Stress Effects on the Mechanisms Regulating Appetite in Teleost Fish

The homeostatic regulation of food intake relies on a complex network involving peripheral and central signals that are integrated in the hypothalamus which in turn responds with the release of orexigenic or anorexigenic neuropeptides that eventually promote or inhibit appetite. Under stress conditions, the mechanisms that control food intake in fish are deregulated and the appetite signals in the brain do not operate as in control conditions resulting in changes in the expression of the appetite-related neuropeptides and usually a decreased food intake. The effect of stress on the mechanisms that regulate food intake in fish seems to be mediated in part by the corticotropin-releasing factor (CRF), an anorexigenic neuropeptide involved in the activation of the HPI axis during the physiological stress response. Furthermore, the melanocortin system is also involved in the connection between the HPI axis and the central control of appetite. The dopaminergic and serotonergic systems are activated during the stress response and they have also been related to the control of food intake. In addition, the central and peripheral mechanisms that mediate nutrient sensing capacity and hence implicated in the metabolic control of appetite are inhibited in fish under stress conditions. Finally, stress also affects peripheral endocrine signals such as leptin. In the present minireview, we summarize the knowledge achieved in recent years regarding the interaction of stress with the different mechanisms that regulate food intake in fish

Unraveling the periprandial changes in brain serotonergic activity and its correlation with food intake-related neuropeptides in rainbow trout Oncorhynchus mykiss

This study explored changes in brain serotonin content and activity together with hypothalamic neuropeptide mRNA abundance around feeding time in rainbow trout, as well as the effect of one-day fasting. Groups of trout fed at two (ZT2) and six (ZT6) hours after lights on were sampled from 90 minutes before to 240 minutes after feeding, while additional groups of non-fed trout were also included in the study. Changes in brain amine and metabolite contents were measured in hindbrain, diencephalon and telencephalon, while in the diencephalon the mRNA abundance of tryptophan hydroxylase ( tph1 , tph2 ), serotonin receptors (5htr1a , 5htr1b and 5htr2c ) and several neuropeptides ( npy , agrp1 , cartpt , pomca1 , crfb ) involved in the control of food intake were also assessed. The results showed changes in the hypothalamic neuropeptides that were consistent with the expected role for each in the regulation of food intake in rainbow trout. Serotonergic activity increased rapidly at the time of food intake in the diencephalon and hindbrain and remained high for much of the postprandial period. This increase in serotonin abundance was concomitant with elevated levels of pomca1 mRNA in the diencephalon, suggesting that serotonin might act on brain neuropeptides to promote a satiety profile. Furthermore, serotonin synthesis and neuronal activity appear to increase already before the time of feeding, suggesting additional functions for this amine before and during food intake. Exploration of serotonin receptors in the diencephalon revealed only small changes for gene expression of 5htr1b and 5htr2c receptors during the postprandial phase. Therefore, the results suggest that serotonin may play a relevant role in the regulation of feeding behavior in rainbow trout during periprandial time, but a better understanding of its interaction with brain centers involved in receiving and processing food-related signals is still needed.Agencia Estatal de Investigación | Ref. PID2022-136288OB-C31Xunta de Galicia | Ref. ED431B 2019/37Agencia Estatal de Investigación | Ref. BES-2017-079708Xunta de Galicia | Ref. ED481B-2022-08

Influence of Stress on Liver Circadian Physiology. A Study in Rainbow Trout, Oncorhynchus mykiss, as Fish Model

