Modifying the physiological responses to handling stress in beluga sturgeon Huso huso (Linnaeus, 1758); Interactive effects of feeding time and dietary fat

The present study investigated the interaction of feeding times with two dietary fat levels on physiological responses to handling stress in juvenile Beluga sturgeon. Fish were fed with two different diets (high energy; HE: 24% fat and low energy; LE: 12% fat) for 8 weeks at two feeding times; 09:00 and 16:00 (during the day) or 21:00 and 04:00 (during the night). At the end of the trial, blood samples were taken and the resting fish were held in a net out of water for 90 s as the handling stressor. Three hours after application of stress, post‐stress blood was taken. Cortisol, glucose, and lactate concentrations were considered as stress indicators. The mean values of cortisol and lactate levels did not reveal a significant difference between pre‐ and 3‐hr post‐stress samples, but the average concentration of glucose showed a significant difference. Cortisol, lactate and glucose concentrations were not influenced by the difference in the diets. Moreover, the cortisol and lactate concentrations were not affected by the different feeding times, while glucose levels were significantly affected by the feeding times with the lowest level in fish fed during the day. With respect to stress indicators, the results revealed that feeding times affected pre‐ and post‐stress secondary response to handling stress, but the rate and magnitude of metabolites (cortisol, glucose and lactate) were not affected by dietary fat levels. Therefore, it is necessary to examine the best feeding times with the interaction of feed ingredients in sturgeon farming.     

Learning-Induced Gene Expression in the Hippocampus Reveals a Role of Neuron -Astrocyte Metabolic Coupling in Long Term Memory

We examined the expression of genes related to brain energy metabolism and particularly those encoding glia (astrocyte)-specific functions in the dorsal hippocampus subsequent to learning. Context-dependent avoidance behavior was tested in mice using the step-through Inhibitory Avoidance (IA) paradigm. Animals were sacrificed 3, 9, 24, or 72 hours after training or 3 hours after retention testing. The quantitative determination of mRNA levels revealed learning-induced changes in the expression of genes thought to be involved in astrocyte-neuron metabolic coupling in a time dependent manner. Twenty four hours following IA training, an enhanced gene expression was seen, particularly for genes encoding monocarboxylate transporters 1 and 4 (MCT1, MCT4), alpha2 subunit of the Na/K-ATPase and glucose transporter type 1. To assess the functional role for one of these genes in learning, we studied MCT1 deficient mice and found that they exhibit impaired memory in the inhibitory avoidance task. Together, these observations indicate that neuron-glia metabolic coupling undergoes metabolic adaptations following learning as indicated by the change in expression of key metabolic genes.     

Local adaptation and embryonic plasticity affect antipredator traits in hatchling pond snails

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Cortisol response and immune-related effects of Atlantic salmon (Salmo salar Linnaeus) subjected to short- and long-term stress

It is generally considered that stress causes decreased immune function in fish. In this study we examined in Atlantic salmon (Salmo salar Linnaeus) the effects of both short- (a single 15s out of water) and long-term (4 weeks of daily handling 15s out of water) stress on plasma cortisol (free and total) and glucose levels, expression of interleukin-1beta (IL-1beta) and survival of head kidney (HK) macrophages under culture with Aeromonas salmonicida. In the short-term study, samples were collected prior to the application of the stressor, and at 1, 3, 6, 12 and 24h post stress. Free and total plasma cortisol levels and the percentage of free cortisol increased significantly in the stressed group at 1 and 3h post stress. Plasma glucose levels were significantly higher than those of control fish at 1, 3 and 6h post stress. Constitutive expression of IL-1beta in macrophages isolated from head kidneys in stressed fish was significantly higher at 1 and 3h post stress. However, lipopolysaccharide (LPS) stimulated expression of IL-1beta in HK macrophages, exhibited significantly higher fold increases in unstressed fish compared to stressed fish. In the long-term study, with the exception of an increase in plasma glucose levels at 1 week, there were no significant differences in stress parameters between groups. There was a significantly higher constitutive IL-1beta expression in macrophages isolated from stressed fish over the first 2 weeks. At weeks 1, 2 and 3 the magnitude of IL-1beta response of isolated HK macrophages to LPS stimulation was reduced in >90% of the stressed fish. At 4 weeks there was no significant difference in inducible IL-1beta expression between the groups. Macrophages isolated from stressed fish also showed significantly decreased survival when exposed to A. salmonicida. This study shows a clear pattern from repeated handling stress, whereby effects on immune cells begin with increased constitutive expression of IL-1beta, followed by decreased stimulation of leucocytes by extracellular antigen, and finally decreased leukocyte survival when exposed to A. salmonicida. The implications of these changes in the immune system will be discussed with respect to the use of classical indicators of stress to predict possible effects on the immune system of fish.     

