Lucrezia Mola

Endogenous morphine levels increase in molluscan neural and immune tissues after physical trauma.

The aim of this study was to demonstrate by biochemical and immunocytochemical methods the presence of endogenous morphine in nervous and immune tissues of the freshwater snail, Planorbarius corneus. High performance liquid chromatography (HPLC) coupled to electrochemical detection performed on tissues from control snails, revealed that the CNS contains 6.20+/-2.0 pmol/g of the alkaloid, the foot tissue contains a much lower level, 0.30+/-0.03 pmol/g, whilst morphine is not detected in the hemolymph and hepatopancreas. In specimens that were traumatized, we detected a significant rise of the CNS morphine level 24 h later (43.7+/-5.2 pmol/g) and an initial decrease after 48 h (19.3+/-4.6 pmol/g). At the same times, we found the appearance of the opiate in the hemolymph (0.38+/-0.04 pmol/ml and 0.12+/-0.03 pmol/ml) but not in the hepatopancreas. Using indirect immunocytochemistry, a morphine-like molecule was localized to a number of neurons and a type of glial cell in the CNS, to some immunocytes in the hemolymph and to amoebocytes in the foot, as well as to fibers in the aorta wall. Simultaneously to the rise of morphine biochemical level following trauma, morphine-like immunoreactivity (MIR) increased in both intensity and the number of structures responding positively, i.e., neurons and fiber terminals. In another mollusc, the mussel Mytilus galloprovincialis, the same pattern of enhanced MIR was found after trauma. Taken together, the data suggest the presence of a morphinergic signaling in invertebrate neural and immune processes resembling those of classical messenger systems and an involvement in trauma response.

Factors affecting DNA preservation from museum‐collected lepidopteran specimens

Recent innovations in molecular genetics made DNA an intriguing molecule not only in molecular biology, but also in ecology and evolutionary and conservation biology. Despite this general interest, several discrepancies have been reported in the literature regarding the techniques for preserving insects for DNA analysis, prompting us to analyse the effects of different storage conditions on lepidopteran DNA preservation. In particular, in the present paper, adults of the cabbage moth, Mamestra brassicae (L.) (Lepidoptera: Noctuidae), were stored under various conditions in order to verify which method is the most suitable to preserve lepidopteran specimens for DNA studies. Mamestra brassicae adults were stored by rapid desiccation with silica gel, by preservation in acetone, 2‐propanol, Carnoys or ethanol (both at 75 and 100% concentrations) solutions, and finally by storage in an ultracold freezer and liquid nitrogen. Adults preserved by each method were used to extract DNA at the aim of verifying the size of the extracted DNAs, the extraction yield and the possibility of using these samples to amplify both short and long DNA sequences by polymerase chain reaction. The results were compared with those obtained using fresh samples acting as controls. Acetone preservation appeared to be the most recommendable method for moth specimens as it proved to be a good storage medium for DNA analysis, it is cost‐effective, and it is applicable not only to field surveys, but also to obtain efficient and low‐cost storage of lepidopteran specimens in museum collections.

Survey of neuropeptide-like immunoreactivity in supramedullary neurons of Coris julis (L.)

The supramedullary neurons of the marine teleost Coris julis (L.) were surveyed for neuropeptide-like immunoreactivity using antisera against 12 peptides. These neurons exhibit positive immunoreactivity to CCK-8, CCK-39 and gastrin(18-34). The presence of gastrin/CCK-like peptides in the supramedullary neurons is discussed.

Macroglial cells of the teleost central nervous system: a survey of the main types

Following our previous review of teleost microglia, we focus here on the morphological and histochemical features of the three principal macroglia types in the teleost central nervous system (ependymal cells, astrocyte-like cells/radial glia and oligodendrocytes). This review is concerned with recent literature and not only provides insights into the various individual aspects of the different types of macroglial cells plus a comparison with mammalian glia, but also indicates the several potentials that the neural tissue of teleosts exhibits in neurobiological research. Indeed, some areas of the teleost brain are particularly suitable in terms of the establishment of a “simple” but complete research model (i.e. the visual pathway complex and the supramedullary neuron cluster in puffer fish). The relationships between neurons and glial cells are considered in fish, with the aim of providing an integrated picture of the complex ways in which neurons and glia communicate and collaborate in normal and injured neural tissues. The recent setting up of successful protocols for fish glia and mixed neuron-glia cultures, together with the molecular facilities offered by the knowledge of some teleost genomes, should allow consistent input towards the achievement of this aim.

Endoreplication: a molecular trick during animal neuron evolution.

