Food choice is a modifiable health factor which involves neural, hormonal, and metabolic signals. The lateral parabrachial nucleus is a brain structure in the pons that integrates multiple aspects of food choice. It receives information from the homeostatic melanocortin hypothalamic system and projects to the mesolimbic dopamine reward system. The lateral parabrachial is molecularly diverse and expresses the acetylcholine synthesis enzyme: choline acetyltransferase (ChAT). In this study, we used ChAT-CRE mice to investigate the anatomical projections, the calcium dynamics, and the causal role of the LPBN ChAT neurons. Anatomical projection results were produced using CRE dependent viral vectors to express mRuby tagged synaptophysin, the highest output being the central amygdala. Calcium dynamics were measured at the amygdala using the genetically encoded calcium indicator GCaMP. The dynamics around the decision to consume food were seen to be different between fasted and sated satiety states. Activation of the circuit showed a pronounced latency to food consumption and time to finish for a single calorie of food. These data demonstrate a possible node that integrates homeostatic feeding information and relays it to higher order brain systems that modify reward value. / Master of Science / Health can be impacted by the food an individual decides to eat, and this choice is controlled by the brain. There are many regions of the brain that are recruited when an individual decides to eat, but the two major circuits recruited are the homeostatic feeding circuit and the reward feeding circuit. The homeostatic feeding circuit involves the hypothalamus, the structure that controls basic essential functions of the body and circulating hunger hormones to signal energy availability. The second circuit is the reward circuitry which uses the neurotransmitter dopamine to signal pleasure and motivation for food. At the middle of the two circuits sits the parabrachial nucleus which expresses choline acetyltransferase, the enzyme that creates the neurotransmitter acetylcholine. To harness the molecular and anatomical specificity, we employed viral dependent protein expression to measure the anatomical output, the activity when a mouse is engaged in feeding behavior, and the causal role of the identified circuit during feeding behavior. The results showed the anatomical output to be the central amygdala, a modifier of food reward and value. The activity of the cells while feeding was seen to be higher when sated, and the activation of the circuit saw an increased latency to eat food and increased the time to consume a calorie. Together, we have demonstrated a circuit from the parabrachial nucleus the amygdala which integrates homeostatic information and projects to a brain structure that modifies food value and reward.

Animal survival depends on the brain’s ability to detect the energetic state of the body and to alter behaviour in order to maintain homeostasis. Current research in the control of food consumption stresses the importance of identifying and establishing the specific roles of homeostatic neurons, which sense the body’s energetic state and elicit complex and flexible food seeking behaviours. Recent developments in optogenetics, molecular genetics, and anatomical techniques have made these investigations possible at the resolution of specific cell types and circuits. These neurons are of particular interest because they serve as key entry points to the identification of downstream circuits and reinforcement mechanisms that control feeding behaviour. This dissertation probes the role of two kinds of homeostatic neurons— agouti-related peptide (AGRP) in the arcuate nucleus (ARC) and leptin receptor (LepRb) neurons in the lateral hypothalamic area (LHA)—in the control of food intake. First, I examined the role of LepRb neurons in the LHA in feeding. Results from electrophysiological studies indicate that these neurons consist of a subpopulation of homeostatic sensing LHA γ-aminobutyric acid (GABA) expressing neurons. In addition to their response to leptin, these neurons are capable of modulating their activity in response to changes in glucose levels, further substantiating their role as homeostatic sensing neurons. Behavioural studies using optogenetic activation of these neurons show that their elevated activity is capable of reducing body weight, although their role in modulating feeding remains unclear. Second, I investigated the reinforcement mechanisms employed by AGRP neurons to elicit voracious food consumption and increased willingness to work for food. Conditioned place avoidance studies under optogenetic activation of AGRP neurons reveal that their increased activity has negative valence and is avoided. In addition, imposition of elevated AGRP neuron activity in an operant task reduced instrumental food seeking with particular sensitivity under high effort requirements. Taken together, these results suggest that AGRP neurons employ a negative reinforcement teaching signal to direct action selection during food seeking and consumption. Third, I systematically analyzed the contribution of specific AGRP neuron projection subpopulations in AGRP neuron mediated evoked-feeding behaviour. Optogenetic activation studies of AGRP neuron axons in downstream projection regions indicate that several, but not all, subpopulations are capable of independently evoke food consumption. This work reveals a parallel and redundant functional circuit organization for AGRP neurons in the control of food intake. Interestingly, all AGRP neuron subpopulations examined displayed similar modulation by states of energy deficit and signals of starvation, despite their apparent divergence in function. As a whole, this dissertation extends our understanding of the role of homeostatic neurons in food consumption and uncovers previously unappreciated functional organization and reinforcement mechanisms employed by neuronal circuits that control feeding behaviour.

