Maddalena Sturla

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.

Immunoreactive localization of vasoactive hormones (atrial natriuretic peptide and endothelin) in the heart of Protopterus annectens, an African lungfish

Abstract.The present study demonstrated, by immunohistochemistry and Western blotting, the presence of immunoreacting atrial natriuretic peptide (ANP) and endothelin in the heart of Protopterus annectens by both light and electron microscopy. The distribution of ANP granules was investigated. ANP granules were localised in myocytes from the atrium, ventricle and conus arteriosus; endothelin-1 (ET-1) was demonstrated in subendocardial myocytes of the atrium and the conus. No ET-1 immunoreactivity was observed in the ventricle wall. At the light-microscopical level, ET-1 appeared to occur in the endocardium, but at the electron-microscopical level no immunogold labelling was seen on the granules of the endocardial cells. It is suggested that ET-1 is produced and stored in the subendocardial cells and released into the subendocardial space to reach the ANP-producing myocytes and the endothelial cells.

Localization of interleukin-1β mRNA in the cerebral ganglion of the protochordate, Styela plicata

There is evidence that interleukin 1 beta (IL-1 beta) plays an important role in several biological functions in mammals where it is synthesized by cells of haematological, dermal and neural origin. Moreover, production of cytokine-like molecules has been demonstrated in some blood cells of non-mammalian vertebrates and invertebrates in which also nerve cells are demonstrated to be IL-1 beta immunoreactive. The purpose of the present study is to demonstrate the IL-1 beta mRNA expression in nerve cells of the ascidian Styela plicata by use of non-isotopic in situ hybridization technique. The expression of IL-1 beta messenger was demonstrated in monopolar neurons in the cortical layer of the cerebral ganglion. The neuronal expression of cytokine-like molecules in tunicates suggests that IL-1 beta is an ancestral and functionally conserved molecule, and that a neuroimmune axis appeared early during the metazoan phylogeny.

Ion transport systems in the kidney and urinary bladder of two Antarctic teleosts, Chionodraco hamatus and Trematomus bernacchii

Abstract. Na+-K+-Cl- cotransport and Na+/K+ATPase were studied by immunohistochemistry in the kidney and urinary bladder of Trematomus bernacchii and Chionodraco hamatus. The activity was correlated to the density of mitochondria. The first segment of the renal proximal tubule was more active than the second one. In T. bernacchii and the temperate marine teleost Pagellus bogaraveo, the immunoreactivity for the antibody to cotransporters and to the α-subunit of the sodium pump was stronger than in the icefish. This difference indicates in the kidney of the icefish, a weaker secretory activity, a consequent lower osmolarity in the lumen and lower water loss, which correlates well with the need for a greater blood volume in the icefish. The epithelium of the urinary bladder in T. bernacchii, where intense immunostaining was observed, was composed of columnar cells. In C. hamatus the columnar cells, where the immunostaining was weaker, lined only a portion of the urinary bladder, the other region being composed of cuboidal cells.

Immunoreactive atrial natriuretic peptide and autoradiographic distribution of atrial natriuretic peptide binding sites in the brain of the Antarctic fish, Chionodraco hamatus

Abstract The anatomical distribution of atrial natriuretic peptide (ANP)-immunoreactive structures and the autoradiographic localization of ANP binding sites were studied in the brain of the Antarctic fish, Chionodraco hamatus. ANP-containing elements were colocated with ANP binding sites in the dorsal medial and lateral subdivisions of the telencephalon, prethalamic nuclear complex, and in the nucleus of the medial longitudinal fasciculus of the mesencephalon. However, mismatching was observed in other brain regions, particularly at mesencephalic and metencephalic levels. In the pituitary, ANP immunoreactivity occurred only in the pars distalis, whereas ANP binding sites were localized in the whole pituitary. In this paper we describe the occurrence of ANP immunoreactivity and ANP binding sites in the brain and pituitary of an Antarctic fish. In particular, in the cerebellum and pituitary of C. hamatus, ANP binding sites are distributed in corresponding brain regions of dipnoans, amphibians and mammals. The immunocytochemical and histoautoradiographic data suggest that ANP acts as neuromodulator in the brain of C. hamatus. Moreover, the presence of ANP-like substances in tanycytes lining the diencephalic ventricle suggests a chemosensorial role for such liquor-contacting cells and a possible modulatory effect of ANP on the osmoregulation of the cerebrospinal fluid.

