Neural activation in photosensitive brain regions of Atlantic salmon (Salmo salar) after light stimulation.

Photoreceptive inputs to the teleost brain are perceived as image of the visual world and as photo-modulation of neuroendocrine and neuronal signals. The retina and pineal organ are major receptive organs with projections to various parts of the brain, but in the past decades deep brain photoreceptors have emerged as candidates for photoreceptive inputs, either independent or in combination with projections from light sensory organs. This study aimed to test the effects of narrow bandwidth light using light-emitting diodes technology on brain neural activity through putative opsin stimulation in Atlantic salmon. The expression of c-fos, a known marker of neural activity, was compared in situ between dark-adapted salmon parr and following light stimulation with different wavelengths. c-fos expression increased with duration of light stimulation and the strongest signal was obtained in fish exposed to light for 120 minutes. Distinct and specific brain regions were activated following dark to light stimulation, such as the habenula, suprachiasmatic nucleus, thalamus, and hypothalamus. The c-fos expression was overlapping with photoreceptors expressing melanopsin and/or vertebrate ancient opsin, suggesting a potential direct activation by light. Interestingly in the habenula, a distinct ring of vertebrate ancient opsin and melanopsin expressing cells is overlapping with c-fos expression after neural activation. Salmon exposed to different spectra had neural activation in similar brain regions. The most apparent difference was melanopsin expression in the lateral cells of the lateral tuberal nuclus in the hypothalamus, which appeared to be specifically activated by red light. Light-stimulated neuronal activity in the deep brain was limited to subpopulations of neurons, mainly in regions with neuronal modulation activity, retinal and pineal innervations and known presence of nonvisual photoreceptors. The overlapping expression patterns of c-fos and nonvisual opsins support direct light stimulation of deep brain photoreceptors and the importance of these systems in light induced brain activity.

Early life stress induces long-term changes in limbic areas of a teleost fish: the role of catecholamine systems in stress coping.

Early life stress (ELS) shapes the way individuals cope with future situations. Animals use cognitive flexibility to cope with their ever-changing environment and this is mainly processed in forebrain areas. We investigated the performance of juvenile gilthead seabream, previously subjected to an ELS regime. ELS fish showed overall higher brain catecholaminergic (CA) signalling and lower brain derived neurotrophic factor (bdnf) and higher cfos expression in region-specific areas. All fish showed a normal cortisol and serotonergic response to acute stress. Brain dopaminergic activity and the expression of the α2Α adrenergic receptor were overall higher in the fish homologue to the lateral septum (Vv), suggesting that the Vv is important in CA system regulation. Interestingly, ELS prevented post-acute stress downregulation of the α2Α receptor in the amygdala homologue (Dm3). There was a lack of post-stress response in the ß2 adrenergic receptor expression and a downregulation in bdnf in the Dm3 of ELS fish, which together indicate an allostatic overload in their stress coping ability. ELS fish showed higher neuronal activity (cfos) post-acute stress in the hippocampus homologue (Dlv) and the Dm3. Our results show clear long-term effects on limbic systems of seabream that may compromise their future coping ability to environmental challenges.

Atlantic salmon (Salmo salar L.) smolts require more than two weeks to recover from acidic water and aluminium exposure.