Nutrient sensing neurons are unique in coupling changes in the concentration of nutrients to changes in neuronal activity. These neurons typically exist in regions of the brain where the blood brain barrier is fenestrated, such as the arcuate nucleus of the hypothalamus. Glucose and leucine are nutrients known to be sensed by neurons in this brain region, but the mechanisms by which they are sensed, and cells that sense them require further study. Using calcium imaging of adult neuron cultures from the mouse mediobasal hypothalamus, I demonstrated that leucine bidirectionally regulates neuronal activity in a neurochemically heterogeneous population of neurons, including AgRP/NPY and POMC neurons. Using pharmacological tools, I demonstrated, unexpectedly, that this acute sensing is independent of mTOR and leucine metabolism, known pathways involved in leucine sensing in vivo. Leucine sensing is LAT1 independent. The response principally relies on calcium entry into the cell across the plasma membrane, but IP3 sensitive calcium stores play a role in neurons inhibited by leucine. Using phosphoTRAP and single cell RNA sequencing, I aimed to identify a molecular marker for leucine sensing cells to allow their manipulation in vivo. PhosphoTRAP, and subsequent pharmacological studies identified a T Type calcium channel may be a marker for leucine sensing cells. AgRP neurons are essential for feeding, and also play roles in controlling glucose homeostasis. Using chemogenetics to selectively activate these neurons, I demonstrated, in contrast to a similar, recently published study, that blood glucose concentrations did not rise upon activation of these neurons. A subpopulation of AgRP neurons express glucokinase, and some AgRP neurons are glucose inhibited, but the role of glucokinase in these neurons has not been characterised. Our lab generated an AgRP neuron specific glucokinase knock out mouse line. Preliminary results suggest 18 – 25 week old female AgRP glucokinase knock out mice may have altered glucose tolerance, but conclusions can only be drawn once further mice have been phenotyped, and the success of the glucokinase knock out from AgRP neurons has been confirmed.

The hypothalamic arcuate nucleus (Arc) is a central unit that controls the appetite through the integration of metabolic, hormonal, and neuronal afferent inputs. Agouti-related protein (AgRP), proopiomelanocortin (POMC), and dopaminergic neurons in the Arc differentially regulate feeding behaviors in response to hunger, satiety, and appetite, respectively. At the time of writing, the anatomical and electrophysiological characterization of these three neurons has not yet been intensively explored. Here, we interrogated the overall characterization of AgRP, POMC, and dopaminergic neurons using genetic mouse models, immunohistochemistry, and whole-cell patch recordings. We identified the distinct geographical location and intrinsic properties of each neuron in the Arc with the transgenic lines labelled with cell-specific reporter proteins. Moreover, AgRP, POMC, and dopaminergic neurons had different firing activities to ghrelin and leptin treatments. Ghrelin led to the increased firing rate of dopaminergic and AgRP neurons, and the decreased firing rate of POMC. In sharp contrast, leptin resulted in the decreased firing rate of AgRP neurons and the increased firing rate of POMC neurons, while it did not change the firing rate of dopaminergic neurons in Arc. These findings demonstrate the anatomical and physiological uniqueness of three hypothalamic Arc neurons to appetite control.

No description available.

