Ethinylestradiol differentially interferes with IGF-I in liver and extrahepatic sites during development of male and female bony fish

Growth and sexual development are closely interlinked in fish; however, no reports exist on potential effects of estrogen on the GH/IGF-I-axis in developing fish. We investigate whether estrogen exposure during early development affects growth and the IGF-I system, both at the systemic and tissue level. Tilapia were fed from 10 to 40 days post fertilization (DPF) with 17alpha-ethinylestradiol (EE(2)). At 50, 75, 90, and 165 DPF, length, weight, sex ratio, serum IGF-I (RIA), pituitary GH mRNA and IGF-I, and estrogen receptor alpha (ERalpha) mRNA in liver, gonads, brain, and gills (real-time PCR) were determined and the results correlated to those of in situ hybridization for IGF-I. Developmental exposure to EE(2) had persistent effects on sex ratio and growth. Serum IGF-I, hepatic IGF-I mRNA, and the number of IGF-I mRNA-containing hepatocytes were significantly decreased at 75 DPF, while liver ERalpha mRNA was significantly induced. At 75 DPF, a transient decline of IGF-I mRNA and a largely reduced number of IGF-I mRNA-containing neurons were observed in the female brain. In both sexes, pituitary GH mRNA was significantly suppressed. A transient downregulation of IGF-I mRNA occurred in ovaries (75 DPF) and testes (90 DPF). In agreement, in situ hybridization revealed less IGF-I mRNA signals in granulosa and germ cells. Our results show for the first time that developmental estrogen treatment impairs GH/IGF-I expression in fish, and that the effects persist. These long-lasting effects both seem to be exerted indirectly via inhibition of pituitary GH and directly by suppression of local IGF-I in organ-specific cells.

Brain CYP1A in seabream, Sparus aurata exposed to benzo(a)pyrene

This study compares basal and induced expression of cytochrome P4501A-CYP1A in the brain of gilthead seabream, Sparus aurata. Larval or adult seabream were exposed to benzo(a)pyrene -B(a)P- and the CYP1A response was assessed by analyzing CYP1A mRNA (RT-PCR), CYP1A protein (expression levels: ELISA, western blotting; cellular localization: immunohistochemistry), and CYP1A catalytic activity (7-ethoxyresorufin-O-deethylase-EROD). In the brain of adult S. aurata, CYP1A immunostaining was generally detected in the vasculature. It was present in the neuronal fibers and glial cells of the olfactory bulbs and the ventral telencephalon. ELISA and RT-PCR analyses confirmed CYP1A expression in the brains of non-exposed seabream. B(a)P exposure led to increased CYP1A staining mainly in neuronal fibers and glial cells of the olfactory bulbs, but also in the vascular endothelia. EROD activity, however, could not be detected in the brain of adult seabream, neither in control nor in exposed fish. In the developing brain of S. aurata larvae, immunohistochemical staining detected CYP1A protein exclusively in endothelia of the olfactory placode and in retina. Staining intensity of CYP1A slightly increases with larval development, especially in vascular brain endothelia. Exposing the larvae to 0.3 or 0.5 microg B(a)P/L from hatching until 15 days post hatching (dph) did not result in enhanced CYP1A immunostaining in the brain. In samples of whole seabream larvae, both from controls and BaP treatments, neither CYP1A mRNA, protein nor catalytic activity were detectable. The results demonstrate that CYP1A is expressed already and inducible in the larval brain, but that the regional and cellular expression differs partly between larval and adult brain. This may have implications for the toxicity of CYP1A-inducing xenobiotics on early and mature life stages of seabream.

A sensory brain map for each behavior?