The detrimental effects of acid rain and aluminium (Al) on salmonids have been extensively studied, yet knowledge about the extent and rate of potential recovery after exposures to acid and Al episodes is limited. Atlantic salmon smolts in freshwater (FW) were exposed for 2 and 7-day episodes (ACID2 and ACID7, respectively) to low pH (5.7±0.2) and inorganic aluminium (Ali; 40±4 µg) and then transferred to good water quality, control water (CW; pH 6.8±0.1; <14±2 µg Ali). Al accumulation on gills after 2 and 7 days of acid/Al exposure was 35.3±14.1 and 26.6±1.8 µg g(-1) dry weight, respectively. These elevated levels decreased 2 days post transfer to CW and remained higher than in control (CON; 5-10 µg Ali) for two weeks. Plasma Na(+) levels in ACID2 and ACID7 smolts decreased to 141±0.8 and 138.6±1.4mM, respectively, and remained significantly lower than CON levels for two weeks post transfer to CW. Similarly, plasma Cl(-) levels in ACID7 smolts (124.3±2.8mM) were significantly lower than in CON, with Cl(-) levels remaining significantly lower in ACID7 (126.2±4.8 mM) and ACID2 (127.6±3.7 mM) than in CON following 9 and 14 days post-transfer to CW, respectively. ACID2 and ACID7 smolts sustained elevated plasma glucose levels post transfer to CW suggesting elevated stress for more than a week following exposure. While gill Na(+), K(+)-ATPase (NKA) activity was only slightly affected in ACID2 and not in ACID7 smolts in FW, acid/Al exposure resulted in a transient decrease in NKA activity following SW exposure in both groups. Acid/Al episodes had limited impact on isoform specific NKA α-subunit mRNA during exposure. However, the transfer of ACID2 and ACID7 smolts to CW showed an increase in NKAα1a mRNA (the FW isoform) and inhibited the up-regulation of NKAα1b (the SW isoform), probably resulting in higher abundance of the enzyme favouring ion uptake. Gill caspase 3B gene transcription did not change in acid/Al treated smolts, indicating no increased apoptosis in gills. ACID2 and ACID7 treatments resulted in lower smolt-related gill transcription of the gene encoding the tight junction protein claudin 10e compared to CON, while the gene encoding claudin 30 showed lower mRNA expression only after 11 days SW exposure in ACID7 fish. Our data suggest that acid/Al conditions affect ion perturbations through a combination of alteration of the preparatory increase in paracellular permeability and negative impact on the SW type NKA α-subunit mRNA transcripts, and raise major concerns regarding the recovery of physiological disruption in smolts following acid/Al exposure. Smolts may require more than two weeks to fully recover from even short moderate episodes of acid/Al exposure. Acid/Al exposure thus probably has greater impact on salmon populations than previously acknowledged.

Effects of acidic water and aluminum exposure on gill Na(+), K(+)-ATPase alpha-subunit isoforms, enzyme activity, physiology and return rates in Atlantic salmon (Salmo salar L.).

Na(+), K(+)-ATPase (NKA) is involved, through its role as a major driving force for electrochemical gradients, in a range of transmembrane transport processes. Maintenance of homeostasis in anadromous salmonids requires modulation of several gill ion secretory proteins as part of the preparatory adaptation and acclimation to marine life. Atlantic salmon smolts were exposed to combinations of low pH and inorganic aluminum (acid/Al(i)) in freshwater (FW) and were then transferred to seawater (SW) for studies of post-smolt performance. Gill mRNA levels of four NKA-alpha isoforms (alpha1a, alpha1b, alpha1c and alpha3) of the catalytic NKA subunit and NKA enzyme activity were measured. Moderate acid/Al treatment (MOD, pH 5.9+/-0.3, 15+/-9microgl(-1)Al(i)) prevented the FW preparatory increase in NKA activity observed in control (CON, pH 6.9+/-0.1, 8+/-3microgl(-1)Al(i)) smolts, while high acid/Al treatment (SEV, pH 5.6+/-0.2, 30+/-7microgl(-1)Al(i)) caused a rapid and persistent reduction in NKA activity. Correspondingly, a 3.3-fold increase in plasma glucose levels in the SEV groups concurrent with a decrease in plasma chloride levels suggest that acid/Al exposed fish were stressed and experienced problems maintaining ion homeostasis. Gill NKA activities in acid/Al exposed groups were re-established after 28 days in SW. Both long (9 days) and short-term (2.5 days) treatments had significant impact on isoform-specific Na(+), K(+)-ATPase alpha-subunit mRNA abundance in the FW period. Acid/Al exposed groups lacked the preparatory increases in all NKA-alpha isoform mRNA levels seen in the CON group, except for alpha1a. In contrast to the other isoforms measured, alpha1a mRNA abundance decreased sharply upon SW transfer, supporting the hypothesis of isozyme shifting as a mechanism of altering the gill from an ion absorbing to an ion excreting tissue during smoltification and SW exposure. Adult return rates to the Imsa river were significantly reduced both in short-term (78% of controls) and long-term (55% of controls) acid/Al exposures, emphasising the physiological and ecological consequences of acid/Al exposure during smoltification.

Characterization, tissue distribution, and regulation of agouti-related protein (AgRP), cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY) in Atlantic salmon (Salmo salar).

