Acute environmental temperature variation affects brain protein expression, anxiety and explorative behaviour in adult zebrafish.

This study investigated the effect of 4-d acute thermal treatments at 18 °C, 26 °C (control) and 34 °C on the nervous system of adult zebrafish (Danio rerio) using a multidisciplinary approach based on behavioural tests and brain proteomic analysis. The behavioural variations induced by thermal treatment were investigated using five different tests, the novel tank diving, light and dark preference, social preference, mirror biting, and Y-Maze tests, which are standard paradigms specifically tailored for zebrafish to assess their anxiety-like behaviour, boldness, social preference, aggressiveness, and explorative behaviour, respectively. Proteomic data revealed that several proteins involved in energy metabolism, messenger RNA translation, protein synthesis, folding and degradation, cytoskeleton organisation and synaptic vesiculation are regulated differently at extreme temperatures. The results showed that anxiety-like behaviours increase in zebrafish at 18 °C compared to those at 26 °C or 34 °C, whereas anxiety-related protein signalling pathways are downregulated. Moreover, treatments at both 18 °C and 34 °C affect the exploratory behaviour that appears not to be modulated by past experiences, suggesting the impairment of fish cognitive abilities. This study is the continuation of our previous work on the effect of 21-d chronic treatment at the same constant temperature level and will enable the comparison of acute and chronic treatment effects on the nervous system function in adult zebrafish.

Increase in environmental temperature affects exploratory behaviour, anxiety and social preference in Danio rerio.

The aim of this work is to investigate the effect of a temperature increase on the behaviour of adult zebrafish (Danio rerio) maintained for 21 days at 34 °C (treatment) and 26 °C (control). The temperatures chosen are within the vital range of zebrafish and correspond to temperatures that this species encounters in the natural environment. Previous results showed that the same treatment affects the brain proteome and the behaviour of adult zebrafish by producing alterations in the proteins involved in neurotransmitter release and synaptic function and impairing fish exploratory behaviour. In this study, we have investigated the performance of treated and control zebrafish during environmental exploration by using four behavioural tests (novel tank diving, light and dark preference, social preference and mirror biting) that are paradigms for assessing the state of anxiety, boldness, social preference and aggressive behaviour, respectively. The results showed that heat treatment reduces anxiety and increases the boldness of zebrafish, which spent more time in potentially dangerous areas of the tank such as the top and the uncovered bright area and at a distance from the social group, thus decreasing protection for the zebrafish. These data suggest that the increase in ambient temperature may compromise zebrafish survival rate in the natural environment.

Review: Assessing fish welfare in research and aquaculture, with a focus on European directives.

The number of farmed fish in the world has increased considerably. Aquaculture is a growing industry that will in the future provide a large portion of fishery products. Moreover, in recent years, the number of teleost fish used as animal models for scientific research in both biomedical and ecological fields has increased. Therefore, it is increasingly important to implement measures designed to enhance the welfare of these animals. Currently, a number of European rules exist as requirements for the establishment, care and accommodation of fish maintained for human purposes. As far as (teleost) fish are concerned, the fact that the number of extant species is much greater than that of all other vertebrates must be considered. Of further importance is that each species has its own specific physical and chemical requirements. These factors make it difficult to provide generalized recommendations or requirements for all fish species. An adequate knowledge is required of the physiology and ecology of each species bred. This paper integrates and discusses, in a single synthesis, the current issues related to fish welfare, considering that teleosts are target species for both aquaculture and experimental models in biological and biomedical research. We first focus on the practical aspects, which must be considered when assessing fish welfare in both research and aquaculture contexts. Next, we address husbandry and the care of fish housed in research laboratories and aquaculture facilities in relation to their physiological and behavioural requirements, as well as in reference to the suggestions provided by European regulations. Finally, to evaluate precisely which parameters described by Directive 2010/63/EU are reported in scientific papers, we analysed 82 articles published by European researchers in 2014 and 2015. This review found that there is a general lack of information related to the optimal environmental conditions that should be provided for the range of species covered by this directive.