Expression of warm temperature acclimation-related protein 65-kDa (Wap65) mRNA, and physiological changes with increasing water temperature in black porgy, Acanthopagrus schlegeli

We isolated the warm temperature acclimation-related protein 65-kDa (Wap65) cDNA from the liver of black porgy and investigated the expression by increasing water temperature in black porgy, Acanthopagrus schlegeli. Black porgy Wap65 full-length cDNA consists of 1,338 nucleotides, including an open reading frame, predicted to encode a protein of 425 amino acids and showed high homology to pufferfish (79%), Medaka (73%), carp (70%), and goldfish (68%) Wap65. Increase in water temperature (20 degrees C --> 30 degrees C; 1 degrees C/day) induced the rise of Wap65 mRNA expression in liver of black porgy. Also, the levels of cortisol and glucose in plasma were significantly higher at 30 degrees C than at 20 degrees C. To determine the high water temperature stressor specificity of the induction of Wap65, black porgy were transferred from seawater (SW) to freshwater (FW) for 24 hr. Wap65 expression was not detected when the fish were transferred from SW to FW (in fish transferred from SW to FW), although the levels of cortisol and glucose in plasma were increased. These results suggest that increase in Wap65 gene is related to high water temperature stress and play important roles in high water temperature environment of black porgy.     

Rapid and Transient Learning-Associated Increase in NMDA NR1 Subunit in the Rat Hippocampus

Several lines of evidence indicate that glutamate NMDA receptors are critically involved in long-term potentiation (LTP) and in certain forms of learning. It was previously demonstrated that memory formation of an inhibitory avoidance task in chick is specifically associated with an increase in the density of NMDA receptor in selected brain regions. Here we report on the effect of a one trial inhibitory avoidance training in rats, a hippocampal-dependent learning task, on the levels of different subunits of the glutamate NMDA receptor in synaptic plasma membranes (SPM) isolated from the hippocampus. Training rats on a one trial inhibitory avoidance task results in a rapid, transient and selective increase (+33 %, p < 0.05) in NMDA NR1 subunit expression in hippocampal SPM of rats sacrificed 30 min posttraining. No changes were observed at 0 or 120 min after training or in shocked animals in comparison to naive control rats. In addition, no training-associated increase in the levels of NMDA NR2A and NR2B or AMPA GluR 2/3 subunits was observed at any timepoint tested. In conclusion, the present findings support the hypothesis that alterations in expression of synaptic NMDA NR1 subunits in the hippocampus are specifically associated with memory formation of an inhibitory avoidance task and strongly suggest that hippocampal NMDA receptors are crucially involved in the neural mechanisms underlying certain forms of learning.These authors contributed equally to this work     

