Behavioural responses in a net restraint test predict interrenal reactivity in Arctic charr Salvelinus alpinus.

In this study, a 1 min net restraint test was evaluated as a method to predict stress-coping style in Arctic charr Salvelinus alpinus, by investigating the relationship between behaviour during the test and levels of plasma cortisol sampled after 30 min confinement. In two separate groups of S. alpinus, general linearized model revealed significant correlations between cortisol levels and principal component scores extracted from principal component analysis, combining measures of activity in the tests. With the use of glmulti, the model selection ruled out any effects of size, sex and order of capture on interrenal reactivity. In general, S. alpinus that were more active in the net restraint test also had low levels of circulating cortisol, suggesting a proactive coping style. The results from two repeated runs were not correlated, but both runs, performed eight days apart, show a negative correlation between post-stress cortisol level and activity in the net. The lack of consistency could be explained by different treatments before each run and individual differences in behavioural plasticity. The net restraint test is thus predictive of stress-coping style in S. alpinus, and has the benefit of being less time-consuming than the commonly used confinement stress test.

Brain monoaminergic activity during predator inspection in female Trinidadian guppies (Poecilia reticulata).

To understand the processes underpinning social decision-making, we need to determine how internal states respond to information gathered from the social environment. Brain monoamine neurotransmitters are key in the appraisal of the social environment and can reflect the internal state underlying behavioural responses to social stimuli. Here we determined the effects of conspecific partner cooperativeness during predator inspection on brain monoamine metabolic activity in Trinidadian guppies (Poecilia reticulata). We quantified the concentration of dopamine, serotonin and their metabolites across brain sections sampled immediately after ostensibly experiencing cooperation or defection from social partners whilst inspecting a predator model, using a familiar object as a control condition. Our results indicate dopaminergic and serotonergic activity differs with the cooperativeness experienced; these different neurotransmission profiles are likely to affect the expression and regulation of downstream behaviours that ultimately contribute to the patterning of cooperative interactions among individuals in a population.

Geographic variation in corticosterone response to chronic predator stress in tadpoles.

Chronic stress often affects growth and development negatively, and these effects are often mediated via glucocorticoid hormones, which elevate during stress. We investigated latitudinal variation in corticosterone (CORT) response to chronic predator stress in Rana temporaria tadpoles along a 1500-km latitudinal cline in Sweden tadpoles, in a laboratory experiment. We hypothesized that more time-constrained high-latitude populations have evolved a lower CORT response to chronic stress to maintain higher growth under stressful conditions. Southern tadpoles had higher CORT content in response to predators after 1 day of exposure, whereas there was no increase in CORT in the northern populations. Two weeks later, there were no predator-induced CORT elevations. Artificially elevated CORT levels strongly decreased growth, development and survival in both northern and southern tadpoles. We suggest that the lower CORT response in high-latitude populations can be connected with avoidance of CORT-mediated reduction in growth and development, but also discuss other possible explanations.

Evidence for small scale variation in the vertebrate brain: mating strategy and sex affect brain size and structure in wild brown trout (Salmo trutta).

The basis for our knowledge of brain evolution in vertebrates rests heavily on empirical evidence from comparative studies at the species level. However, little is still known about the natural levels of variation and the evolutionary causes of differences in brain size and brain structure within-species, even though selection at this level is an important initial generator of macroevolutionary patterns across species. Here, we examine how early life-history decisions and sex are related to brain size and brain structure in wild populations using the existing natural variation in mating strategies among wild brown trout (Salmo trutta). By comparing the brains of precocious fish that remain in the river and sexually mature at a small size with those of migratory fish that migrate to the sea and sexually mature at a much larger size, we show, for the first time in any vertebrate, strong differences in relative brain size and brain structure across mating strategies. Precocious fish have larger brain size (when controlling for body size) but migratory fish have a larger cerebellum, the structure in charge of motor coordination. Moreover, we demonstrate sex-specific differences in brain structure as female precocious fish have a larger brain than male precocious fish while males of both strategies have a larger telencephalon, the cognitive control centre, than females. The differences in brain size and structure across mating strategies and sexes thus suggest the possibility for fine scale adaptive evolution of the vertebrate brain in relation to different life histories.