Multiple brain maps are commonly found in virtually every vertebrate sensory system. Although their functional significance is generally relatively little understood, they seem to specialize in processing distinct sensory parameters. Nevertheless, to yield the stimulus features that ultimately elicit the adaptive behavior, it appears that information streams have to be combined across maps. Results from current lesion experiments in the electrosensory system, however, suggest an alternative possibility. Inactivations of different maps of the first-order electrosensory nucleus in electric fish, the electrosensory lateral line lobe, resulted in markedly different behavioral deficits. The centromedial map is both necessary and sufficient for a particular electrolocation behavior, the jamming avoidance response, whereas it does not affect the communicative response to external electric signals. Conversely, the lateral map does not affect the jamming avoidance response but is necessary and sufficient to evoke communication behavior. Because the premotor pathways controlling the two behaviors in these fish appear to be separated as well, this system illustrates that sensory–motor control of different behaviors can occur in strictly segregated channels from the sensory input of the brain all through to its motor output. This might reflect an early evolutionary stage where multiplication of brain maps can satisfy the demand on processing a wider range of sensory signals ensuing from an enlarged behavioral repertoire, and bridging across maps is not yet required.

Assessment of fight outcome is needed to activate socially driven transcriptional changes in the zebrafish brain

Group living animals must be able to express different behavior profiles depending on their social status. Therefore, the same genotype may translate into different behavioral phenotypes through socially driven differential gene expression. However, how social information is translated into a neurogenomic response and what are the specific cues in a social interaction that signal a change in social status are questions that have remained unanswered. Here, we show for the first time, to our knowledge, that the switch between status-specific neurogenomic states relies on the assessment of fight outcome rather than just on self- or opponent-only assessment of fighting ability. For this purpose, we manipulated the perception of fight outcome in male zebrafish and measured its impact on the brain transcriptome using a zebrafish whole genome gene chip. Males fought either a real opponent, and a winner and a loser were identified, or their own image on a mirror, in which case, despite expressing aggressive behavior, males did not experience either a victory or a defeat. Massive changes in the brain transcriptome were observed in real opponent fighters, with losers displaying both a higher number of differentially expressed genes and of coexpressed gene modules than winners. In contrast, mirror fighters expressed a neurogenomic state similar to that of noninteracting fish. The genes that responded to fight outcome included immediate early genes and genes involved in neuroplasticity and epigenetic modifications. These results indicate that, even in cognitively simple organisms such as zebrafish, neurogenomic responses underlying changes in social status rely on mutual assessment of fighting ability.

The brain of the fur seal, Callorhinus ursinus : with a comparative description of those of Zalophus californianus, Phoca vitulina, Ursus americanus and Monachus tropicalis /

Caption title.

Seawater carbonate chemistry and fish Amphiprion percula behaviour during experiments, 2012

Predicted future CO2 levels have been found to alter sensory responses and behaviour of marine fishes. Changes include increased boldness and activity, loss of behavioural lateralization, altered auditory preferences and impaired olfactory function. Impaired olfactory function makes larval fish attracted to odours they normally avoid, including ones from predators and unfavourable habitats. These behavioural alterations have significant effects on mortality that may have far-reaching implications for population replenishment, community structure and ecosystem function. However, the underlying mechanism linking high CO2 to these diverse responses has been unknown. Here we show that abnormal olfactory preferences and loss of behavioural lateralization exhibited by two species of larval coral reef fish exposed to high CO2 can be rapidly and effectively reversed by treatment with an antagonist of the GABA-A receptor. GABA-A is a major neurotransmitter receptor in the vertebrate brain. Thus, our results indicate that high CO2 interferes with neurotransmitter function, a hitherto unrecognized threat to marine populations and ecosystems. Given the ubiquity and conserved function of GABA-A receptors, we predict that rising CO2 levels could cause sensory and behavioural impairment in a wide range of marine species, especially those that tightly control their acid-base balance through regulatory changes in HCO3 and Cl levels.