Key enzymes of the kallikrein-kinin system in Antarctic teleosts

Abstract In this study, some of the mammalian kallikrein-kinin system (KKS)-like components were identified in two species of Antarctic notothenioid [Chionodraco hamatus (Channichthydae) and Trematomus bernacchii (Nothothenidae)]. The kidney and heart were assayed for kallikrein-like activity using the synthetic substrate d-Val-Leu-Arg-paranitroanilide. Values expressed as nmol p-nitroanilide/mg proteins, were in C. hamatus 15.5±1.5 and 15.2±1.4 in kidney and heart, respectively, and 15.8±2.2 and 15.9±1 in kidney and heart of T. bernacchii. Kallikrein-like activity was inhibited bvy aprotinin and phenylmethylsulfonyl fluoride (PMSF). The assay was stable at 20°C. Kininase II-like activity was performed on kidney, gills and heart using the substrate hippuryl-lhistidyl-l-leucine. The activity was inhibited by captopril, and in kidney and gills by high temperatures (20°C and 37°C); in the heart the enzymatic activity was measurable also at 20°C. Bradykinin-like immunoreactive cells were localized by immunohistochemistry in the nephron, in the gills, and in the arterial walls of the heart. These results show that Antarctic teleosts possess elements comparable to those of the KKS, including kallikrein-like, and kininase II-like activities, and the end product of the enzymatic cascade, bradykinin. The enzymatic cascade appears to be fully active only at low temperatures.

Identification and distribution of nitric oxide synthase in the brain of adult Antarctic teleosts

We investigated the presence of nitric oxide synthase (NOS) in brain of adult Antarctic teleosts by indirect immunofluorescence technique using a synthetic rat neuronal NOS (nNOS) antibody. The following species were examined: Trematomus bernacchii, Gymnodraco acuticeps, Histiodraco velifer, Cygnodraco mawsoni (haemoglobin-rich), Chionodraco hamatus and Pagetopsis macropterus (haemoglobin-free). Immunoreactive cell bodies were localized in dorsal telencephalon, in hypothalamus, in optic tectum of the mesencephalon as well as in Purkinje cells of the cerebellum. No differences were observed in the localization of the nNOS immunopositivity in the Antarctic teleosts brains examined and NOS distribution was similar to that described in other teleosts, suggesting that nitric oxide (NO) may also function as a neurotransmitter in the brain of Antarctic teleosts. A strong immunopositivity was observed in the cerebral blood vessels of the icefishes suggesting that NO may play a pivotal role in the regulation of the cerebral blood flow especially in these haemoglobin-free species.

Atrial Natriuretic Peptides in Antarctic Fish

Atrial natriuretic factors (ANFs) belong to a family of peptides originally described in mammalian heart. Beside ANPs, two other different types of natriuretic peptides with high sequence similarity have been identified, B-type natriuretic peptide (BNP) in the heart of chicken and mammals, and C-type natriuretic peptide (CNP) in the brain [1]. Their physiological and pharmacological effects have been extensively studied, their structure is known [2,3], their primary effects in mammals are natriuretic, diuretic and vasorelaxant [4,5]. ANFs are also present in nonmammalian vertebrates including birds [6], reptiles [7,8], amphibians [1,9,10], jawed [1,7,11,12] and jawless fishes [13]. Moreover, there are specific binding sites for ANP in several vertebrates’ tissues. Extracts from fish heart stimulate ion excretion in the trout [14] and are vasodepressor in the quail [15]. In addition, rat ANP stimulates chloride secretion from the killifish opercular membrane in vitro [16]. Together these data indicate that ANFs regulate body water, electrolyte balance and cardiovascular dynamics in vertebrates. To inhabit deep cold water, Antarctic and subAntarctic fish have developed remarkable cardiovascular adaptations like reduction of the number of red blood cells: this adaptation is extreme in Channichthyidae which have erythrocyte-free blood. To compensate for the lack of hemic pigments, the icefish possess a hypertrophic heart [17,18] that generates a high cardiac output [19]; the blood volume is also very large [20].