Vaginal breech delivery at term and neonatal morbidity and mortality - a population-based cohort study in Sweden.

INTRODUCTION: The routine to deliver almost all term breech cases by elective cesarean section (CS) has continued to be debated due to the risk of maternal and neonatal complications. The aims of the study were (1) to investigate if mode of delivery impacts on the risk of morbidity and mortality among term infants in breech presentation and (2) to compare the rates of severe neonatal complications and mortality in relation to presentation and mode of delivery. METHODS: This population-based cohort study used data from the Swedish Medical Birth Register. All women (and their newborn infants) with singleton pregnancies who gave birth at term to an infant in breech (n = 27,357) or cephalic presentation (n = 837,494) between 2001 and 2012 were included. Births with vacuum extraction and induced labors were excluded, as well as antepartum stillbirths, births with infants diagnosed with congenital malformations and multiple births. RESULTS: On one hand, the rates of neonatal complications and mortality were higher among infants born in vaginal breech compared to the vaginal cephalic group. On the other hand, after CS, the rates of all neonatal complications under study and neonatal mortality were lower among infants in breech presentation than in those in cephalic presentation. After adjustment for confounders, infants delivered in vaginal breech had 23.8 times higher odds AOR (ratio) for brachial plexus injury, 13.3 times higher odds ratio for Apgar score <7 at 5 min, 6.7 times higher odds of intracranial hemorrhage (ICH), or convulsions and 7.6 higher odds ratio for perinatal mortality than those delivered by elective CS. CONCLUSIONS: Despite a probable selection of women who before-hand were considered at low risk and, therefore, could be recommended vaginal breech delivery, infants delivered in vaginal breech faced substantially increased risks of severe neonatal complications compared with infants in breech presentations delivered by elective CS. Key message Vaginal breech delivery is associated with increased risk for severe neonatal complications.

Serotonergic characteristics of rainbow trout divergent in stress responsiveness.

Juvenile rainbow trout divergent in their cortisol response to confinement stress (HR: high responsive or LR: low responsive fish) were exposed to either 1 or 3 h of confinement stress. Untreated fish served as control. After the exposure blood and brain samples were collected. From the blood samples, the levels of cortisol and catecholamines were determined, while the brain serotonergic and monoamineoxidase (MAO) activity was determined in four different brain areas (brain stem, hypothalamus, telencephalon and optic tectum). Our results show that the LR fish responds to handling stress with a higher increase in plasma epinephrine compared to HR fish. Our results also show that confinement stress leads to a larger increase in the serotonergic activity in the brain stem and telencephalon in LR fish compared to HR fish. These results support the hypothesis that stress coping strategies similar to those described in mammals also exists in fish. Further, our results have shown that the MAO activity increases in optic tectum and hypothalamus of rainbow trout during confinement stress, while it remains unchanged or decreases in brain stem and telencephalon. Moreover, the MAO activity does not differ between the two selection lines. This indicates that MAO participates actively in the stress response without contributing to the differences in stress coping strategies.

Relationships between sex and the size and number of forebrain gonadotropin-releasing hormone-immunoreactive neurones in the ballan wrasse (Labrus berggylta), a protogynous hermaphrodite.

This study is the first to examine the brain gonadotropin-releasing hormone (GnRH) cell population phenotype in a protogynous and monandric sequentially hermaphroditic fish. Male ballan wrasse (Labrus berggylta) had on average higher numbers of GnRH-immunoreactive (GnRH-ir) cells within the brain preoptic area (POA) than females, a difference not found in GnRH-ir cells in other brain regions. Furthermore, in males, but not females, the number of these POA GnRH-ir cells correlated with body size. Maturational state (prespawning or postspawning) had marked effects on mean profile sizes (but not numbers) of both GnRH-ir cell bodies and cell nuclei, even when existing differences in body size and allometric relationships had been taken into account. Postspawning males tended to have larger GnRH-ir profiles in all brain regions relative to both prespawning males and females. Moreover, the GnRH-ir cell number in POA, and the cell body profile size in both POA and at the level of the anterior commissure, correlated with gonad size in spermiated prespawning males, indicating a relationship between both size and number of GnRH cells and male gonadal development. These results suggest that temporary changes in the size of brain GnRH-ir neurones are coupled to the male spawning cycle, and that permanent POA GnRH-ir cell number changes are involved in the process of sex change in sequential hermaphrodites. However, smaller males had no more preoptic GnRH-ir cells than equally sized females, which may argue against a proximate inducing role of GnRH cell number changes in naturally occurring sex reversal.