Agonistic interactions elicit rapid changes in brain nonapeptide levels in zebrafish

The teleost fish nonapeptides, arginine vasotocin (AVT) and isotocin (IT), have been implicated in the regulation of social behavior. These peptides are expected to be involved in acute and transient changes in social context, in order to be efficient in modulating the expression of social behavior according to changes in the social environment. Here we tested the hypothesis that short-term social interactions are related to changes in the level of both nonapeptides across different brain regions. For this purpose we exposed male zebrafish to two types of social interactions: (1) real opponent interactions, from which a Winner and a Loser emerged; and (2) mirror-elicited interactions, that produced individuals that did not experience a change in social status despite expressing similar levels of aggressive behavior to those of participants in real-opponent fights. Non-interacting individuals were used as a reference group. Each social phenotype (i.e. Winners, Losers, Mirror-fighters) presented a specific brain profile of nonapeptides when compared to the reference group. Moreover, the comparison between the different social phenotypes allowed to address the specific aspects of the interaction (e.g. assessment of opponent aggressive behavior vs. self-assessment of expressed aggressive behavior) that are linked with neuropeptide responses. Overall, agonistic interactions seem to be more associated with the changes in brain AVT than IT, which highlights the preferential role of AVT in the regulation of aggressive behavior already described for other species.

Vocal differentiation parallels development of auditory saccular sensitivity in a highly soniferous fish

Vocal differentiation is widely documented in birds and mammals but has been poorly investigated in other vertebrates, including fish, which represent the oldest extant vertebrate group. Neural circuitry controlling vocal behaviour is thought to have evolved from conserved brain areas that originated in fish, making this taxon key to understanding the evolution and development of the vertebrate vocal-auditory systems. This study examines ontogenetic changes in the vocal repertoire and whether vocal differentiation parallels auditory development in the Lusitanian toadfish Halobatrachus didactylus (Batrachoididae). This species exhibits a complex acoustic repertoire and is vocally active during early development. Vocalisations were recorded during social interactions for four size groups (fry: <2 cm; small juveniles: 2-4 cm; large juveniles: 5-7 cm; adults >25 cm, standard length). Auditory sensitivity of juveniles and adults was determined based on evoked potentials recorded from the inner ear saccule in response to pure tones of 75-945 Hz. We show an ontogenetic increment in the vocal repertoire from simple broadband-pulsed 'grunts' that later differentiate into four distinct vocalisations, including low-frequency amplitude-modulated 'boatwhistles'. Whereas fry emitted mostly single grunts, large juveniles exhibited vocalisations similar to the adult vocal repertoire. Saccular sensitivity revealed a three-fold enhancement at most frequencies tested from small to large juveniles; however, large juveniles were similar in sensitivity to adults. We provide the first clear evidence of ontogenetic vocal differentiation in fish, as previously described for higher vertebrates. Our results suggest a parallel development between the vocal motor pathway and the peripheral auditory system for acoustic social communication in fish.

[the Use Of Fish On Buccal Smear To Investigate Mosaicism With A 45,x Cell Line: Study On Healthy Men And Patients With Disorders Of Sex Development].

To verify whether fluorescence in situ hybridization (FISH) of cells from the buccal epithelium could be employed to detect cryptomosaicism with a 45,X lineage in 46,XY patients. Samples of nineteen 46,XY healthy young men and five patients with disorders of sex development (DSD), four 45,X/46,XY and one 46,XY were used. FISH analysis with X and Y specific probes on interphase nuclei from blood lymphocytes and buccal epithelium were analyzed to investigate the proportion of nuclei containing only the signal of the X chromosome. The frequency of nuclei containing only the X signal in the two tissues of healthy men did not differ (p = 0.69). In all patients with DSD this frequency was significantly higher, and there was no difference between the two tissues (p = 0.38), either. Investigation of mosaicism with a 45,X cell line in patients with 46,XY DSD or sterility can be done by FISH directly using cells from the buccal epithelium.

Feasibility And Reproducibility Of Diffusion-weighted Magnetic Resonance Imaging Of The Fetal Brain In Twin-twin Transfusion Syndrome.