Regulatory peptides and physiological adaptations to the cold environment in Antarctic teleosts

Abstract Physiological adaptation of Antarctic teleosts to cold environment was studied with regard to the presence of regulatory peptides involved in (i) cardiac and ion‐water homeostasis, (ii) brain, (iii) intestine, and (iv) gonads. Immunoreactivity for different regions of the Atrial Natriuretic Peptide was detected in the heart of Trematomus bernacchii, Chionodraco hamatus, Cryodraco antarcticus, and Champsocephalus gunnari, while immunoreactivity for other regulatory peptides (i.e., endothelin‐1, somatostatin‐14, and galanin) appeared to be widely distributed in the cardio‐vascular system. Osmoregulatory peptides (somatostatin‐14, prolactin, atrial natriuretic peptide, galanin, and urotensin II) were localised by immunohistochemistry in the urinary bladder and gills. From studies on the distribution of the pituitary specific transcription factor 1 and adenohypophysial hormones (growth hormone, prolactin, and gonadotropin releasing hormone) in the brain and pituitary of Trematomus bernacchii and Pagothenia coriiceps, it was evident that the pituitary specific transcription factor 1 acts as a developmental regulator of the anterior pituitary, responsible for growth hormone and prolactin cell commitment, differentiation, and gene expression. This coexistence appeared to be a unique trait of Antarctic teleosts. Analyses of the distribution and localisation of gastrointestinal hormones and neuropeptides (insulin, glucagon, pancreatic polypeptide, somatostatin, vasoactive intestinal polypeptide, pituitary adenylate cyclase activating peptide, and peptide histidine isoleucine) in adults of Chionodraco hamatus, Pagetopsis mascropterus, and Notothenia coriiceps, and larvae of Pleuragramma antarcticum showed that most of the immunoreactive nerve fibres in Notothenioids are of extrinsic type. Ultrastructural analysis of Chionodraco hamatus, Champsocephalus gunnari, and Notothenia coriiceps male gametes showed some peculiar aspects in the head of the spermatozoa. Studies on the spermatogenesis indicated a shifted circannual cycle in the icefishes and in the red blooded fishes: when spermato‐genensis is completed in the icefish, in the red blooded teleosts the testis is not in reproductive activity as shown by the positive immunoreaction for Fibroblast Growth Factor and its receptor 1 in spermatogonia and Sertoli cells of Chionodraco hamatus and the lack of positivity in Trematomus bernacchii.

Immunohistochemical localisation of FMRF-amide-like peptide in the brain of icefish and red-blooded Antarctic Teleosts

The distribution of Phe-Met-Arg-Phe-NH2 (FMRF-amide) -like immunoreactivity was investigated by indirect immunofluorescence technique using the molluscan FMRF-amide antibody in the brain of icefish (Pagetopsis macropterus and Chionodraco hamatus used as positive control) and red blooded (Trematomus bernacchii, Gymnodraco acuticeps, Histiodraco velifer, Cygnodraco mawsoni) Antarctic Teleosts. Immunoreactive perikarya were localised in the ventral thalamus, in the hypothalamus (preoptic and periventricular regions) and in the intermedioventral rhombencephalon (vagal motor nucleus) as well as in the telencephalon and in the mesencephalon. Positive nerve fibres were seen to project towards the caudal brainstem to reach the rhombencephalon. No differences were observed in the immunopositivity of FMRF-amide new distribution in the Antarctic Teleosts examined. In the icefishes the immunoreaction was stronger than in the hemoglobin-rich Teleosts. The distribution patterns of the FMRF-amide immunostaining suggest that this peptide may play a pivotal role in the cardiovascular regulation in the Antarctic Teleosts.