The central nervous system (CNS) melanocortin signaling pathway plays a critical role in the regulation of metabolism. However, the regulatory effects of CNS melanocortin signaling on hepatic lipid metabolism and fatty liver disease have not been well established. Although the activity of the CNS melanocortin system is regulated by metabolic signals, the mechanism for this regulation is not fully understood. Variants of the FTO (fat mass and obesity-associated) gene are associated with obesity and FTO is expressed in the hypothalamic neurons including proopiomelanocortin (POMC) neurons. Therefore, it is hypothesized that hypothalamic FTO plays a role in the regulation of metabolism by mediating the effect of metabolic signals on hypothalamic melanocortinergic neurons, and that impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease. Intracerebroventricular (i.c.v.) treatment with SHU9119, a melanocortin antagonist, increased hepatic lipid accumulation and the expression of genes encoding lipogenic enzymes in lean mice. Conversely, i.c.v. treatment with MTII, a melanocortin agonist, reduced the expression of hepatic lipogenic genes in association with reduction in body weight in ob/ob mice, a mouse model of fatty liver disease. Immunohistochemical analysis demonstrated that Fto is co-expressed in both POMC and agouti-related protein (AgRP) neurons in the mouse hypothalamus. Fto mRNA and protein expression was reduced by fasting and increased by glucose treatment in nutritionally important hypothalamic nuclei. Fasting-induced reduction in hypothalamic Fto expression was observed in both lean wild-type and obese ob/ob mice, while the stimulatory effect of glucose on hypothalamic Fto expression was absent in ob/ob mice. These findings support the hypothesis that central melanocortin signaling regulates hepatic lipid metabolism in part by regulating de novo lipogenesis. Impairments in the central melanocortin signaling lead to the development of hepatic steatosis, while enhanced melanocortin signaling may be beneficial in reversing abnormal hepatic lipid metabolism in fatty liver disease (Poritsanos et al., 2008). These findings also support the hypothesis that Fto is expressed in the hypothalamic melanocortinergic neurons and is regulated by metabolic signals involving changes in CNS glucose availability and/or glucose action. Impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease.

The central nervous system (CNS) melanocortin signaling pathway plays a critical role in the regulation of metabolism. However, the regulatory effects of CNS melanocortin signaling on hepatic lipid metabolism and fatty liver disease have not been well established. Although the activity of the CNS melanocortin system is regulated by metabolic signals, the mechanism for this regulation is not fully understood. Variants of the FTO (fat mass and obesity-associated) gene are associated with obesity and FTO is expressed in the hypothalamic neurons including proopiomelanocortin (POMC) neurons. Therefore, it is hypothesized that hypothalamic FTO plays a role in the regulation of metabolism by mediating the effect of metabolic signals on hypothalamic melanocortinergic neurons, and that impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease. Intracerebroventricular (i.c.v.) treatment with SHU9119, a melanocortin antagonist, increased hepatic lipid accumulation and the expression of genes encoding lipogenic enzymes in lean mice. Conversely, i.c.v. treatment with MTII, a melanocortin agonist, reduced the expression of hepatic lipogenic genes in association with reduction in body weight in ob/ob mice, a mouse model of fatty liver disease. Immunohistochemical analysis demonstrated that Fto is co-expressed in both POMC and agouti-related protein (AgRP) neurons in the mouse hypothalamus. Fto mRNA and protein expression was reduced by fasting and increased by glucose treatment in nutritionally important hypothalamic nuclei. Fasting-induced reduction in hypothalamic Fto expression was observed in both lean wild-type and obese ob/ob mice, while the stimulatory effect of glucose on hypothalamic Fto expression was absent in ob/ob mice. These findings support the hypothesis that central melanocortin signaling regulates hepatic lipid metabolism in part by regulating de novo lipogenesis. Impairments in the central melanocortin signaling lead to the development of hepatic steatosis, while enhanced melanocortin signaling may be beneficial in reversing abnormal hepatic lipid metabolism in fatty liver disease (Poritsanos et al., 2008). These findings also support the hypothesis that Fto is expressed in the hypothalamic melanocortinergic neurons and is regulated by metabolic signals involving changes in CNS glucose availability and/or glucose action. Impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease.