The occurrence of endoreplication has been repeatedly reported in many organisms, including protists, plants, worms, arthropods, molluscs, fishes, and mammals. As a general rule, cells possessing endoreplicated genomes are largesized and highly metabolically active. Endoreplication has not been frequently reported in neuronal cells that are typically considered to be fully differentiated and nondividing, and which normally contain a diploid genome. Despite this general statement, various papers indicate that giant neurons in molluscs, as well as supramedullary and hypothalamic magnocellular neurons in fishes, contain DNA amounts larger than 2C. In order to study this issue in greater detail here, we review the available data about endoreplication in invertebrate and vertebrate neurons, and discuss its possible functional significance. As a whole, endoreplication seems to be a sort of molecular trick used by neurons in response to the high functional demands that they experience during evolution.

Immunolocalisation of vasoactive intestinal peptide and substance P in the developing gut of Dicentrarchus labrax (L.)

This study was carried out on the sea bass (Dicentrarchus labrax) to follow, during development, the appearance and distribution of substance P (SP) and vasoactive intestinal peptide (VIP), which act on gut motility. The results suggest that SP and VIP play an important role as neuromodulators, influencing the motility of the digestive tract starting from the early stages of gut development, even prior to exotrophic feeding. In the peptidergic nervous system, the appearance of immunoreactivity to SP began at the rectum and followed a distal to proximal gradient, whereas for VIP, it began proximally and progressed along a proximal to distal gradient. The two peptides also appeared in gut epithelial cells. In some regions, all the cells were positive. From this distribution of positive cells, we conclude that these peptides may also have other roles, besides being neurotransmitters in the enteric nervous system and hormones of the gastro-entero-pancreatic system. VIP and SP might have paracrine and/or autocrine activity in the physiological maturation of the gut epithelium, as it has already been hypothesised for other peptides.

Microglia of teleosts: facing a challenge in neurobiology

This review is concerned with recent literature on teleost fish CNS microglia. It covers not only various aspects of these cells, notably comparing them with mammalian microglia, but also points out the several potentialities neural tissue of teleosts exhibits in neurobiological research. The relationships between neurons and glial cells are considered in fish, aiming at an integrated picture of the complex ways neurons and glia communicate and collaborate in normal and injured neural tissues. In addition, attention has been paid to different teleost models according to their availability, easy maintenance in experimental conditions, possibilities of embryos manipulation and sequenced genome. The recent setting up of successful protocols for fish glia and mixed neuron-glia cultures, together with the molecular facilities offered from genome knowledge, should provide a new boost to studies about microglia and neuron-microglia relationships.

Histochemical and immunocytochemical localization of nitric oxide synthase in the supramedullary neurons of the pufferfish Tetraodon fluviatilis

The presence of the nitric oxide (NO) converting enzyme, constitutive neuronal NO synthase (nNOS), was investigated in the supramedullary neurons (SN) cluster of the pufferfish Tetraodon fluviatilis. The identification of NADPH diaphorase- (NADPHd-) positivity and the demonstration of nNOS with the BAS technique and with immunofluorescence together, strongly indicate the presence of a constitutive NO converting enzyme in SN cellular bodies and axons, and provides evidence that the SN cluster represents a distinct nitrergic neuronal system in the vertebrate CNS. The possible roles of NO in the cluster are discussed, including an involvement in communication among neurons and between neurons and the glial cells in the cluster.

Neuroanatomical and immunohistochemical studies on the dorsal neurons in the spinal cord of Trigla lucerna L. and Scorpaena porcus L. (Scorpaeniformes)

Peculiar dorsal neurons present in the dorsal spinal cord of Trigla lucerna L. and Scorpaena porcus L. were investigated by neuroanatomical and immunohistochemical methods. These neurons were previously defined as commissural cells, but have now been identified as supramedullary neurons. The following fundamental criteria for identifying the supramedullary neurons of Teleosts are proposed: a) dorsomedial location in the spinal cord; b) large size of the soma and axon; c) immunoreactivity to gastrin/CCK-like peptides.

The supramedullary neurons of fish: present status and goals for the future

In this paper, we report the recent findings on supramedullary neurons of fish, with special attention to the studies, which made the nature of this neuronal system clear. Indeed, immunohistochemical, physiological and neuroanatomical data, taken together, point out that this neuronal system is a component of the autonomic nervous system. New goals have been opened by the more recent research, especially in comparative neurobiology. Indeed, the supramedullary neurons, owing to some characteristics, like the DNA endoreplication, the large size, the accessible localization and the relationship with glial cells, may be utilised as a very suitable model in several fields of neurobiology of vertebrates, such as molecular genetic, electrophysiology, nervous system ageing, glial-neuron interactions.