The role of social learning in conservation and fisheries reintroductions

The plight of the world fish stocks is all too well documented. As part of an ongoing attempt to bolster fish stocks for both commercial and conservation purposes, many fish are reared in captivity and released into the wild. It is well known that hatchery‐reared fish have low post‐release survival compared with wild fish of similar age. Part of the reason for this high mortality is that hatchery fish show deficits in virtually all aspects of their behaviour, including prey selection and predator avoidance. Much behaviour requires repeated experience so that it may become fine‐tuned to prevailing circumstances via learning during development. It has been suggested that inappropriate behaviour is encouraged when fish are reared in the unnatural surroundings of the hatchery. However, hatchery fish can be taught to recognise live, novel prey items and predators and the rate of learning is increased in the presence of a more knowledgeable conspecifics. Here we present data showing how social learning protocols can be used to dramatically increase foraging success in juvenile Atlantic salmon. We also outline related aspects of our ongoing research and discuss some of the possibilities for altering hatchery practices to maximize post‐release survival.     

Cortisol response to waterborne 4-nonylphenol exposure leads to increased brain POMC and HSP70 mRNA expressions and reduced total antioxidant capacity in juvenile sole (Solea solea)

4-Nonylphenol (4-NP) is a breakdown product of alkylphenolpolyethoxylates and can be found in almost all environmental water matrices. 4-NP can act as environmental stressor on fish, typically causing modulation of hypothalamic-pituitary-interrenal axis (HPI). To examine the effects of the xenoestrogen 4-NP or 17β-estradiol (E2) on induction of stress response mechanisms by evaluating the levels of proopiomelanocortin (POMC) mRNA, heat shock protein 70 (HSP70) mRNA and plasma cortisol, we exposed juvenile sole (Solea solea), under static condition for 7day, to either 10(-6) or 10(-8)M 4-NP, or 10(-8)M E2. In addition, plasma cortisol titers were correlated to the total antioxidant capacity (TAC), one of the oxidative stress parameters. 4-NP treatments resulted in high levels of POMC mRNA, HSP70 mRNA and plasma cortisol. On the contrary, E2 basically down-regulated POMC expression. Moreover, elevated cortisol levels in fish exposed to the highest dose of 4-NP were accompanied by low TAC. These results suggest that 4-NP modulates the sole HPI axis inducing a cortisol-mediated stress response. Specifically, we suggest that 4-NP affects brain POMC mRNA levels via non-estrogen receptor (ER)-mediated mechanism further supporting the ability of 4-NP to target multiple receptor systems.     

Levels of arginine-vasopressin in cerebrospinal fluid during passive avoidance behavior in rats

The concentration of immunoreactive arginine-vasopressin (IR-AVP) was measured in the cerebrospinal fluid (CSF) during acquisition and retention of passive avoidance behavior. IR-AVP level in CSF of male Wistar rats immediately after the learning trial was increased; the rate of which was related to the intensity of the electric footshock during the learning trial and the avoidance latency as measured 1 day after the learning trial. Immediately after the 24 h retention test IR-AVP levels were significantly increased in rats subjected to the low (0.25 mA) shock intensity during the learning trial, but IR-AVP levels of rats exposed to the high shock (1.0 mA) were under the limit of detection. If the retention test was postponed till 5 days after the learning trial, the increase of IR-AVP level in the CSF was related to avoidance latencies which reflect the intensity of aversive stimulation (electric footshock). The results suggest an association between central AVP release and passive avoidance behavior and may be indicative of the role of this peptide in neuronal mechanisms underlying learning and memory processes.     

Agonistic encounters and cellular angst: social interactions induce heat shock proteins in juvenile salmonid fish

Juvenile salmonid fish readily form dominance hierarchies when faced with limited resources. While these social interactions may result in profound behavioural and physiological stress, it is unknown if this social stress is evident at the level of the cellular stress response—specifically, the induction of stress or heat shock proteins (Hsps). Thus, the goal of our study was to determine if Hsps are induced during hierarchy formation in juvenile rainbow trout (Oncorhynchus mykiss). To this end, we measured levels of three Hsps, Hsp70, Hsc (heat shock cognate)70 and Hsp90 in the white muscle, liver and brain of trout that had been interacting for 36 h, 72 h or 6 days. Our data indicate that Hsps are induced in both dominant and subordinate fish in a time- and tissue-specific manner. In further mechanistic experiments on fasted and cortisol-treated fish, we demonstrated that high plasma cortisol does not affect Hsp induction in trout white muscle or liver, but both conditions may be part of the mechanism for Hsp induction with social stress in the brain. We conclude that the behavioural and physiological stress experienced by juvenile rainbow trout in dominance hierarchies can be extended to the induction of Hsps.     