Effects of Schistocephalus solidus infection on brain monoaminergic activity in female three-spined sticklebacks Gasterosteus aculeatus.

The three-spined stickleback Gasterosteus aculeatus is an intermediate host of the tapeworm Schistocephalus solidus. Changes in predator avoidance, foraging and shoaling behaviour have been reported in sticklebacks infested with S. solidus, but the mechanisms underlying parasite-induced behavioural changes are not understood. Monoamine neurotransmitters are involved in the control of behaviour and central monoaminergic systems are sensitive to various stressors. Thus, the behavioural effects of S. solidus infestation might be a reflection of changes in brain monoaminergic activity in the stickleback host. The concentrations of 5-hydroxytryptamine (5-HT), dopamine (DA), norepinephrine (NE) and their metabolites 5-hydroxy-indoleacetic acid (5-HIAA), homovanilic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured in the telencephalons, hypothalami and brainstems of parasitized and non-parasitized female sticklebacks held in the laboratory. The ratios of 5-HIAA:5-HT were significantly elevated in both the hypothalami and brainstems of infected sticklebacks. The concentrations of 5-HT and NE were significantly reduced in the telencephalons of infected fish as compared with controls, but there was no elevation of metabolite concentrations. The results are consistent with chronic stress in infected fish, but may also reflect other alterations of neuroendocrine status resulting from parasite infection.

Induction of social dominance by L-dopa treatment in Arctic charr.

The effect of L-dopa on social dominance was studied in the juvenile Arctic charr (Salvelinus alpinus). L-dopa is the immediate precursor of dopamine, a neurotransmitter that has been connected with aggressive behaviour in fish as well as mammals. Arctic charr were placed in pairs. One individual in each pair was given L-dopa orally, while the other was given vehicle. The results showed that 18 out of 22 fish given 10 mg L-dopa kg-1 became dominant (p = 0.004, binomial test). A higher dose of L-dopa (200 mg kg-1) induced dyskinesia. L-dopa was found to cause a dose-dependent increase in the brain levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC, a major dopamine metabolite), as well as an increase in the DOPAC/dopamine ratio (an index of dopaminergic activity).

Predator exposure alters brain serotonin metabolism in bicolour damselfish.

The effect of predator exposure on brain serotonin utilization was studied in bicolour damselfish (Pomacentrus partitus). Predator exposure (lasting 2 h), which took place in an aquarium where a transparent wall separated the damselfish from the predator (a graysby, Epinephelus cruentatus), resulted in increased concentrations of 5-hydroxyindoleacetic acid (5-HIAA, the main serotonin metabolite) and 1.6-1.8 fold elevations of 5-HIAA/serotonin ratios (an index of serotonergic activity) in telencephalon, hypothalamus and brain stem. The results show that predator exposure, like intraspecific social stress, induces increased brain serotonergic activity in fish. Different types of stress also elevate brain serotonergic activity in mammals, indicating that this is a phylogenetically very old stress response, possibly helping the animal's coping response.

Multiple high-affinity binding sites for

Binding of [3H]serotonin (5-HT) to membranes prepared from Arctic charr brain homogenates was most consistent with a one-site model for [3H]5-HT binding, with KD and Bmax values of 5.7±0.3 nmol l-1 and 60.7±7.3 fmol mg-1 protein, respectively. Similarly, 5-HT displacement of [3H]5-HT was best explained by a monophasic model with an apparent Ki of 4.3±0.7 nmol l-1. The ability of a number of synthetic 5-HT receptor ligands to displace [3H]5-HT was studied. 8OH-DPAT was found to interact with three [3H]5-HT binding sites, whereas buspirone, TFMPP, spiperone and mianserin all distinguish two sites. In the presence of 300 nmol l-1 buspirone, 8OH-DPAT and mianserin distinguished two [3H]5-HT binding sites, whereas spiperone interacted with only one. Moreover, 8OH-DPAT differentiated three [3H]5-HT binding sites even in the presence of 0.5 mmol l-1 GTP, making it unlikely that these sites represent different affinity states of G-protein-coupled receptors. GTP had no effect on apparent Ki values for 8OH-DPAT, but reduced the Bmax value of the high-affinity site by 60 %. GTP had a similar effect on the saturation binding curve for [3H]5-HT, reducing Bmax by approximately 50 %, whereas KD was unaffected. The results provide evidence for at least three different high-affinity [3H]5-HT binding sites, one of them showing a pharmacological profile strikingly similar to that of the mammalian 5-HT1A receptor.