Tesis por compendio / [ES] El sistema de melanocortina es una estructura clave en la regulación de una amplia gama de funciones fisiológicas que incluyen la melanogénesis, la respuesta al estrés y el equilibrio energético, mediante la unión a una familia de receptores acoplados a la proteína G específicos (MC1R-MC5R). La sobreexpresión de agonistas inversos, la proteína de señalización agutí (Asip) y la proteína relacionada con agutí (Agrp) da como resultado un aumento de la ingesta de alimentos, de crecimiento lineal y de peso corporal. Asip regula la polaridad de pigmentación dorsoventral a través del MC1R, y la sobreexpresión induce obesidad en ratones al unirse al Mc4r central. La sobreexpresión de asip1 en el pez cebra transgénico (asip1-Tg) mejora el crecimiento, sin afectar la acumulación lipídica (obesidad), incluso cuando se alimentan bajo regímenes inductores severos. Los peces asip1-Tg no necesitan comer más para crecer más y más rápido, lo que sugiere una mayor eficiencia alimentaria. Además, los peces asip1-Tg criados en alta densidad son capaces de crecer mucho más que los peces de tipo salvaje (WT) criados en baja densidad, aunque los peces asip1-Tg parecen ser más sensibles al estrés por hacinamiento que los peces WT. El análisis transcriptómico comparativo del intestino de asip1-Tg refleja una expresión diferencial de transportadores aminoácidicos, monocarboxilatos, transportadores iónicos y de vitaminas. La sobreexpresión reduce la integridad del epitelio intestinal aumentando su permeabilidad paracelular y potencia el transporte electrogénico de aminoácidos. Así, los peces transgénicos poseen mayor capacidad para la absorción de nutrientes y, por extensión una mejora en la eficiencia alimenticia que podría explicar, en parte, ese crecimiento diferencial bajo tasas de ingesta similares. Esta tesis tuvo también como objetivo investigar si los asip1-Tg mantienen un fenotipo dominante asociado con una mayor tasa de alimentación. Los resultados muestran, por el contrario, un carácter reactivo/subordinado en los asip1-Tg que aboga por una participación del sistema de melanocortinas en la regulación del comportamiento de peces. El perfil subordinado de los animales asip1-Tg, junto con una activación del eje del estrés, sugiere que estos animales pueden mostrar un comportamiento de ansiedad. Los resultados indicaron que los peces asip1-Tg muestran un comportamiento de ansiedad que además relacionado con una severa disminución de los niveles centrales de serotonina (5HT) y dopamina y elevación de su recaptación neuronal y degradación. La administración de un inhibidor de la recaptación de 5HT, recupera el fenotipo comportamental salvaje, mitigando el comportamiento de ansiedad en los peces transgénicos y rescatando los niveles de 5HT. Esta ansiedad podría repercutir en una alteración del comportamiento locomotor de los animales, por ello estudiamos los ritmos circadianos de actividad locomotora. Los resultados muestran que los animales asip1-Tg exhiben una disrupción completa del ritmo de actividad, con una actividad muy elevada, especialmente durante la noche. Esta disrupción es concomitante con una desaparición del ritmo diario de serotonina y melatonina. Además, los resultados muestran una pérdida de ritmos de expresión de genes reloj (per1a y clock1a). La incubación, in vitro, de glándulas pineales con Asip1 produjo una inhibición de la secreción de melatonina replicando los resultados obtenidos in vivo y demostrando un efecto directo de Asip1, sobre la fisiología de la pineal. En esta tesis, se utilizó el pez cebra como modelo para investigar los efectos de la obesidad sobre la ansiedad y la memoria. La obesidad no tuvo ningún efecto sobre la ansiedad, pero produjo una disminución de la memoria a corto plazo, estudiada mediante test de condicionamiento aversivo. Este estudio proporciona, un protocolo fiable para evaluar el efecto de las enfermedades metabólica en la función cognitiva y conductual. / [CA] El sistema de melanocortina és una estructura clau en la regulació d'una ampla gamma de funcions fisiològiques que inclouen la melanogènesi, la resposta a l'estrès i l'equilibri energètic, mitjançant la unió a una família de receptors acoblats a la proteïna G específics (MC1R-MC5R). La sobreexpressió d'agonistes inversos, la proteïna de senyalització agutí (Asip) y la proteïna relacionada con agutí (Agrp) dona com a resultat