The aim of this study is to test the feasibility and reproducibility of diffusion-weighted magnetic resonance imaging (DW-MRI) evaluations of the fetal brains in cases of twin-twin transfusion syndrome (TTTS). From May 2011 to June 2012, 24 patients with severe TTTS underwent MRI scans for evaluation of the fetal brains. Datasets were analyzed offline on axial DW images and apparent diffusion coefficient (ADC) maps by two radiologists. The subjective evaluation was described as the absence or presence of water diffusion restriction. The objective evaluation was performed by the placement of 20-mm(2) circular regions of interest on the DW image and ADC maps. Subjective interobserver agreement was assessed by the kappa correlation coefficient. Objective intraobserver and interobserver agreements were assessed by proportionate Bland-Altman tests. Seventy-four DW-MRI scans were performed. Sixty of them (81.1%) were considered to be of good quality. Agreement between the radiologists was 100% for the absence or presence of diffusion restriction of water. For both intraobserver and interobserver agreement of ADC measurements, proportionate Bland-Altman tests showed average percentage differences of less than 1.5% and 95% CI of less than 18% for all sites evaluated. Our data demonstrate that DW-MRI evaluation of the fetal brain in TTTS is feasible and reproducible.

Evidences Of Endocytosis Via Caveolae Following Blood-brain Barrier Breakdown By Phoneutria Nigriventer Spider Venom.

Spider venoms contain neurotoxic peptides aimed at paralyzing prey or for defense against predators; that is why they represent valuable tools for studies in neuroscience field. The present study aimed at identifying the process of internalization that occurs during the increased trafficking of vesicles caused by Phoneutria nigriventer spider venom (PNV)-induced blood-brain barrier (BBB) breakdown. Herein, we found that caveolin-1α is up-regulated in the cerebellar capillaries and Purkinje neurons of PNV-administered P14 (neonate) and 8- to 10-week-old (adult) rats. The white matter and granular layers were regions where caveolin-1α showed major upregulation. The variable age played a role in this effect. Caveolin-1 is the central protein that controls caveolae formation. Caveolar-specialized cholesterol- and sphingolipid-rich membrane sub-domains are involved in endocytosis, transcytosis, mechano-sensing, synapse formation and stabilization, signal transduction, intercellular communication, apoptosis, and various signaling events, including those related to calcium handling. PNV is extremely rich in neurotoxic peptides that affect glutamate handling and interferes with ion channels physiology. We suggest that the PNV-induced BBB opening is associated with a high expression of caveolae frame-forming caveolin-1α, and therefore in the process of internalization and enhanced transcytosis. Caveolin-1α up-regulation in Purkinje neurons could be related to a way of neurons to preserve, restore, and enhance function following PNV-induced excitotoxicity. The findings disclose interesting perspectives for further molecular studies of the interaction between PNV and caveolar specialized membrane domains. It proves PNV to be excellent tool for studies of transcytosis, the most common form of BBB-enhanced permeability.

Bile And Liver Metallothionein Behavior In Copper-exposed Fish.

The present study analyzed metallothionein (MT) excretion from liver to bile in Nile Tilapia (Oreochromis niloticus) exposed to sub-lethal copper concentrations (2mgL(-1)) in a laboratory setting. MTs in liver and bile were quantified by spectrophotometry after thermal incubation and MT metal-binding profiles were characterized by size exclusion high performance liquid chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS). Results show that liver MT is present in approximately 250-fold higher concentrations than bile MT in non-exposed fish. Differences between the MT profiles from the control and exposed group were observed for both matrices, indicating differential metal-binding behavior when comparing liver and bile MT. This is novel data regarding intra-organ MT comparisons, since differences between organs are usually present only with regard to quantification, not metal-binding behavior. Bile MT showed statistically significant differences between the control and exposed group, while the same did not occur with liver MT. This indicates that MTs synthesized in the liver accumulate more slowly than MTs excreted from liver to bile, since the same fish presented significantly higher MT levels in liver when compared to bile. We postulate that bile, although excreted in the intestine and partially reabsorbed by the same returning to the liver, may also release MT-bound metals more rapidly and efficiently, which may indicate an efficient detoxification route. Thus, we propose that the analysis of bile MTs to observe recent metal exposure may be more adequate than the analysis of liver MTs, since organism responses to metals are more quickly observed in bile, although further studies are necessary.

Triggering Of Protection Mechanism Against Phoneutria Nigriventer Spider Venom In The Brain.

