Effects of nanosilver exposure on cholinesterase activities, CD41, and CDF/LIF-like expression in zebrafish (Danio rerio) larvae.

Metal nanosolicoparticles are suspected to cause diseases in a number of organisms, including man. In this paper, we report the effects of nanosilver (Ag, 1-20 nm particles) on the early development of the zebrafish, a well-established vertebrate model. Embryos at the midgastrula stage were exposed to concentrations ranging from 100 to 0.001 mg/L to verify the effects on different endpoints: lethality, morphology, expression of cholinergic molecules, and development of the immune system. (1) Relative risk of mortality was exponential in the range between 0.001 and 10 mg/L. Exposure to 100 mg/L caused 100% death of embryos before reaching the tail-bud stage. (2) Developmental anomalies were present in the 72 h larvae obtained from embryos exposed to nanosilver: whole body length, decreased eye dimension, and slow response to solicitation by gentle touch with a needle tip, with a significant threshold at 0.1 mg/L. (3) Dose-dependent inhibition of acetylcholinesterase activity was significant among the exposures, except between 1 mg/L and 10 mg/L. (4) The distribution of CD41+ cells and of CDF/LIF-like immunoreactivity was altered according to the Ag concentration. The possible effect of nanosilver in impairing immune system differentiation through the inhibition of molecules related to the cholinergic system is discussed.

Immunolocalization of G protein α subunits in the olfactory system of Polypterus senegalus (Cladistia, Actinopterygii).

In vertebrates, the receptor neurons of the olfactory/vomeronasal systems express different receptor gene families and related G-protein types (in particular the G protein alpha subunit). There are no data in the literature about the molecular features of the olfactory/vomeronasal systems of Cladistia thus, in this work, the presence and distribution of different types of G protein alpha subunits were investigated in the olfactory organs of the bichir Polypterus senegalus, using immunohistochemistry. Gαo-like immunoreactivity was detected in the microvillous receptor neurons, with the cell body in the basal zone of the sensory epithelium, and in the crypt neurons. Gαo-like ir glomeruli were mainly localized in the anterior part of the olfactory bulb. Gαolf-like immunoreactivity in the sensory epithelium was detected in the ciliated receptor neurons, while the immunoreactive glomeruli in the olfactory bulb were mainly localized in the ventral-posterior part. No Gαq nor Gαi3 immunoreactivity was detected. These data are partially in agreement with studies that show the distribution of G protein alpha subunits in teleosts, allowing to hypothesize a common organization of the olfactory/vomeronasal systems in the group of Actinopterigians.

First detection of neuropeptide Y (NPY)-like immunoreactivity in the lateral line: presence and distribution in the neuromasts of the Antarctic notothenioid fish Trematomus bernacchii.

The mechanosensory lateral line (LL) is involved in many fish and amphibian behaviors, however little is known about the molecules involved in the signal transmission. Neuropeptide Y (NPY) has a number of functions in vertebrate physiology and also plays important roles in different sensory systems. The Antarctic nototheniods are a monophyletic radiation of fishes that have evolved under the extreme environmental conditions of low light and cold, where non-visual sensory structures, such as LL, are of importance. In this study we describe the presence of NPY-like immunoreactivity (IR) in LL of the Antarctic nototheniod fish, Trematomus bernacchii Boulenger. Differences in size and cellular composition between the two neuromasts were in compliance with previous descriptions of these sensory organs. Despite structural and functional differences between canal and superficial neuromasts, the distribution of NPY-like IR was similar within both the receptors classes. In particular, NPY IR was observed in all three cell types which constitute these sensory organs, allowing us to hypothesize the involvement of this molecule in the processing of the sensory information.

Gamma-aminobutyric acid and related molecules in the sea fan Eunicella cavolini (Cnidaria: Octocorallia): a biochemical and immunohistochemical approach.

The aim of this study has been the biochemical demonstration of the presence of gamma-aminobutyric acid (GABA) in the Mediterranean sea fan Eunicella cavolini by means of high-performance liquid chromatography, and the description of the distribution pattern of GABA and its related molecules, glutamic acid decarboxylase (GAD), vesicular GABA transporter (VGAT) and one of the GABA receptors (GABA(B) R) by immunohistochemical methods. The interrelationships of GABA, GAD and GABA receptor immunoreactivity have been established by using double-immunohistochemical methods and confocal microscopy. The immunodetection of monoclonal and/or polyclonal antibodies has revealed GABA immunoreactivity throughout the polyp tissue, both in neuronal and non-neuronal elements. GAD immunoreactivity has been mostly localized in the neuronal compartment, contacting epithelial and muscular elements. GABA(B) R immunoreactivity appears particularly intense in the nematocytes and in the oocyte envelope; its presence in GAD-immunoreactive neurons in the tentacles suggests an autocrine type of regulation. Western blot analysis has confirmed that a GABA(B) R, with a molecular weight of 142 kDa, similar to that of rat brain, is present in E. cavolini polyp tissue. The identification of the sites of the synthesis, vesicular transport, storage and reception of GABA strongly suggests the presence of an almost complete set of GABA-related molecules for the functioning of the GABAergic system in this simple nervous system. The distribution of these different immunoreactivities has allowed us to hypothesize GABA involvement in nematocyst discharge, in body wall and enteric muscular contraction, in neuronal integration and in male gametocyte differentiation.

Clinorotation-induced weightlessness influences the cytoskeleton of glial cells in culture.

During and after spaceflight astronauts experience neurophysiological alterations. To investigate if the impairment observed might be traced back to cytomorphology, we undertook a ground based research using a random positioning machine (clinostat) as a simulation method for microgravity. The outcome of the study was represented by cytoskeletal changes occurring in cultured glial cells (C(6) line) after 15 min, 30 min, 1 h, 20 h and 32 h under simulated microgravity. Glia is fundamental for brain function and it is essential for the normal health of the entire nervous system. Our data showed that after 30 min under simulated microgravity the cytoskeleton was damaged: microfilaments (F-actin) and intermediate filaments (Vimentin, Glial Fibrillary Acidic Proteins GFAP) were highly disorganised, microtubules (alpha-tubulin) lost their radial array, the overall cellular shape was deteriorated, and the nuclei showed altered chromatin condensations and DNA fragmentation. This feature got less dramatic after 20 h of simulated microgravity when glial cells appeared to reorganise their cytoskeleton and mitotic figures were present. The research was carried out by immunohistochemistry using antibodies to alpha-tubulin, vimentin and GFAP, and cytochemical labelling of F-actin (Phalloidin-TRIC). The nuclei were stained with propidium iodide or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). The cells were observed at the conventional and/or the confocal laser scanning microscope. Samples were also observed at the scanning electron microscope (SEM). Our data showed that in weightlessness alterations occur already visible at the scale of the single cell; if this may lead to the neurophysiological problems observed in flight is yet to be established.