Environmental temperature variation affects brain protein expression and cognitive abilities in adult zebrafish (Danio rerio): A proteomic and behavioural study.

Water temperature is an important environmental parameter influencing the distribution and the health of fishes and it plays a central role in ectothermic animals. The aim of this study is to determine the effects of environmental temperature on the brain proteome and the behavioural responses in zebrafish, a widely used animal model for environmental "omics" studies. Adult specimens of wild-type zebrafish were kept at 18 °C, 34 °C and 26 °C (control) for 21 days. Proteomic data revealed that several proteins involved in cytoskeletal organization, mitochondrial regulation and energy metabolism are differently regulated at the extreme temperatures. In particular, the expression of proteins associated to synapses and neurotransmitter release is down-regulated at 18 °C and 34 °C. In both thermal conditions, fish exhibited a reduced interest for the novel environment and an impairment of cognitive abilities during Y-Maze behavioural tests. The observed pathways of protein expression are possibly associated to functional alterations of the synaptic transmission that may result in cognitive functions impairment at central nervous system level as those revealed by behavioural tests. This study indicates that temperature variations can elicit biochemical changes that may affect fish health and behaviour. This combined approach provides insights into mechanisms supporting thermal acclimation and plasticity in fishes. SIGNIFICANCE: Environmental temperature variation may impact on all levels of biological life. Understanding the impact of thermal variation on the nervous system and animal behaviour is of primary importance since the results obtained can be applied from the ecological to the biomedical fields.

HIV-1 gp120 increases the permeability of rat brain endothelium cultures by a mechanism involving substance P.

OBJECTIVE: To analyse whether an HIV-1 envelope protein might play a role in damaging the blood-brain barrier as a fundamental step in the early invasion of the central nervous system by HIV-1. DESIGN: Analysis of permeability of rat brain endothelium cultures to albumin, to assess the functional integrity of the vascular component of the blood-brain barrier. METHODS: Rat brain endothelium cultures prepared by cerebral microvessels were exposed to recombinant gp120IIIB on microporous membranes and passage of biotin-labelled albumin was analysed. Scanning electron microscopy was used to analyse cell culture morphology. Some cultures were preincubated with N-nitro-L-arginine methyl ester (L-NAME), a selective inhibitor of nitric oxide synthase, or with spantide, a selective substance P antagonist. RESULTS: HIV-1 gp120 increased the permeability of rat brain endothelial cells to albumin in a dose-dependent manner. Scanning electron microscopy revealed profound gp120-induced alterations in cell morphology accounting for the increased permeability to macromolecules. These alterations were neutralized by anti-gp120 monoclonal antibody but not by isotype control antibody or L-NAME. By contrast, spantide and anti-substance P polyclonal antibody completely blocked the gp120-induced increase in albumin permeability. Control cultures exposed to measles virus nucleoprotein showed an increase in permeability that was not blocked by spantide. Brain endothelial cells, exposed to gp120, displayed cell surface immunoreactivity for substance P, suggesting that substance P is secreted by brain endothelium in response to gp120 stimulation and binds to brain endothelial cells through a receptor-mediated mechanism. CONCLUSIONS: These findings suggest a role for substance P in the gp120-induced increase in permeability of brain endothelium.

Stimulated growth hormone (GH) secretion in children with delays in pubertal development before and after the onset of puberty: relationship with peripheral plasma GH-releasing hormone and somatostatin levels.