In vertebrates stress negatively affects body homeostasis and triggers a battery of metabolic responses, with liver playing a key role. This organ responds with altered metabolism, leading the animal to cope with the stress situation, which involves carbohydrate and lipid mobilization. However, metabolism among other physiological functions is under circadian control within the liver. Then, metabolic homeostasis at system level involves circadian timing systems within tissues and cells, and collaborate with each other. During chronic stress, cortisol maintains the liver metabolic response by modulating carbohydrate- and lipid-related metabolism. Stress also disrupts the circadian oscillator within the liver in mammals, whereas little information is available in other vertebrates, such as fish. To raise the complexity of this process, other candidates may mediate in such effect of stress. In fact, sirtuin1, a link between cellular sensing of energy status and circadian clocks, participates in the response to stress in mammals, but no information is available in fish. Considering the role played by liver in providing energy for the animal to deal with an adverse situation, and the existence of a circadian oscillator within this tissue, jeopardized liver circadian physiology during stress exposure might be expected. Whether the physiological response to stress is a well conserved process through the phylogeny and the mechanisms involved in such response is a question that remains to be elucidated. Then, we provide information at this respect in mammals and show comparable results in rainbow trout as fish animal model. Similar to that in mammals, stress triggers a series of responses in fish that leads the animal to cope with the adverse situation. Stress influences liver physiology in fish, affecting carbohydrate and lipid metabolism-related parameters, and the circadian oscillator as well. In a similar way than that of mammals different mediators participate in the response of liver circadian physiology to stress in fish. Among them, we confirm for the teleost rainbow trout a role of nuclear receptors (rev-erbβ), cortisol, and sirt1. However, further research is needed to evaluate the independent effect of each one, or the existence of any interaction among them

Papel dos sistemas monoaminérxicos cerebrais (catecolaminas e serotonina) no comportamento alimentario e influenza do estrés en peixes teleósteos