Serotonin as a regulator of hypothalamic-pituitary-interrenal activity in teleost fish.

Evidence for the presence of a serotonin1A (5-HT1A) receptor subtype in the salmonid fish brain has recently been presented. In the present study the potent 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) was tested for its effect on plasma cortisol concentrations in rainbow trout (Oncorhynchus mykiss). Blood was sampled and 8-OH-DPAT administered through a catheter in the dorsal aorta. Thirty minutes after the injection of 40 microg of 8-OH-DPAT/kg, plasma cortisol levels had increased from 12 to 149 ng/ml, whereupon they fell, reaching baseline levels after 4 h. The effect of 1-40 microg 8-OH-DPAT/kg on plasma cortisol concentrations was dose-dependent. The results lends further support to the hypothesis that the brain serotonergic system plays a key role in integrating autonomic, behavioral and neuroendocrine stress-responses in fish as well as mammals, suggesting that not only the structural and biochemical organization, but also the function of the serotonergic system has been conserved during vertebrate evolution.

Effects of L-thyroxine on brain monoamines during parr-smolt transformation of Atlantic salmon (Salmo salar L.).

During spring, seaward migrating juvenile Atlantic salmon (Salmo salar) undergo parr-smolt transformation (PST) which involves changes in physiology, including one or two peaks in plasma thyroxine (T4). To investigate if changes in plasma T4 influence neural function, we measured levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and also measured serotonin (5-hydroxytryptamine, 5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in brain regions of two groups of Atlantic salmon parr on an 8:16 h light/dark photoperiod. One group was treated with ambient T4 to simulate the natural smolt peak in plasma T4. T4 treatment depressed DOPAC levels as well as DOPAC/DA and 5-HIAA/5-HT ratios in the olfactory system but with no changes in the optic tectum. We conclude that during PST monoaminergic functions in specific brain regions of juvenile Atlantic salmon are affected by T4 treatment.

Stress-induced changes in brain serotonergic activity, plasma cortisol and aggressive behavior in Arctic charr (Salvelinus alpinus) is counteracted by L-DOPA.

Arctic charr (Salvelinus alpinus) were tested for aggressive behavior using intruder tests, before and after 2 days of dyadic social interaction. Following social interaction, half of the dominant and half of the subordinate fish were given L-DOPA (10 mg/kg, orally), whereas the remaining dominant and subordinate fish were given vehicle. One hour following drug treatment, the fish were tested for aggressive behavior again in a third and final intruder test, after which blood plasma and brain tissue were sampled for analysis of plasma cortisol concentrations and brain levels of monoamines and monoamine metabolites. Subordinate fish showed a reduction in the number of attacks launched against the intruder, as well as an increase in attack latency, as compared to prior to dyadic social interactions. Social subordination also resulted in an elevation of brain serotonergic activity. Fish receiving L-DOPA prior to the final intruder test showed shorter attack latency than vehicle controls. Drug treatment was a stressful experience and vehicle controls showed elevated plasma cortisol levels and longer attack latency as compared to before treatment. L-DOPA-treated fish showed lower plasma levels of cortisol and lower serotonergic activity in certain brain areas than vehicle controls. These results suggest that L-DOPA counteracts the stress-induced inhibition of aggressive behavior, and at the same time inhibits stress-induced effects on brain serotonergic activity and plasma cortisol concentrations.

Context-dependent responses to novelty in Rainbow trout (Oncorhynchus mykiss), selected for high and low post-stress cortisol responsiveness.