A reduced GH secretion has often been shown in prepubertal children with delays in pubertal development. In order to study the mechanism underlying this finding, we evaluated peripheral circulating levels of GH, GHRH, and somatostatin (SRIH) before and after the onset of sexual development in a group of eight late maturing children (six boys, two girls), comparing the results with those obtained in two groups of five prepubertal and four pubertal short children with familial short stature. GH was measured by a two-site immunoradiometric assay. Both GHRH and SRIH were assayed by specific RIAs after an acetone-petrolether extraction from plasma. Our data showed a significant increase (P less than 0.001) in GH, GHRH, and SRIH levels (peak vs. basal values) in response to L-dopa administration in all groups. In pubertal children with delays in pubertal development GH and GHRH peak values (15.8 +/- 2.2 micrograms/L and 120 +/- 18 pg/mL, respectively) were significantly greater (P less than 0.001) than in the same subjects before puberty (8.2 +/- 0.9 micrograms/L and 79 +/- 9 pg/mL, respectively), whereas SRIH peak values did not significantly change (41 +/- 6 vs. 41 +/- 5 pg/mL; P = NS). Furthermore, prepubertal subjects with delays in pubertal development showed GH and GHRH peak values lower (P less than 0.001) than those of prepubertal subjects with FSS (13.3 +/- 1.8 micrograms/L and 120 +/- 13 pg/mL, respectively), whereas no statistical difference was present between the two groups of subjects after pubertal development (18.2 +/- 2.9 micrograms/L and 128 +/- 11 pg/mL, respectively). In conclusion, these findings support the assumption that in late maturing subjects during prepubertal period the decreased GH secretion may be ascribed to a reduced GHRH secretion, reversible with the onset of puberty, without change in circulating SRIH levels.

Morphology of afferent synapses in the Mauthner cell of larval Xenopus laevis.

The fine structure of the afferent synapses on the Mauthner cell of larval Xenopus laevis has been studied as a first step toward comparing the fine structure of the afferent synaptic apparatus before and after metamorphosis. There are various types of afferent endings on this cell, some of which are confined to specific cellular regions, while others are distributed over most of the large surface of the neuron. Four different main types of endings have been observed: club endings, round-vesicle end bulbs, flattened-vesicle end bulbs and spiral fibers endings. While the myelinated club endings and the spiral fibers endings are located at the distal end of the lateral dendrite and in the axon cap, respectively, the end bulbs are widely distributed over the whole cell. A further type of ending has been observed, although rarely, on the Mauthner cell soma and dendrites: end bulbs characterized by an unusually dense presynaptic substance. Results obtained in the present research suggest that, as in fish, different endings on the anuran Mauthner neuron correspond to different synaptic inputs. The possible origin of some of these inputs is discussed.

Enhanced secretion of substance P by cytokine-stimulated rat brain endothelium cultures.

Substance P (SP) was analyzed in rat brain endothelium cultures after cytokine stimulation. SP secretion was found after stimulation with high doses of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). High doses of interferon-gamma (INF-gamma) had no effect on this secretion. Elevated SP release was found to be associated with mRNA expression of beta-preprotachykinin (beta-PPT), precursor of SP, in the cells. Under cytokine stimulation, part of SP was bound to brain endothelial cell surface, suggesting the existence of an autocrine network for this neuropeptide. These findings suggest that SP may have an immunomodulatory action at the blood-brain barrier during inflammatory and autoimmune processes in the central nervous system.

Norepinephrine and vasoactive intestinal peptide induce IL-6 secretion by astrocytes: synergism with IL-1 beta and TNF alpha.