Egg Cortisol Exposure Enhances Fearfulness in Larvae and Juvenile Rainbow Trout

We investigated the effects of an early boost of cortisol exposure in rainbow trout (Oncorhynchus mykiss) eggs during fertilisation on subsequent behavioural responses when exposed to a sudden stimulus in larvae and juveniles. At 55 d post‐fertilisation (dpf), treatment had no effect on high accelerations occurring after a sudden event. At 146 dpf, these high accelerations were more frequent in cortisol‐treated fish than in controls. At 146 dpf also, swimming activity was increased in cortisol‐treated fish both before and after the sudden stimulus. This study underlines the important behavioural modifications in both larvae and juveniles, linked to a change in the surrounding environment of the embryo. Indeed, fish exposed to cortisol as eggs showed a higher level of fearfulness later in life. Our findings are of major interest for stress management in an aquaculture context and also allow for a better understanding of the long‐lasting effects of a permanent and/or acute stress – mediated by cortisol – that could be encountered by females, affecting population's life history trajectory.     

Changes in free and total plasma cortisol levels in juvenile haddock (Melanogrammus aeglefinus) exposed to long-term handling stress

We measured changes in free and total plasma cortisol levels, plasma glucose, gill hsp70 levels, and growth in haddock (Melanogrammus aeglefinus) subjected to a long-term handling stress (15 s out of water, each day, for 4 weeks), and the effect of this long-term stress on the ability of haddock to respond to an acute stressor. The acute stressor was a single handling stress, and fish were sampled at 1, 6, and 12 h post-stress. During the long-term stress study, free and total plasma cortisol levels increased significantly (10-fold) in the stressed group after the second week. However, the percentage of free cortisol was already significantly elevated by the first week (control 17%, stressed 55%), and remained high during the second week (control 35% and stressed 65%). After 3 and 4 weeks of handling, both free and total cortisol declined in stressed fish to levels that were not significantly different from pre-stress values. Control fish grew significantly more than stressed fish (by 32% and 18%, respectively) over the 4 week study, and condition factor only increased in control fish. Although fish from the control group showed elevated total plasma cortisol levels (to 47 ng mL(-1)) 1 h after the acute stress, and the levels in stressed fish were comparable to those for the control fish, no significant increase in plasma cortisol was measured in the group subjected to the long-term stress. Free plasma cortisol levels did not increase significantly in either group following the acute stress. However, free plasma cortisol levels were significantly higher in long-term stress group, as compared with the control group, at 6 h post-stress. Plasma glucose and gill hsp70 levels were not altered by either the long-term stress or acute stressor. Our data indicate that cortisol (free and total), but not glucose or hsp70, appears to be adequate to assess short- and long-term stress in haddock.     

The impact of mineralocorticoid receptor ISO/VAL genotype (rs5522) and stress on reward learning