A regulación da inxestión é un dos aspectos máis interesantes a estudar desde o punto de vista da acuicultura a fin de mellorar as prácticas acuícolas e optimizar os rendementos produtivos. Neste proceso interveñen un sistema central regulador do apetito e un periférico, regulador da saciedade. O hipotálamo integra os sinais periféricos e controla a inxestión mediante neuropéptidos orexixénicos (NPY, AGRP) e anorexixénicos (CART, POMC, CRF). Ademais, sábese que os neurotransmisores monoaminérxicos (catecolaminas -noradrenalina, dopamina-, serotonina) modulan a inxestión actuando sobre as neuronas neuropeptidérxicas hipotalámicas. Con todo, a acción das monoaminas na regulación da alimentación é moi ampla, xa que afecta a aspectos tales como o comportamento social e xerárquico, a agresividade, o efecto recompensa do alimento e a propia regulación do apetito e a saciedade. Estudos previos demostraron que as monoaminas exercen unha acción fundamentalmente anorexixénica, establecéndose a participación dalgúns receptores específicos que median as respostas a dopamina e serotonina. Por outra banda, a alimentación organízase rítmicamente no tempo de forma que os peixes adaptan a inxestión a determinados momentos do día, o que engade complexidade á regulación neuroendocrina deste proceso. As situacións que xeran estrés son outro dos condicionantes negativos da inxestión nos peixes. Na resposta de estrés interveñen dous eixos endocrinos (hipotálamo-sistema simpático-células cromafins e hipotálamo-hipófisis-células interrenais) que producen a liberación ao sangue das hormonas catecolaminas e cortisol, as cales desencadean fortes cambios fisiolóxicos e metabólicos. Traballos previos do grupo de UVI no que se desenvolverá a tese doutoral demostraron que as monoaminas cerebrais teñen un papel esencial no inicio da resposta de estrés, podendo actuar sobre os neuropéptidos anorexixénicos para desencadear a inhibición da inxestión. Tamén se hipotetizou que os niveis de cortisol aumentados en situacións de estrés poderían exercer un efecto modulador das aminas cerebrais e do comportamento de inxesta. Con todo, nos peixes falta información de cómo se establecen estas relacións funcionais entre os neurotransmisores monoaminérxicos, o estrés e a regulación do comportamento de alimentación. Tendo en conta estes antecedentes, o obxectivo da presente tese doutoral será o de profundar no papel dos sistemas monoaminérxicos cerebrais na regulación do comportamento alimentario nos peixes teleósteos e na influencia negativa de diferentes tipos de estrés. O plan de investigación inclúe varios obxectivos experimentais que se desenvolverán na troita arco da vella (Oncorhynchus mykiss) como modelo de peixe teleósteo: • Avaliar a actividade dos sistemas dopaminérxico e serotoninérxico en diferentes rexións cerebrais nos tempos peri-prandiales. Relación con parámetros de estrés • Mediante estudos farmacolóxicos, completar o mapa de receptores serotoninérxicos e dopaminérxicos implicados na regulación da inxestión de alimento en peixes teleósteos • Establecer as bases para o estudo do papel dos sistemas monoaminérxicos no comportamento de recompensa ao alimento. Analizarase a actividade monoaminérxica mediante estímulos condicionados ao alimento, a preferencia por dietas con composicións nutricionais específicas e o efecto de sinais orexixénicas e anorexixénicas mediadas por hormonas periféricas (ghrelina, leptina) • Profundar no papel mediador dos neurotransmisores monoaminérxicos na resposta de estrés e nos seus efectos anorexixénicos. Establecer os tipos de receptores implicados • Aplicación das técnicas de microdiálisis cerebral in vivo para ampliar o coñecemento dos mecanismos básicos que regulan a actividade das neuronas monoaminérxicas en peixes teleósteosLa regulación de la ingesta es uno de los aspectos más interesantes a estudiar desde el punto de vista de la acuicultura a fin de mejorar las prácticas acuícolas y optimizar los rendimientos productivos. En este proceso intervienen un sistema central regulador del apetito y uno periférico, regulador de la saciedad. El hipotálamo integra las señales periféricas y controla la ingesta mediante neuropéptidos orexigenicos (NPY, AGRP) y anorexigénicos (CART, POMC, CRF). Además, se sabe que los neurotransmisores monoaminérgicos (catecolaminas -noradrenalina, dopamina-, serotonina) modulan la ingesta actuando sobre las neuronas neuropeptidérgicas hipotalámicas. No obstante, la acción de las monoaminas en la regulación de la alimentación es muy amplia, ya que afecta a aspectos tales como el comportamiento social y jerárquico, la agresividad, el efecto recompensa del alimento y la propia regulación del apetito y la saciedad. Estudios previos han demostrado que las monoaminas ejercen una acción fundamentalmente anorexigénica, habiéndose establecido la participación de algunos receptores específicos que median las respuestas a dopamina y serotonina. Por otra parte, la alimentacion se organiza rítmicamente en el tiempo de forma que los peces adaptan su ingesta a determinados momentos del día, lo que añade complejidad a la regulación neuroendocrina de este proceso. Las situaciones que generan estrés son otro de los condicionantes negativos de la ingesta en los peces. En la respuesta de estrés intervienen dos ejes endocrinos (hipotálamo-sistema simpático-células cromafines e hipotálamo-hipófisis-células interrenales) que producen la liberación a la sangre de las hormonas catecolaminas y cortisol, las cuales desencadenan fuertes cambios fisiológicos y metabólicos. Trabajos previos del grupo de UVI en el que se desarrollará la tesis doctoral han demostrado que las monoaminas cerebrales tienen un papel esencial en el