensory characterization

ABSTRACT The cephalopod olfactory organ was described for the first time in 1844 by von Kölliker, who was attracted to the pair of small pits of ciliated cells on each side of the head, below the eyes close to the mantle edge, in both octopuses and squids. Several functional studies have been conducted on decapods but very little is known about octopods. The morphology of the octopus olfactory system has been studied, but only to a limited extent on post-hatching specimens, and the only paper on adult octopus gives a minimal description of the olfactory organ. Here, we describe the detailed morphology of young male and female Octopus vulgaris olfactory epithelium, and using a combination of classical morphology and 3D reconstruction techniques, we propose a new classification for O. vulgaris olfactory sensory neurons. Furthermore, using specific markers such as olfactory marker protein (OMP) and proliferating cell nuclear antigen (PCNA) we have been able to identify and differentially localize both mature olfactory sensory neurons and olfactory sensory neurons involved in epithelium turnover. Taken together, our data suggest that the O. vulgaris olfactory organ is extremely plastic, capable of changing its shape and also proliferating its cells in older specimens. Summary: A 3D reconstruction of the Octopus vulgaris olfactory organ, as well as the localization of PCNA and (for the first time) OMP-like proteins in cephalopod mature olfactory chemosensory neurons are described.

Volatile secondary metabolites as aposematic olfactory signals and defensive weapons in aquatic environments

Significance This report gives empirical evidence indicating that chemoreception of volatile/odorant lipophilic compounds, almost insoluble in water, can occur in aquatic environments, by means of “tactile” forms of olfaction. This thesis has been proved by exploring the defensive role of terpenes isolated from benthic invertebrates. The isolated metabolites were found to act both as defensive toxic weapons and as olfactory signals. In addition, the most abundant compound induced avoidance learning in crustaceans and fish that experienced postingestive negative effects. Olfaction is considered a distance sense; hence, aquatic olfaction is thought to be mediated only by molecules dissolved in water. Here, we challenge this view by showing that shrimp and fish can recognize the presence of hydrophobic olfactory cues by a “tactile” form of chemoreception. We found that odiferous furanosesquiterpenes protect both the Mediterranean octocoral Maasella edwardsi and its specialist predator, the nudibranch gastropod Tritonia striata, from potential predators. Food treated with the terpenes elicited avoidance responses in the cooccurring shrimp Palaemon elegans. Rejection was also induced in the shrimp by the memory recall of postingestive aversive effects (vomiting), evoked by repeatedly touching the food with chemosensory mouthparts. Consistent with their emetic properties once ingested, the compounds were highly toxic to brine shrimp. Further experiments on the zebrafish showed that this vertebrate aquatic model also avoids food treated with one of the terpenes, after having experienced gastrointestinal malaise. The fish refused the food after repeatedly touching it with their mouths. The compounds studied thus act simultaneously as (i) toxins, (ii) avoidance-learning inducers, and (iii) aposematic odorant cues. Although they produce a characteristic smell when exposed to air, the compounds are detected by direct contact with the emitter in aquatic environments and are perceived at high doses that are not compatible with their transport in water. The mouthparts of both the shrimp and the fish have thus been shown to act as “aquatic noses,” supporting a substantial revision of the current definition of the chemical senses based upon spatial criteria.

Neuroanatomical relationships between FMRFamide- immunoreactive components of the nervus terminalis and the topology of olfactory bulbs in teleost fish

The nervus terminalis (NT) is the most anterior of the vertebrate cranial nerves. In teleost fish, the NT runs across all olfactory components and shows high morphological variability within this taxon. We compare the anatomical distribution, average number and size of the FMRFamide-immunoreactive (ir) NT cells of fourteen teleost species with different positions of olfactory bulbs (OBs) with respect to the ventral telencephalic area. Based on the topology of the OBs, three different neuroanatomical organizations of the telencephalon can be defined, viz., fish having sessile (Type I), pseudosessile (short stalked; Type II) or stalked (Type III) OBs. Type III topology of OBs appears to be a feature associated with more basal species, whereas Types I and II occur in derived and in basal species. The displacement of the OBs is positively correlated with the peripheral distribution of the FMRFamide-ir NT cells. The number of cells is negatively correlated with the size of the cells. A dependence analysis related to the type of OB topology revealed a positive relationship with the number of cells and with the size of the cells, with Type I and II topologies of OBs showing significantly fewer cells and larger cells than Type III. A dendrogram based on similarities obtained by taking into account all variables under study, i.e., the number and size of the FMRFamide-ir NT cells and the topology of OBs, does not agree with the phylogenetic relationships amongst species, suggesting that divergent or convergent evolutionary phenomena produced the olfactory components studied.