Severe accidents caused by the armed spider Phoneutria nigriventer cause neurotoxic manifestations in victims. In experiments with rats, P. nigriventer venom (PNV) temporarily disrupts the properties of the BBB by affecting both the transcellular and the paracellular route. However, it is unclear how cells and/or proteins participate in the transient opening of the BBB. The present study demonstrates that PNV is a substrate for the multidrug resistance protein-1 (MRP1) in cultured astrocyte and endothelial cells (HUVEC) and increases mrp1 and cx43 and down-regulates glut1 mRNA transcripts in cultured astrocytes. The inhibition of nNOS by 7-nitroindazole suggests that NO derived from nNOS mediates some of these effects by either accentuating or opposing the effects of PNV. In vivo, MRP1, GLUT1 and Cx43 protein expression is increased differentially in the hippocampus and cerebellum, indicating region-related modulation of effects. PNV contains a plethora of Ca(2+), K(+) and Na(+) channel-acting neurotoxins that interfere with glutamate handling. It is suggested that the findings of the present study are the result of a complex interaction of signaling pathways, one of which is the NO, which regulates BBB-associated proteins in response to PNV interference on ions physiology. The present study provides additional insight into PNV-induced BBB dysfunction and shows that a protective mechanism is activated against the venom. The data shows that PNV has qualities for potential use in drug permeability studies across the BBB.

Behavioral Changes Caused By Austrodiplostomum Spp. In Hoplias Malabaricus From The São Francisco River, Brazil.

Traira (Hoplias malabaricus) is a neotropical fish that is widely distributed in freshwater environments in South America. In the present study, we documented the occurrence of metacercariae of Austrodiplostomum spp. (Diplostomidae) in the eyes and cranial cavity of H. malabaricus and described parasite-induced behavioral changes in the host. The fish were collected from the upper São Francisco River, in the Serra da Canastra mountain range, Minas Gerais, transported alive to the laboratory, observed for 2 weeks, and subsequently examined for parasites. Of the 35 fish examined, 28 (80 %) had free metacercariae in the vitreous humor (mean intensity=95.4; mean abundance=76.3), and 24 (68.57 %) had free metacercariae in the cranial cavity, mainly concentrated below the floor of the brain, at the height of the ophthalmic lobe (mean intensity=12.91; mean abundance=8.85). Specimens of H. malabaricus with a high intensity of infection in the brain displayed changes in swimming behavior.

Vascular Endothelial Growth Factor Increases During Blood-brain Barrier-enhanced Permeability Caused By Phoneutria Nigriventer Spider Venom.

Phoneutria nigriventer spider accidental envenomation provokes neurotoxic manifestations, which when critical, results in epileptic-like episodes. In rats, P. nigriventer venom (PNV) causes blood-brain barrier breakdown (BBBb). The PNV-induced excitotoxicity results from disturbances on Na(+), K(+) and Ca(2+) channels and glutamate handling. The vascular endothelial growth factor (VEGF), beyond its angiogenic effect, also, interferes on synaptic physiology by affecting the same ion channels and protects neurons from excitotoxicity. However, it is unknown whether VEGF expression is altered following PNV envenomation. We found that adult and neonates rats injected with PNV showed immediate neurotoxic manifestations which paralleled with endothelial occludin, β-catenin, and laminin downregulation indicative of BBBb. In neonate rats, VEGF, VEGF mRNA, and Flt-1 receptors, glutamate decarboxylase, and calbindin-D28k increased in Purkinje neurons, while, in adult rats, the BBBb paralleled with VEGF mRNA, Flk-1, and calbindin-D28k increases and Flt-1 decreases. Statistically, the variable age had a role in such differences, which might be due to age-related unequal maturation of blood-brain barrier (BBB) and thus differential cross-signaling among components of the glial neurovascular unit. The concurrent increases in the VEGF/Flt-1/Flk-1 system in the cerebellar neuron cells and the BBBb following PNV exposure might imply a cytokine modulation of neuronal excitability consequent to homeostatic perturbations induced by ion channels-acting PNV neuropeptides. Whether such modulation represents neuroprotection needs further investigation.