un augment de la ingesta d'aliments, de creixement lineal i de pes corporal. Asip regula la polaritat de pigmentació dors-ventral a través del MC1R, y la sobreexpressió indueix obesitat en ratolins en unir-se al MC4R central. La sobreexpressió de asip1 en el peix zebra transgènic (asip1-Tg) millora el creixement, sense afectar l'acumulació lipídica (obesitat), inclús quan s'alimenten sota règims inductors severs. Los peces asip1-Tg no necessiten menjar més per a créixer més i més ràpid, lo qual suggereix una major eficiència alimentària. A més a més, els peixos asip1-Tg criats en alta densitat són capaces de créixer molt més que els peixos de tipus salvatge (WT) criats en baixa densitat, malgrat que els peixos asip1-Tg semblen ser més sensibles a l'estrès per amuntegament que els peixos WT. L'anàlisi transcriptòmic comparatiu de l'intestí de asip1-Tg reflecteix una expressió diferencial de transportadors aminoacídics, monocarboxilats, transportadors iònics i de vitamines. La sobreexpressió redueix la integritat de l'epiteli intestinal augmentant la seua permeabilitat paracel·lular i potencia el transport electrogènic d'aminoàcids. Per tant, els peixos transgènics posseeixen major capacitat per l'absorció de nutrients i, per extensió una millora en la eficiència alimentària que podria explicar, en part, eixe creixement diferencial sota taxes d'ingesta similars. Aquesta tesi tingué també com a objectiu investigar si els asip1-Tg mantenien un fenotip dominant associat amb una major taxa d'alimentació. Els resultats mostren, pel contrari, un caràcter reactiu/subordinat en los asip1-Tg que advoca per una participació del sistema de melanocortines en la regulació del comportament de peixos. El perfil subordinat dels animals asip1-Tg, junt amb una activació de l'eix de l'estrès, suggereix que aquests animals poden mostrar un comportament d'ansietat. Els resultats indicaren que els peixos asip1-Tg mostren un comportament d'ansietat relacionat amb una severa disminució dels nivells centrals de serotonina (5HT) i dopamina i elevació de la seua recaptació neuronal i degradació. L'administració de un inhibidor de la recaptació de 5HT recupera el fenotip comportamental salvatge, mitigant el comportament d'ansietat en els peixos transgènics i rescatant els nivells centrals de 5HT. Esta ansietat podria repercutir en una alteració del comportament locomotor dels animals, per la qual cosa vam estudiar els ritmes circadians d'activitat locomotora. Els resultats mostren que els animals asip1-Tg exhibeixen una disrupció completa del ritme d'activitat, amb una activitat molt elevada durant tot el cicle diari, especialment durant la nit. Esta disrupció es concomitant amb una desaparició del ritme diari de serotonina i melatonina. A més a més, els resultats mostren una pèrdua de ritmes de expressió de gens rellotge (per1a y clock1a). La incubació, in vitro, de glàndules pineals con Asip1 va produir una inhibició de la secreció de melatonina replicant els resultats obtinguts in vivo y demostrant un efecte directe de Asip1 sobre la fisiologia de la pineal. En esta tesi, se va utilitzar el peix zebra com a model per investigar els efectes de la obesitat sobre la ansietat i la memoria. L'obesitat no va tindre cap efecte sobre l'ansietat, però va produir una disminució de la memòria a curt termini, estudiada mitjançant tests de condicionament aversiu. Aquest estudi proporciona, un protocol fiable per a avaluar l'efecte de les malalties metabòliques en la funció cognitiva i conductual. / [EN] The melanocortin system plays a key role in the regulation of a wide range of physiological functions including melanogenesis, stress response and energy balance, through binding to a family of specific G protein-coupled receptors (MC1R-MC5R). Overexpression of inverse agonists, agouti-signalling protein (Asip) and agouti-related protein (Agrp) results in increased food intake, linear growth and body weight. Asip regulates dorso-ventral pigmentation polarity through MC1R, and over-expression induces obesity in mice by binding to the central MC4R. Overexpression of asip1 in transgenic zebrafish (asip1-Tg) enhances growth, without affecting lipid accumulation (obesity), even when fed under severe inducing regimens. The asip1-Tg fish do not need to eat more to grow bigger and faster, suggesting increased feed efficiency. In addition, asip1-Tg fish reared at high density are able