Research suggests that stress disrupts reinforcement learning and induces anhedonia. The mineralocorticoid receptor (MR) determines the sensitivity of the stress response, and the missense iso/val polymorphism (Ile180Val, rs5522) of the MR gene (NR3C2) has been associated with enhanced physiological stress responses, elevated depressive symptoms and reduced cortisol‐induced MR gene expression. The goal of these studies was to evaluate whether rs5522 genotype and stress independently and interactively influence reward learning. In study 1, participants (n = 174) completed a probabilistic reward task under baseline (i.e. no‐stress) conditions. In study 2, participants (n = 53) completed the task during a stress (threat‐of‐shock) and no‐stress condition. Reward learning, i.e. the ability to modulate behavior as a function of reinforcement history, was the main variable of interest. In study 1, in which participants were evaluated under no‐stress conditions, reward learning was enhanced in val carriers. In study 2, participants developed a weaker response bias toward a more frequently rewarded stimulus under the stress relative to no‐stress condition. Critically, stress‐induced reward learning deficits were largest in val carriers. Although preliminary and in need of replication due to small sample size, findings indicate that psychiatrically healthy individuals carrying the MR val allele, gene, which has been recently linked to depression, showed a reduced ability to modulate behavior as a function of reward when facing an acute, uncontrollable stressor. Future studies are warranted to evaluate whether rs5522 genotype interacts with naturalistic stressors to increase the risk of depression and whether stress‐induced anhedonia might moderate such risk.     

Baseline cortisol levels and social behavior differ as a function of handedness in marmosets (Callithrix jacchus)

Population hand preferences are rare in nonhuman primates, but individual hand preferences are consistent over a lifetime and considered to reflect an individual's preference to use a particular hemisphere when engaged in a specific task. Previous findings in marmosets have indicated that left‐handed individuals tend to be more fearful than their right‐handed counterparts. Based on these findings, we tested the hypotheses that left‐handed marmosets are (a) more reactive to a social stressor and (b) are slower than right‐handed marmosets in acquiring a reversal learning task. We examined the hand preference of 27 male and female marmosets (ages of 4–7 years old) previously tested in a social separation task and a reversal learning task. Hand preference was determined via a simple reaching task. In the social separation task, monkeys were separated from their partner and the colony for a single 7‐hr session. Urinary cortisol levels and behavior were assessed at baseline, during the separation and 24 hr postseparation. Hand preferences were equally distributed between left (n = 10), right‐handed (n = 10), and ambidextrous (n = 7) individuals. The separation phase was associated with an increase in cortisol levels and behavioral changes that were similar across handedness groups. However, cortisol levels at baseline were positively correlated with right‐handedness, and this relationship was stronger in females than in males. In addition, the occurrence of social behaviors (pre‐ and postseparation) was positively correlated with right‐handedness in both sexes. Baseline cortisol levels did not correlate significantly with social behavior. Acquisition of the reversals was poorer in females than males but did not differ as a function of handedness. We conclude that (a) both stress reactivity and cognitive flexibility are similar across handedness groups and (b) left‐handers exhibit less social behavior and have lower basal cortisol levels than ambidextrous and right‐handed subjects. The underlying causes for these differences remain to be established.     

Genetic disruptions of Drosophila Pavlovian learning leave extinction learning intact

Individuals who experience traumatic events may develop persistent posttraumatic stress disorder. Patients with this disorder are commonly treated with exposure therapy, which has had limited long‐term success. In experimental neurobiology, fear extinction is a model for exposure therapy. In this behavioral paradigm, animals are repeatedly exposed in a safe environment to the fearful stimulus, which leads to greatly reduced fear. Studying animal models of extinction already has lead to better therapeutic strategies and development of new candidate drugs. Lack of a powerful genetic model of extinction, however, has limited progress in identifying underlying molecular and genetic factors. In this study, we established a robust behavioral paradigm to study the short‐term effect (acquisition) of extinction in Drosophila melanogaster. We focused on the extinction of olfactory aversive 1‐day memory with a task that has been the main workhorse for genetics of memory in flies. Using this paradigm, we show that extinction can inhibit each of two genetically distinct forms of consolidated memory. We then used a series of single‐gene mutants with known impact on associative learning to examine the effects on extinction. We find that extinction is intact in each of these mutants, suggesting that extinction learning relies on different molecular mechanisms than does Pavlovian learning.