Key peptide hormones involved in the control of appetite in vertebrates were identified, their genes characterized and their regulation studied in Atlantic salmon: two agouti-related proteins (AgRP), cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY). The AgRP-1 and AgRP-2 genes encode prepro-proteins of 142- and 117-amino acids, respectively. The deduced AgRP-2 protein has 10 cysteine residues in the C-terminal polycysteine domain, while the AgRP-1 lacks the 6th and 7th cysteine residues observed in other species. AgRP-1 was principally expressed in the pituitary and skin, while AgRP-2 was highly expressed in the mid-gut, red muscle and gonads. The CART gene, encoding 118-amino acids, was strongly expressed in the brain and eye. In addition to salmon CART, we identified three to six variants of the CART gene in lower vertebrates by mining available databases. The salmon NPY gene, encoding 100-amino acids, was mainly expressed in the brain and eye. AgRP-1 and CART mRNA levels in the brain decreased after 6 days of fasting while AgRP-2 and NPY showed no significant change, suggesting that AgRP-1 and CART are involved in feeding regulation in Atlantic salmon. The identification of multiple variants of these appetite-regulating genes emphasizes the importance to further investigate the complex regulation of these genes.

Differential expression of gill Na+,K+-ATPase alpha- and beta-subunits, Na+,K+,2Cl- cotransporter and CFTR anion channel in juvenile anadromous and landlocked Atlantic salmon Salmo salar.

This study examines changes in gill Na(+),K(+)-ATPase (NKA) alpha- and beta-subunit isoforms, Na(+),K(+),2Cl(-) cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR I and II) in anadromous and landlocked strains of Atlantic salmon during parr-smolt transformation, and after seawater (SW) transfer in May/June. Gill NKA activity increased from February through April, May and June among both strains in freshwater (FW), with peak enzyme activity in the landlocked salmon being 50% below that of the anadromous fish in May and June. Gill NKA-alpha1b, -alpha3, -beta(1) and NKCC mRNA levels in anadromous salmon increased transiently, reaching peak levels in smolts in April/May, whereas no similar smolt-related upregulation of these transcripts occurred in juvenile landlocked salmon. Gill NKA-alpha1a mRNA decreased significantly in anadromous salmon from February through June, whereas alpha1a levels in landlocked salmon, after an initial decrease in April, remained significantly higher than those of the anadromous smolts in May and June. Following SW transfer, gill NKA-alpha1b and NKCC mRNA increased in both strains, whereas NKA-alpha1a decreased. Both strains exhibited a transient increase in gill NKA alpha-protein abundance, with peak levels in May. Gill alpha-protein abundance was lower in SW than corresponding FW values in June. Gill NKCC protein abundance increased transiently in anadromous fish, with peak levels in May, whereas a slight increase was observed in landlocked salmon in May, increasing to peak levels in June. Gill CFTR I mRNA levels increased significantly from February to April in both strains, followed by a slight, though not significant increase in May and June. CFTR I mRNA levels were significantly lower in landlocked than anadromous salmon in April/June. Gill CFTR II mRNA levels did not change significantly in either strain. Our findings demonstrates that differential expression of gill NKA-alpha1a, -alpha1b and -alpha3 isoforms may be important for potential functional differences in NKA, both during preparatory development and during salinity adjustments in salmon. Furthermore, landlocked salmon have lost some of the unique preparatory upregulation of gill NKA, NKCC and, to some extent, CFTR anion channel associated with the development of hypo-osmoregulatory ability in anadromous salmon.

Cardiovascular disease and risk factors in three Alaskan Eskimo populations: the Alaska-Siberia project.

OBJECTIVES: To determine the prevalence of CVD and to identify and characterize associated risk factors in three distinct Eskimo populations. STUDY DESIGN: Cross-sectional. METHODS: A slightly modified Strong Heart Study protocol was followed to examine 454 participants, aged 25-91, from four villages. RESULTS: Overall, 6% of the participants under 55 years of age and 26% of those > or = 55 years of age showed evidence of CHD by ECG, or in patient records. The prevalence of "definite coronary heart disease" (CHD) in women with glucose intolerance (GI) was 21.0%, compared to 2.4% in those with normal glucose tolerance (NGT). Men had comparable values of 26.7% and 6.3%. In addition, comparable values for "possible CHD" were 29.7% vs 6.0% for women and 21.4% vs 8.0% for men. GI was associated with relatively higher prevalences of CHD in women than in men (prevalence ratio = 8.5 vs 4.3). CHD was significantly related to age, glucose intolerance and insulin. Hypertension and obesity were significantly associated with CHD only in some ethnic groups. The prevalence of current smokers was 56%. CONCLUSIONS: Recent changes in lifestyle and diet of Alaskan Eskimos, leading to obesity, hypertension, insulin resistance and DM, contribute to an increased risk for cardiovascular disease.

A successful diabetes prevention study in Eskimos: the Alaska Siberia project.