inicio de la respuesta de estrés, pudiendo actuar sobre los neuropéptidos anorexigénicos para desencadenar la inhibición de la ingesta. También se ha hipotetizado que los niveles de cortisol aumentados en situación de estrés podrían ejercer un efecto modulador de las aminas cerebrales y del comportamiento de ingesta. Sin embargo, en los peces falta información de como se establecen estas relaciones funcionales entre los neurotransmisores monoaminérgicos, el estrés y la regulación del comportamiento de alimentación. Teniendo en cuenta estos antecedentes, el objetivo de la presente tesis doctoral será el de profundizar en el papel de los sistemas monoaminérgicos cerebrales en la regulación del comportamento alimentario en los peces teleósteos y en la influencia negativa de diferentes tipos de estrés. El plan de investigación incluye varios objetivos experimentales que se desarrollarán en la trucha arco iris (Oncorhynchus mykiss) como modelo de pez teleósteo: • Evaluar la actividad de los sistemas dopaminérgico y serotoninérgico en diferentes regiones cerebrales en los tiempos peri-prandiales. Relación con parámetros de estrés • Mediante estudios farmacológicos, completar el mapa de receptores serotoninérgicos y dopaminérgicos implicados en la regulación de la ingesta de alimento en peces teleósteos • Establecer las bases para el estudio del papel de los sistemas monoaminérgicos en el comportamiento de recompensa al alimento. Se analizará la actividad monoaminérgica mediante estímulos condicionados al alimento, la preferencia por dietas con composiciones nutricionales específicas y el efecto de señales orexigénicas y anorexigénicas mediadas por hormonas periféricas (ghrelina, leptina) • Profundizar en el papel mediador de los neurotransmisores monoaminérgicos en la respuesta de estrés y sus efectos anorexigénicos. Tipos de receptores implicados • Aplicación de las técnicas de microdiálisis cerebral in vivo para ampliar el conocimiento de los mecanismos básicos que regulan a actividade de las neuronas monoaminérgicas en peces teleóThe regulation of intake is one of the most interesting aspects to be studied from the point of view of aquaculture in order to improve aquaculture practices and optimize yields. This process involves a central regulatory system of appetite and a peripheral one, which regulates saciety. The hypothalamus integrates peripheral signals and controls ingestion by orexigenic (NPY, AGRP) and anorexigenic neuropeptides (CART, POMC, CRF). In addition, monoaminergic neurotransmitters (catecholamines -noradrenaline, dopamine-, serotonin) are known to modulate food intake by acting on hypothalamic neuropeptidergic neurons. However, the action of monoamines in regulating feeding is very extensive, as it affects aspects such as social and hierarchical behavior, aggressiveness, reward effect of food and regulation of appetite and satiety. Previous studies have shown that monoamines basically exert an anorexic action, with the participation of some specific receptors mediating the responses to dopamine and serotonin. On the other hand, feeding is organized rhythmically over time so that fish adapt their diet at certain times of the day, which adds complexity to the neuroendocrine regulation of intake. The situations that generate stress in fish are another of the negative conditioning factors of food intake. The stress response involves two endocrine axes (hypothalamus-sympathetic system-chromaffin cells and hypothalamus-pituitary-interrenal cells) that produce the release into the blood of the hormones catecholamines and cortisol, which trigger strong physiological and metabolic changes. Previous work by the UVI group in which the doctoral thesis will be developed have shown that the cerebral monoamines play an essential role in the onset of the stress response, being able to act on the anorexigenic neuropeptides to trigger the inhibition of the food intake. It has also been hypothesized that increased levels of cortisol under stress could exert a modulatory effect on brain amines and on intake behavior. However, knowledge is lacking in fish on how these functional relationships between monoaminergic neurotransmitters, stress and regulation of feeding behavior are established. Taking into account this background, the objective of this doctoral thesis will be to deepen the role of brain monoaminergic systems in regulating feeding behavior in teleost fishes and in the negative influence of different types of stress. The research plan includes several experimental objectives that will be developed in rainbow trout (Oncorhynchus mykiss) as a model of teleost fish: • To evaluate the activity of dopaminergic and serotonergic systems in different brain regions during the peri-prandial times. Relationship with stress parameters • By using pharmacological studies, to complete the map of serotonergic and dopaminergic receptors involved in regulation of food intake in teleost fish • To establish the basis for the study of the role of monoaminergic systems in rewarding food behavior. Monoaminergic activity will be analyzed during food-conditioned stimuli, on preference for diets with specific nutritional compositions, and on the effect of orexigenic and anorexigenic signals mediated by peripheral hormones (ghrelin, leptin) • To deepen the role of monoaminergic neurotransmitters in the stress response and its anorectic effects. To establish the types of receptors involved in this action • Application of the brain microdialysis in vivo techniques to expand the knowledge of the basic mechanisms that regulate the activity of monoaminergic neurons in teleost fishMinisterio de Ciencia, Inovación y Universidades | Ref. BES-2017-07970