Resident glial cells and invading inflammatory cells are responsible for cytokine production within the brain. Astrocytes are known to secrete a variety of cytokines upon stimulation with cytokines themselves, protein kinase C activators, bacterial or viral constituents. Astrocytes also have surface receptors for a wide number of neurotransmitters and neuropeptides and some of these substances affect astrocyte immune functions, such as major histocompatibility complex (MHC) class II antigen expression. To elucidate the activity of neuromediators on cytokine secretion by glial cells, we studied the secretion of interleukin-6 (IL-6) by cultured rat astrocytes after incubation with various neurotransmitters and neuropeptides. Norepinephrine (NE) and the beta-adrenergic agonist isoproterenol (IPT) induced IL-6 secretion in a dose-dependent fashion. NE effect was predominantly mediated by beta 2-adrenergic receptors with a minor contribution of alpha 1-adrenergic receptors. The induction of IL-6 release by dibutyryl-cAMP indicated that IL-6 secretion secondary to beta 2-adrenergic receptor activation probably occurs through cAMP signalling pathways. Vasoactive intestinal peptide (VIP) was the sole neuropeptide able to induce IL-6 secretion. NE and VIP promoted IL-6 mRNA synthesis and both substances synergized with interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) in inducing IL-6 release. Our findings provide further evidence that neurons modulate astrocyte cytokine production and thereby regulate central nervous system immune functions.

Nitric oxide synthase in the caudal neurosecretory system of the teleost Oreochromis niloticus.

This study provides evidence that, within the caudal neurosecretory system of the teleost Oreochromis niloticus, neurons express nitric oxide synthase (NOS)-like molecules. The presence of NOS-like molecules was demonstrated by means of NADPH-diaphorase (NADPHd) staining and NOS immunohistochemistry. In the caudal spinal cord, NOS-positive neurosecretory cell bodies and neurosecretory fibers were observed. In addition, NOS-positive structures were found in the urophysis which correspond to neurosecretory axon terminals. Cellular co-localization of NOS and ovine corticotropin-releasing factor (oCRF) immunoreactivities confirmed that the NOS-positive structures belong to the caudal neurosecretory system. The present results suggest that NO may participate in the caudal neuroendocrine function.

Partial biochemical characterization of nitric oxide synthase in the caudal spinal cord of the teleost Oreochromis niloticus.

The present study demonstrates that a NADPH/Ca2+-dependent nitric oxide synthase (NOS) activity is present in the soluble and in the particulate fractions of fish caudal spinal cord homogenates, both activities being inhibited by calmodulin inhibitors (W7 and/or TFP) and by the NOS inhibitor L-NAME. Moreover, Western blot analysis using either anti-NOS I or anti-NOS III antibodies shows that the soluble enzyme corresponds to the brain NOS (NOS-I) of mammals, whereas the particulate one is likely attributable to NOS I and/or NOS III (ecNOS) enzymes. To confirm the nitric oxide (NO) production by the caudal spinal cord homogenates, the NO-mediated conversion of oxyhemoglobin to methemoglobin was monitored spectroscopically. The present results are consistent with a constitutive, Ca2+-calmodulin-dependent, NO production by the caudal neurosecretory system.

Serum anti-brain endothelium antibodies and cognitive assessment in patients with Binswanger's encephalopathy.

The pathogenic mechanism underlying the vascular changes in Binswanger's encephalopathy (BE) is unknown. To test whether alterations of the humoral immunity may lead to endothelium damage, we analyzed serum levels of anti-brain endothelium antibodies (ABEA) (IgG and IgM) in 16 BE patients, 19 subjects with ischemic vascular disease without mental deterioration and 18 normal healthy subjects. ABEA IgM were found elevated in 1/16 (6%) BE patients and in 4/19 (21%) patients with cerebrovascular diseases; an increase in ABEA IgG was found in 6/16 (38%) BE patients and in 7/19 (37%) cerebrovascular patients. Association with anti-cardiolipin antibodies (IgG and/or IgM) was found in 50% of BE patients with elevated ABEA and only 10% of those with no increase, whereas high titres of anti-neurofilament antibodies (1:10,000) were detected in 40% and 71% respectively. In BE, ABEA IgG but not IgM showed a trend, although not significant, towards a correlation with the duration of the disease (rs = 0.47; p = 0.07) and significantly correlated with the cognitive function as assessed by the Mini mental state (MMS) score (rs = 0.56; p = 0.02). Higher mean values of the MMS score were found in BE patients with elevated ABEA than in those without (p = 0.04). This difference was not due to language disorders neither to an association with stroke risk factors or anti-neurofilament antibodies. However, there were no significant differences in MMS scores between cerebrovascular patients with ABEA and those without. A "neuro-protective" role is hypothesized for the ABEA in the development of dementia in BE.