Previous studies in a rainbow trout model, selectively bred for high (HR) and low (LR) post stress plasma cortisol levels, have yielded data that are indicative of contrasting stress coping styles. Fish from the HR line have been suggested to display a more diverse behavioral repertoire in challenging situations than the LR counterpart. The present study addressed whether such variation in behavioral flexibility traits was evident in different experimental settings using these selection lines. The fish were subjected to three sets of challenges (novel object test, resident-intruder test and confinement stressor test), all which were repeated a week later. Introducing a novel object evoked a divergent behavioral response in association with feeding: fish from the LR line displayed consistently suppressed feed intake while the HR fish remained unaffected. This observation was found to be repeatable along with attack latency and movement activity from the resident-intruder and confinement stressor tests. These results indicate that the behavioral responses in this animal model are context-dependent and shed new light on the expression of behavioral flexibility.

Aggression in rainbow trout is inhibited by both MR and GR antagonists.

The present study has investigated the effect of exogenous cortisol on aggression in juvenile rainbow trout, along with the involvement of mineralocorticoid (MR) and glucocorticoid receptors (GR) mediating the effects of cortisol. Fish were fed pellets supplemented with cortisol, the GR antagonist mifepristone (RU486) in combination with cortisol, the MR antagonist spironolactone (SA) in combination with cortisol or both antagonists in combination with cortisol. Aggressive behaviour was then assessed 1h subsequent to feeding. Our results showed that the attack latency was increased by exogenous cortisol, an effect that was not abolished by the antagonists. The intensity of aggression was not changed by exogenous cortisol. However, the intensity of aggression was significantly reduced by both antagonists. These results are discussed with regard to cortisol affecting aggressive behaviour through genomic and non-genomic pathways. Our results have demonstrated the involvement of both MR and GR in regulating behavioural responses during social interactions in teleost fish. The intensity of aggression seen in control and cortisol treated fish is probably mediated by the basal levels of cortisol through the intracellular MRs and GRs. We conclude that the initiative to engage in social confrontations is mediated through a non-genomic pathway, which could involve extracellular corticoid receptors. Further, the majority of arguments lean towards the MR and GR antagonists blocking the effect of cortisol on aggressive intensity through intracellular receptors. If this is the case, then it is probable that these two aspects of aggressive behaviour are based on different neuronal mechanisms.

Elevation of brain 5-HT activity, POMC expression, and plasma cortisol in socially subordinate rainbow trout.

Agonistic behavior, brain concentrations of serotonin (5-hydroxytryptamine, 5-HT), and 5-hydroxyindoleacetic acid (5-HIAA, the main 5-HT metabolite), plasma cortisol levels, and the pituitary expression of pro-opiomelanocortin (POMC) A and B mRNA were determined in socially dominant and subordinate rainbow trout after 1 or 7 days of social interaction. Telencephalic and brain stem 5-HIAA/5-HT ratios, plasma cortisol levels, and pituitary POMC mRNA concentrations were elevated in fish being subordinate for 1 day. Furthermore, neither telencephalic 5-HIAA/5-HT ratios nor pituitary POMC A or POMC B mRNA expression showed any decline after 7 days of social interaction. By contrast, plasma cortisol concentrations of subordinate fish declined after 7 days but were still significantly higher than in dominant fish. Furthermore, in subordinate fish, hypothalamic 5-HIAA/5-HT ratios and plasma cortisol levels were highly correlated, suggesting an important role of hypothalamic 5-HT in the regulation of the teleost hypothalamic-pituitary-interrenal (HPI) axis. The number of aggressive acts received and plasma cortisol levels were highly correlated in 1-day subordinates, a relationship not seen in fish subjected to 1 wk of subordination. Thus the chronic stress experienced by subordinates in established dominance hierarchies appears to be more closely related to the threat imposed by the presence of the dominant fish than to actual aggressive encounters. The sustained elevation of pituitary POMC mRNA expression, an effect mainly related to an increase of melanotropic POMC expression, in subordinates could be a mechanism serving to maintain HPI axis excitability and promote acclimation in these individuals.

Learning and sibling odor preference in juvenile arctic char,Salvelinus alpinus (L.).