Obesity Impairs Cognitive Function with No Effects on Anxiety-like Behaviour in Zebrafish

Over the last decade, the zebrafish has emerged as an important model organism for behavioural studies and neurological disorders, as well as for the study of metabolic diseases. This makes zebrafish an alternative model for studying the effects of energy disruption and nutritional quality on a wide range of behavioural aspects. Here, we used the zebrafish model to study how obesity induced by overfeeding regulates emotional and cognitive processes. Two groups of fish (n = 24 per group) were fed at 2% (CTRL) and 8% (overfeeding-induced obesity, OIO) for 8 weeks and tested for anxiety-like behaviour using the novel tank diving test (NTDT). Fish were first tested using a short-term memory test (STM) and then trained for four days for a long-term memory test (LTM). At the end of the experiment, fish were euthanised for biometric sampling, total lipid content, and triglyceride analysis. In addition, brains (eight per treatment) were dissected for HPLC determination of monoamines. Overfeeding induced faster growth and obesity, as indicated by increased total lipid content. OIO had no effect on anxiety-like behaviour. Animals were then tested for cognitive function (learning and memory) using the aversive learning test in Zantiks AD units. Results show that both OIO and CTRL animals were able to associate the aversive stimulus with the conditioned stimulus (conditioned learning), but OIO impaired STM regardless of fish sex, revealing the effects of obesity on cognitive processes in zebrafish. Obese fish did not show a deficiency in monoaminergic transmission, as revealed by quantification of total brain levels of dopamine and serotonin and their metabolites. This provides a reliable protocol for assessing the effect of metabolic disease on cognitive and behavioural function, supporting zebrafish as a model for behavioural and cognitive neuroscience

Differential circadian and light-driven rhythmicity of clock gene expression and behaviour in the turbot, Scophthalmus maximus

In fish, the circadian clock represents a key regulator of many aspects of biology and is controlled by combinations of abiotic and biotic factors. These environmental factors are frequently manipulated in fish farms as part of strategies designed to maximize productivity. The flatfish turbot, Scophthalmus maximus, represents one of the most important species within the aquaculture sector in Asia and Europe. Despite the strategic importance of this species, the function and regulation of the turbot circadian system remains poorly understood. Here, we have characterized the core circadian clock genes, clock1, per1, per2 and cry1 in turbot and have studied their daily expression in various tissues under a range of lighting conditions and feeding regimes. We have also explored the influence of light and feeding time on locomotor activity. Rhythmic expression of the four core clock genes was observed in all tissues studied under light dark (LD) cycle conditions. Rhythmicity of clock gene expression persisted upon transfer to artificial free running, constant conditions confirming their endogenous circadian clock control. Furthermore, turbot showed daily cycles of locomotor activity and food anticipatory activity (FAA) under LD and scheduled-feeding, with the activity phase as well as FAA coinciding with and being dependent upon exposure to light. Thus, while FAA was absent under constant dark (DD) conditions, it was still detected in constant light (LL). In contrast, general locomotor activity was arrhythmic in both constant darkness and constant light, pointing to a major contribution of light, in concert with the circadian clock, in timing locomotor activity in this species. Our data represents an important contribution to our understanding of the circadian timing system in the turbot and thereby the optimization of rearing protocols and the improvement of the well-being of turbot within fish farming environments

Obesity Impairs Cognitive Function with No Effects on Anxiety-like Behaviour in Zebrafish

Over the last decade, the zebrafish has emerged as an important model organism for behavioural studies and neurological disorders, as well as for the study of metabolic diseases. This makes zebrafish an alternative model for studying the effects of energy disruption and nutritional quality on a wide range of behavioural aspects. Here, we used the zebrafish model to study how obesity induced by overfeeding regulates emotional and cognitive processes. Two groups of fish (n = 24 per group) were fed at 2% (CTRL) and 8% (overfeeding-induced obesity, OIO) for 8 weeks and tested for anxiety-like behaviour using the novel tank diving test (NTDT). Fish were first tested using a short-term memory test (STM) and then trained for four days for a long-term memory test (LTM). At the end of the experiment, fish were euthanised for biometric sampling, total lipid content, and triglyceride analysis. In addition, brains (eight per treatment) were dissected for HPLC determination of monoamines. Overfeeding induced faster growth and obesity, as indicated by increased total lipid content. OIO had no effect on anxiety-like behaviour. Animals were then tested for cognitive function (learning and memory) using the aversive learning test in Zantiks AD units. Results show that both OIO and CTRL animals were able to associate the aversive stimulus with the conditioned stimulus (conditioned learning), but OIO impaired STM regardless of fish sex, revealing the effects of obesity on cognitive processes in zebrafish. Obese fish did not show a deficiency in monoaminergic transmission, as revealed by quantification of total brain levels of dopamine and serotonin and their metabolites. This provides a reliable protocol for assessing the effect of metabolic disease on cognitive and behavioural function, supporting zebrafish as a model for behavioural and cognitive neuroscience.De två sista författarna delar sistaförfattarskapet.</p