The caudal neurosecretory system and its afferent synapses in the goldfish, Carassius auratus: morphology, immunohistochemistry, and fine structure.

Morphological features of the goldfish caudal neurosecretory system were investigated by means of immunohistochemical localization of urotensins I and II (UI and UII) and electron microscopic examination of the caudal neurosecretory neurons, the urophysis, and the synaptic neuropil. The aim of the work is to provide a detailed morphological description of the afferent synapses to the caudal neurons and to analyze their distribution through the rostrocaudal extension of the caudal neurosecretory system. Three morphologically different types of neurosecretory cells have been identified according to size and shape: large, medium, and small Dahlgren cells. The three different-sized cells share similar patterns of immunoreactivity with the UI (or oCRF) and the UII antisera. Electron microscopic examination of the synaptic neuropil throughout the caudal system revealed the presence of four types of terminals: dense-cored-vesicle end bulbs (DC), spherical-vesicle end bulbs (S), flattened-vesicle end bulbs (F), and granular-vesicle end bulbs (G). The present study demonstrates that the small Dahlgren cells receive different synaptic inputs from the large and the medium neurosecretory cells. Indeed, G terminals are only found on the small Dahlgren cells, whereas DC, S, and F terminals are distributed on the large, medium, and small Dahlgren cell bodies and proximal processes.

Development of the caudal neurosecretory system of the nile tilapia Oreochromis niloticus: an immunohistochemical and electron microscopic study.

The development of the caudal neurosecretory system (CNSS) of the Nile tilapia, Oreochromis niloticus, has been investigated by means of UI/oCRF (urotensin I/ovine corticotropin-releasing factor) immunohistochemistry and transmission electron microscopy. UI-like immunoreactive perikarya and fibers are first detected in the caudal spinal cord of larval fish about 4 days after hatching (stage 21). In the region of the future urophysis two bundles of strongly immunoreactive neurosecretory fibers are observed. At this stage, neurosecretory axons terminate on the meninx sheath of the spinal cord with immature neurosecretory terminals. The histogenesis of the urophysis begins at stage 24. The future neurohemal organ consists of a small ventral swelling of the spinal cord, which is associated with dilated vessels. At this stage, neurosecretory axons terminate on the basal lamina of the ingrowing blood vessels. Further development occurs by means of progressive branching of vessels and the concomitant increase in the number of neurosecretory terminals. In the caudal spinal cord, immunoreactive neurons also increase in number and progressively differentiate morphologically. Typical features of the mature CNSS are recognizable in 4-month-old juveniles. Data suggest that in tilapia both the synthesis and the release of urophysial hormones begin before morphogenesis of the neurohemal organ takes place.

[Thyroid and thyrotropin functions in subjects with pituitary nanism treated with growth hormone]. / Funzione tiroidea e tireotropinica nei soggetti con nanismo ipofisario in trattamento con ormone della crescita.

We have evaluated thyroid and thyrotropin functions before the beginning and during Growth Hormone (GH) treatment for a 2-6-year period in a group composed of 21 children (age: 6.6 +/- 1.1 years, m +/- SD) suffering from classic GH deficiency. Circulating levels of thyroxine, and basal thyroid-stimulating hormone (TSH) always resulted in the normal range. TSH response to thyreotropin-releasing hormone administration showed in some subjects (one out of 21 before the start of treatment, 2 out of 16 after 2 years, 3 out of 12 after 4 years and 2 out of 10 after 6 years) a delayed (after 90-120 minutes) and higher peak in comparison to that of normal subjects. All these high and delayed values have been showed in only one occasion by different children, with the exception of a child who has presented the higher values in two occasions. Growth response to GH treatment has not been modified by the change in thyrotropin response, as subjects with high TSH peak have had a height velocity similar to that of the other children in the corresponding periods of study.

