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

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

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

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

The endocannabinoid system is affected by a high-fat-diet in rainbow trout

The endocannabinoid system (ECs) is a well known contributor to the hedonic regulation of food intake (FI) in mammals whereas in fish, the knowledge regarding hedonic mechanisms that control FI is limited. Previous studies reported the involvement of ECs in FI regulation in fish since anandamide (AEA) treatment induced enhanced FI and changes of mRNA abundance of appetite-related neuropeptides through cannabinoid receptor 1 (cnr1). However, no previous studies in fish evaluated the impact of palatable food like high-fat diets (HFD) on mechanisms involved in hedonic regulation of FI including the possible involvement of ECs. Therefore, we aimed to evaluate the effect of feeding a HFD on the response of ECs in rainbow trout (Oncorhynchus mykiss). First, we demonstrated a higher intake over 4 days of HFD compared with a control diet (CD). Then, we evaluated the postprandial response (1, 3 and 6 h) of components of the ECs in plasma, hypothalamus, and telencephalon after feeding fish with CD and HFD. The results obtained indicate that the increased FI of HFD occurred along with increased levels of 2-arachidonoylglycerol (2-AG) and AEA in plasma and in brain areas like hypothalamus and telencephalon putatively involved in hedonic regulation of FI in fish. Decreased mRNA abundance of EC receptors like cnr1, gpr55 and trpv1 suggest a feed-back counter-regulatory mechanism in response to the increased levels of EC. Furthermore, the results also suggest that neural activity players associated to FI regulation in mammals as cFOS, γ-Amino butyric acid (GABA) and brain derived neurotrophic factor (BDNF)/neurotrophic receptor tyrosine kinase (NTRK) systems could be involved in the hedonic eating response to a palatable diet in fish.Agencia Estatal de Investigación de España | Ref. PID2019-103969RB-C31Xunta de Galicia | Ref. ED431B 2019/37Xunta de Galicia | Ref. ED481A-2019/291Ministerio de Ciencia e Innovación de España | Ref. BES-2017-079708Ministerio de Educación, Cultura y Deporte de España | Ref. FPU16/00045Xunta de Galicia | Ref. ED481B2018/01