The importance of learning for sibling odor preference in juvenile Arctic char was analyzed in the present study. Fish were reared in the following eight conditions: (1) communally with siblings for 15 months; (2) communally with siblings for 17 months; (3) in isolation since fertilization; (4) in isolation since fertilization and exposed to sibling scent during the whole rearing period; (5) in isolation since fertilization and exposed to sibling scent from time of free swimming; (6) in isolation since fertilization and exposed to sibling scent during the whole rearing period, except two months without scent until testing; (7) in isolation since fertilization and exposed to sibling scent from time of free swimming, except two months without scent until testing; and (8) communally with siblings followed by a two-month isolation until testing. Char were followed individually in a Y-maze (fluviarium test) with a video-computer-based image analysis system for 12 hr. Sibling-scented water was supplied to one lateral half of the test area and water from non-siblings on the opposite half. Isolated individuals without any preexposure to siblings showed no significant preference. Test fish reared with siblings and those that had been reared in isolation but exposed to sibling scent until testing preferred water conditioned by their own siblings. Isolated fish that had been exposed to sibling scent since fertilization, or since free swimming, followed by a two-month period with only pure water, showed no significant preference. Char isolated for two months after being communally reared preferred water scented by siblings. The results demonstrated that behavioral discrimination between siblings and nonsibling odors occurred after total isolation (isolated both from siblings and sibling odors) only in individuals that had been communally reared. This may suggest that social interactions are important for learning and long-term memory of sibling odors in Arctic char.

Suppression of aggression in rainbow trout (Oncorhynchus mykiss) by dietary L-tryptophan.

Juvenile rainbow trout Oncorhynchus mykiss were isolated in individual compartments in observation aquaria and allowed to acclimate for 1 week, during which they were fed commercial trout feed. Thereafter, the fish were tested for aggressive behaviour using a resident/intruder test. Following this first resident/intruder test, the feed was exchanged for an experimental wet feed supplemented with 0.15 % or 1.5 % L-tryptophan (by wet mass). Controls received the same feed but without L-tryptophan supplementation. The fish were fed to satiety daily, and their individual feed intake was recorded. Aggressive behaviour was quantified again after 3 and 7 days of L-tryptophan feeding using the resident/intruder test. Feeding the fish L-tryptophan-supplemented feed for 3 days had no effect on aggressive behaviour, whereas feeding the fish L-tryptophan-supplemented feed for 7 days significantly suppressed aggressive behaviour in the fish, an effect seen at both levels of L-tryptophan supplementation. Fish fed L-tryptophan-supplemented feed showed elevated plasma and brain levels of L-tryptophan. The amino acid L-tryptophan is the precursor of serotonin, and supplementary dietary L-tryptophan was found to elevate levels of 5-hydroxyindoleacetic acid (5-HIAA) and the 5-HIAA/serotonin concentration ratio in the brain. Neither feed intake nor plasma cortisol level was significantly affected by dietary L-tryptophan. Central serotonin is believed to have an inhibitory effect on aggressive behaviour, and it is suggested that the suppressive effect of dietary L-tryptophan on aggressive behaviour is mediated by an elevation of brain serotonergic activity.

Changes in the brain levels of GABA and related amino acids in anoxic shore crab (Carcinus maenas).

The effects of anoxia on the brain concentrations of gamma-aminobutyric acid (GABA), glutamate, aspartate, glutamine, alanine, and taurine were measured in the shore crab (Carcinus maenas) and compared with data previously obtained from anoxia-tolerant vertebrates. C. maenas was found to survive 12 h in nitrogen-bubbled water. The changes found in brain amino acid levels were strikingly similar to those seen in anoxia-tolerant vertebrates. Thus, during anoxia, the brain of C. maenas displayed considerable increases in the concentrations of GABA (2.4-fold increase after 12 h) and alanine (8-fold increase after 12 h). By contrast, the brain levels of glutamate, aspartate, and glutamine fell significantly during anoxia, whereas the taurine level remained unchanged. Because GABA is a major inhibitory neurotransmitter in arthropods (as well as in most animal phyla), it is suggested that the increased level of GABA could promote the anoxic metabolic depression displayed by C. maenas and thus prolong anoxic survival. It is also possible that the decreases in glutamate and aspartate levels could play similar roles.