Immunosuppressive monoclonal antibody to CD64 from patients with long-term stable multiple sclerosis.

High intrathecal levels of anti-myelin basic protein (MBP) IgM were previously found to be significantly associated with early favorable course in a cohort of patients with multiple sclerosis (MS). A mAb to MBP 105-120 recognizing the 222-228 epitope of the extracellular domain of high affinity immunoglobulin gamma Fc-receptor I (CD64) was isolated from EBV(+) B cell clones of long-term stable RRMS patients. This mAb exerted immunosuppressive activity on MS-derived T cell lines through induction and release of high amounts of interleukin-10 and decreased levels of interleukin-12 from activated monocytes providing the biological basis for a potential new treatment for MS and other immune-mediated neurological disorders.

Lens formation from cornea in the presence of the old lens in larval Xenopus laevis.

Three different types of experiments were carried out to investigate the role of the lens in lens-forming transformations of the outer cornea of Xenopus laevis tadpoles (at stage 51, as defined by Nieuwkoop and Faber, '56): 1) simple lentectomy; 2) incision of outer and inner cornea in the region opposite the dorsal iris without lens removal; and 3) incision of outer and inner cornea and perforation of dorsal iris without lens removal. The results indicate that the outer cornea of Xenopus laevis larvae has lens-forming competence, even in the presence of the old lens, only when direct communication has been set up between the outer cornea and the environment of the vitreous chamber. These data indicate that the inhibition exerted by the lens on lens-forming transformations of the outer cornea is mechanical in nature.

Inhibition of lens regeneration in larval Xenopus laevis.

The present research aims at showing the role played by the lens in inhibiting lens-forming transformations of the outer cornea of Xenopus laevis tadpoles. Two types of experiment were carried out: 1) lentectomy and insertion of a Millipore filter disk at the side of the lens, and 2) lentectomy and insertion of a Millipore disk with a central hole. Results indicate that when the lens is replaced by a mechanical obstacle capable of preventing direct communication between vitreous and anterior chambers, definite inhibition of the lens-forming transformations of the outer cornea occurs. These data strongly suggest that the inhibition exerted by the lens on lens-forming transformations of the outer cornea in Xenopus laevis eyes is mechanical rather than chemical in nature.

[The GHRH test in Turner's syndrome]. / Il GHRH-test nella sindrome di Turner.

GHRH test was performed in 11 girls suffering from Turner's syndrome ranging in age from 5.6-13.5 years. GH peak resulted lower than 10 ng/ml in three subjects, who had also shown reduced GH values after two conventional pharmacological stimuli (L-dopa- and insulin-induced hypoglycemia) and a value of mean GH concentration over 24 hours lower than 3 ng/ml. Both GH peak and area under the curve were not correlated with height, height velocity, bone age/chronological age ratio, GH peak after conventional pharmacological stimuli and mean GH value of spontaneous secretion. The comparison with the results of GHRH test in other kinds of short stature evidenced in girls with Turner's syndrome the presence of GH values (peak and area under the curve) higher than those in subjects with "classical" GH deficiency, lower than those in "short normal stature" and similar to those in subjects with "non classical" GH deficiency. In conclusion, our data suggest, even if within a certain variability of the responses, a possible involvement of GH deficiency to the pathogenesis of short stature in Turner's syndrome, suggesting the existence of a prevalent hypothalamic nature of GH deficiency.

Transdifferentiation of larval Xenopus laevis iris under the influence of the pituitary.

Fragments of larval Xenopus laevis dorsal iris implanted together with the pituitary into the tail fin transdifferentiate into neural retina. On the contrary, in the control experiments the implanted tissues, dorsal iris alone, pituitary, or dorsal iris with liver fragments, do not undergo any retinal transformation.