to grow much larger than wild-type (WT) fish reared at low density, although asip1-Tg fish appear to be more sensitive to overcrowding stress than WT fish. Comparative transcriptomic analysis of asip1-Tg gut reflects differential expression of amino acid, monocarboxylate, ionic and vitamin transporters. Overexpression reduces the integrity of the intestinal epithelium by increasing its paracellular permeability and enhances electrogenic amino acid transport. Thus, transgenic fish possess a greater capacity for nutrient absorption and, by extension, an improvement in feed efficiency that could explain, in part, this differential growth under similar intake rates. This thesis also aimed to investigate whether asip1-Tg maintain a dominant phenotype associated with a higher feeding rate. Experimental results show, on the contrary, a reactive/subordinate character in asip1-Tg which argues for an involvement of the melanocortin system in the regulation of fish behaviour. Improving feeding motivation without promoting aggression in fish, thus avoiding the threat to native populations in case of an escape, makes the inhibition of the melanocortin system, through the overexpression of asip1, a feasible target for the development of genetically modified lines. The subordinate profile of the asip1-Tg animals, together with an activation of the stress axis, suggests that these animals may exhibit anxiety-like behaviour. The results indicated that asip1-Tg fish show a behaviour similar to our concept of anxiety related to a severe decrease in central serotonin (5HT) and dopamine levels as well as the elevation of their neuronal reuptake and degradation. The administration of fluoxetine, a serotonin reuptake inhibitor, recovers the wild-type behavioural phenotype, mitigating anxiety behaviour in transgenic fish and restoring central 5HT levels. This anxiety could have repercussions on the locomotor behaviour of the animals, so we studied circadian rhythms of locomotor activity. The results show that asip1-Tg animals exhibit a complete disruption of the activity rhythm, with very high activity levels throughout the daily cycle, especially during the night. This disruption is concomitant with a disappearance of the daily rhythm of serotonin and melatonin. In addition, the results show a loss of clock gene expression rhythms (per1a and clock1a). Incubation, in vitro, of pineal glands with Asip1 produced an inhibition of melatonin secretion replicating the results obtained in vivo and demonstrating a direct effect of Asip1 on pineal physiology. In this PhD thesis, zebrafish was used as a model to investigate the effects of overfeeding-induced obesity on anxiety-like behaviour and memory. Obesity had no effect on anxiety, but produced a decrease in short-term memory, studied by means of aversive conditioning tests. This study also provides a reliable protocol for assessing the effect of metabolic diseases on cognitive and behavioural function, supporting zebrafish as a model for cognitive and behavioural neuroscience. / Esta tesis ha sido realizada a través del programa de ‘Ayudas para la formación de personal investigador’ (FPI) BES‐2017‐082424 de la Agencia Estatal de Investigación, en Instituto de Acuicultura Torre de la Sal (IATS) del Consejo Superior de Investigaciones Científicas (CSIC) en el Grupo de investigación de Control de la Ingesta en Peces dirigido por José Miguel Cerdá Reverter, director de esta tesis. Los trabajos llevados a cabo en esta tesis han sido financiados por Ministerio de Ciencia, Innovación y Universidades (MICIU) a través de los siguientes proyectos: MELANOCONDUCT: Implicación del sistema de melanocortinas en la regulación de los mecanismos temporales y conductuales de peces AGL2016-74857-C3-3-R; Cronopeces: Red temática de cronobiología de peces y sus aplicaciones en acuicultura RED2018-102487-T; MacForFish: Nuevos aspectos homeostáticos y comportamentales de la regulación de la ingesta en peces PID2019-103969RB-C33; FISHTASTE: Implicación de los mecanismos sensoriales del gusto en la regulación de la ingesta de peces - Involvement of taste sensing mechanisms in the regulation of feed intake of fish PID2022-136288OB-C33 / Godino Gimeno, A. (2024). Involvement of the Melanocortin System in the Regulation of Circadian and Behavioural Mechanisms in Zebrafish [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203148 / Compendio