OBJECTIVES: To test the efficacy of a simple intervention method to reduce risk factors for type 2 diabetes (DM) and cardiovascular disease (CVD) in Alaskan Eskimos. STUDY DESIGN: The study consisted of 1) a comprehensive screening for risk factors of 454 individuals in 4 villages, 2) a 4-year intervention and 3) a repetition of the screening in year 5 to test the efficacy of the intervention. METHODS: Personal counseling (1hr/year) stressed the consumption of more traditional foods high in omega-3 fatty acids and less of certain specific store-bought foods high in palmitic acid, which was identified as being associated with glucose intolerance. RESULTS: The intervention resulted in significant reductions in plasma concentrations of total cholesterol (p = 0.0001), LDL cholesterol (p = 0.0001), fasting glucose (p = 0.0001), diastolic blood pressure (p = 0.0007) and improved glucose tolerance (p = 0.0006). This occurred without loss of body weight. Sixty percent of the participants had improved glucose tolerance; only one of the 44 originally identified with impaired glucose tolerance (IGT) developed DM during the study. CONCLUSIONS: Dramatic improvements of risk factors for DM and CVD were achieved in the intervention by primarily stressing the need for changes in the consumption of specific fats. The results suggest that fat consumption is an important risk factor for DM.

Nitric oxide synthase in the gill of Atlantic salmon: colocalization with and inhibition of Na+,K+-ATPase.

We investigated the relationship between nitric oxide (NO) and Na(+),K(+)-ATPase (NKA) in the gill of anadromous Atlantic salmon. Cells containing NO-producing enzymes were revealed by means of nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, which can be used as an indicator of NOS activity, i.e. NO production. Antibodies against the two constitutive NOS isoforms, neuronal and endothelial NOS, both produced immunoreactivity restricted to large cells at the base and along the secondary lamellae. NADPHd-positive cells showed a corresponding distribution. Antibodies against the inducible NOS isoform only labeled small cells located deep in the filament. Using in situ hybridization and NKA immunoreactivity, cells expressing Na(+),K(+)-ATPase alpha-subunit mRNA were found to have a similar distribution to the NOS cells. Double labeling for NOS immunoreactivity and NKA alpha-subunit mRNA revealed cellular colocalization of NKA alpha-subunit mRNA and nNOS protein in putative chloride cells at the base of the lamellae and interlamellar space. Along the lamellae, some NOS- or NKA-immunoreactive cells possessed a relatively lower expression of NKA alpha-subunit mRNA in smolts. A clear increase in NADPHd staining in the gill was demonstrated from parr to smolt. The regulatory role of NO on gill NKA activity was studied in vitro using sodium nitroprusside (SNP; 1 mmol l(-1)) and PAPA-NONOate (NOC-15; 0.5 mmol l(-1)) as NO donors. Both SNP and NOC-15 inhibited gill NKA activity by 30% when compared to controls. The study shows that NO systems are abundant in the gill of Atlantic salmon, that NO may be produced preferentially by a constitutive NOS isoform, and suggests that NO influence on gill functions is mediated via intracellular, possibly both auto/paracrine, inhibition of Na(+),K(+)-ATPase activity in chloride cells. Furthermore, the increase in NADPHd in the gill during smoltification suggests a regulatory role of NO in the attenuation of the smoltification-related increase in Na(+),K(+)-ATPase activity prior to entering seawater.

Developmentally regulated thyroid hormone distributor proteins in marsupials, a reptile, and fish.

Thyroid hormones are essential for vertebrate development. There is a characteristic rise in thyroid hormone levels in blood during critical periods of thyroid hormone-regulated development. Thyroid hormones are lipophilic compounds, which readily partition from an aqueous environment into a lipid environment. Thyroid hormone distributor proteins are required to ensure adequate distribution of thyroid hormones, throughout the aqueous environment of the blood, and to counteract the avid partitioning of thyroid hormones into the lipid environment of cell membranes. In human blood, these proteins are albumin, transthyretin and thyroxine-binding globulin. We analyzed the developmental profile of thyroid hormone distributor proteins in serum from a representative of each order of marsupials (M. eugenii; S.crassicaudata), a reptile (C. porosus), in two species of salmonoid fishes (S. salar; O. tshawytsch), and throughout a calendar year for sea bream (S. aurata). We demonstrated that during development, these animals have a thyroid hormone distributor protein present in their blood which is not present in the adult blood. At least in mammals, this additional protein has higher affinity for thyroid hormones than the thyroid hormone distributor proteins in the blood of the adult. In fish, reptile and polyprotodont marsupial, this protein was transthyretin. In a diprotodont marsupial, it was thyroxine-binding globulin. We propose an hypothesis that an augmented thyroid hormone distributor protein network contributes to the rise in total thyroid hormone levels in the blood during development.