Central administration of endocannabinoids exerts bimodal effects in food intake of rainbow trout

The endocannabinoid system (ECs) is known to participate in several processes in mammals related to synaptic signaling including regulation of food intake, appetite and energy balance. In fish, the relationship of ECs with food intake regulation is poorly understood. In the present study, we assessed in rainbow trout Oncorhynchus mykiss the effect of intracerebroventricular administration (ICV) of low and high doses of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) on food intake. We assessed endocannabinoid levels in hypothalamus, telencephalon and plasma as well as the effect of AEA and 2-AG administration at central level on gene expression of receptors involved in ECs (cnr1, gpr55 and trpv1) and markers of neural activity (fos, ntrk2 and GABA-related genes). The results obtained indicate that whereas high doses of endocannabinoids did not elicit changes in food intake levels, low doses of the endocannabinoids produce an orexigenic effect that could be due to a possible inhibition of gabaergic neurotransmission and the modulation of neural plasticity in brain areas related to appetite control, such as hypothalamus and telencephalon.Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGXunta de Galicia | Ref. ED431B 2019/37Xunta de Galicia | Ref. ED481A-2019/291Xunta de Galicia | Ref. ED481B2018/018Agencia Estatal de Investigación | Ref. BES-2017-079708Agencia Estatal de Investigación | Ref. PID2019-103969RB-C31Ministerio de Educación, Cultura y Deporte (España) | Ref. FPU16/0004

Differential circadian and light-driven rhythmicity of clock gene expression and behaviour in the turbot, Scophthalmus maximus.

In fish, the circadian clock represents a key regulator of many aspects of biology and is controlled by combinations of abiotic and biotic factors. These environmental factors are frequently manipulated in fish farms as part of strategies designed to maximize productivity. The flatfish turbot, Scophthalmus maximus, represents one of the most important species within the aquaculture sector in Asia and Europe. Despite the strategic importance of this species, the function and regulation of the turbot circadian system remains poorly understood. Here, we have characterized the core circadian clock genes, clock1, per1, per2 and cry1 in turbot and have studied their daily expression in various tissues under a range of lighting conditions and feeding regimes. We have also explored the influence of light and feeding time on locomotor activity. Rhythmic expression of the four core clock genes was observed in all tissues studied under light dark (LD) cycle conditions. Rhythmicity of clock gene expression persisted upon transfer to artificial free running, constant conditions confirming their endogenous circadian clock control. Furthermore, turbot showed daily cycles of locomotor activity and food anticipatory activity (FAA) under LD and scheduled-feeding, with the activity phase as well as FAA coinciding with and being dependent upon exposure to light. Thus, while FAA was absent under constant dark (DD) conditions, it was still detected in constant light (LL). In contrast, general locomotor activity was arrhythmic in both constant darkness and constant light, pointing to a major contribution of light, in concert with the circadian clock, in timing locomotor activity in this species. Our data represents an important contribution to our understanding of the circadian timing system in the turbot and thereby the optimization of rearing protocols and the improvement of the well-being of turbot within fish farming environments

Periprandial changes in brain serotonergic system and food intake related neuropeptides [Dataset]

In this work, we assessed periprandial serotonin and 5-hydroxyindolacetic acid abundance in several brain areas of rainbow trout, in parallel with the evaluation of gene expression of tryptophan hydroxilase 1 and 2, neuropeptides involved in the central control of food intake, and the quantification of plasma glucose and cortisol. The results showed severe changes in serotonergic systems around mealtime, the most prominent being the increase in their activity just after food intake, suggesting that serotonin has a relevant role in relation to the daily timing of food intake, probably triggering satiety signals. In addition, a temporal adjustment of neuropeptide expression and plasma cortisol was found in relation to food intake, supporting its role in the regulation of feeding behaviour.Agencia Estatal de Investigación, Award: PID2019-103969RBC31-C33. Xunta de Galicia, Award: ED431B 2019/37Peer reviewe