Thesis advisor: Gorica D. Petrovich / The drive to eat, like most motivated behaviors, is controlled by both intrinsic signals from the body as well as extrinsic signals from the environment. Although these factors often act in concert, in some instances environmental cues can override the body’s homeostatic signals. Prior work investigating the ability of learned cues to promote overeating in the absence of hunger identified a critical forebrain network composed of the amygdala, medial prefrontal cortex (mPFC), and lateral hypothalamus (LHA). We hypothesized that a similar forebrain network may also be critical when learned fear-cues inhibit eating despite hunger. The amygdala, mPFC and LHA are each anatomically and functionally positioned to influence feeding, and evidence suggests they could work together to support the fear-cue’s ability to inhibit feeding by overriding homeostatic hunger signals triggered by food-deprivation. Prior anatomical work identified direct pathways between these three large, heterogeneous regions; however, less is known about the organization of the underlying circuitries, especially between distinct nuclei and/or subdivisions that comprise these structures. Study 1 used a dual retrograde tract tracing design to map the topographical organization of the connections between the amygdala, mPFC, and LHA in detail, and to determine whether amygdalar pathways to the mPFC and to LHA originated from the same or different neurons. We found evidence for multiple, topographically organized, direct pathways from the amygdala to the LHA, and separate pathways from the amygdala to areas of the mPFC that send direct projections to the LHA. Importantly, nearly all amygdalar projections to the mPFC and to the LHA originated from different neurons, suggesting that amygdala and amygdala-mPFC processing influence the LHA independently. Study 2 used immediate early gene induction to map the patterns of functional activation within this amygdala-prefrontal-lateral hypothalamic network during the expression of fear-cue inhibited feeding behavior, and to assess whether these patterns were similar in males and females. We found differential activation across the network, and activation patterns related to the presentation of fear-cues, the presence of food-related cues, and the amount of food consumed were associated within distinct cell groups in the amygdala, mPFC, and LHA. Together, the studies presented in this dissertation provide anatomical and functional maps for future interrogation of the circuitry underlying fear-cue inhibited feeding. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Psychology.

O comportamento agressivo maternal é desenvolvido por ratas lactantes com o intuito de preservar a vida de sua prole. Nesse estudo demonstramos que a Área Hipotalâmica de Ataque (HAA), particularmente a porção ventrolateral do núcleo ventromedial (VMHvl) e a parte tuberal do hipotálamo lateral (LHAtu), apresentam um papel crítico para a expressão do comportamento, ao correlacionarmos os níveis de agressão maternal com a ativação de tais setores, e detectarmos prejuízo no comportamento em um grupo submetido a lesões citotóxicas bilaterais por NMDA circunscritas a 1/3 caudal da HAA, e em outro grupo lesões que se estenderam por 2/3 caudais do eixo rostro caudal da HAA. Os parâmetros não agressivos não apresentaram diferenças entre os grupos, assim como os comportamentos maternais, indicando que as lesões não afetaram o cuidado das fêmeas com a sua prole. Em uma análise da densidade de células Fos positivas de possíveis alvos dessas estruturas hipotalâmicas estudadas, não detectamos diferenças estatísticas entre os grupos com lesão e o grupo controle. / The maternal aggression is developed by lactant dams to preserve their offsprings. In the present study we show that Hypothalamic Attack Area (HAA), particularly the ventrolateral part of ventromedial nucleus (VMHvl) and the tuberalis part of the lateral hypothalamus (LHAtu), have a critical role in the expression of this behavior. We have correlated the maternal aggression levels with the activation of this parts and we have detected behavioral damage at both groups with bilateral NMDA lesions at 1/3 caudal of HAA or 2/3 caudal of HAA, compared with control group. The non-agressive parameters did not show diferences among groups, as well as the maternal behavior, indicating that the lesions did not affect the maternal care. We have further analyzed the pattern of Fos expression in the brainstem targets of these hypothalamic regions in the NMDA lesioned dams, but couldnt find any difference between lesioned and intact groups.

Cerca de metade dos brasileiros adultos apresentam obesidade ou sobrepeso, sendo o excesso de ganho de peso durante a gestação um dos mais importantes fatores de risco para o desenvolvimento da obesidade em mulheres. Além do ganho de peso excessivo durante a gravidez, o diabetes gestacional é outro problema metabólico enfrentado frequentemente por gestantes. No presente trabalho demonstramos que a expressão de SOCS3 em células que expressam o receptor de leptina (LepR) é essencial para causar plenamente a hiperfagia decorrente da gestação e lactação, que consequentemente, reduz o ganho de peso e o acúmulo de gordura durante estes períodos, garantindo menor retenção do peso pós-parto. Finalmente, a deleção do gene SOCS3 em células que expressam o LepR melhorou significantemente a homeostase glicêmica durante a gestação protegendo as fêmeas contra o desenvolvimento do diabetes gestacional. / About half of brazilian adults are obese or overweight, and excess weight gain during pregnancy among the most important risk factors for the development of obesity in women. Besides the excessive weight gain during pregnancy, gestational diabetes is another metabolic problem often faced by pregnant women. The present work we demonstrated that the expression of SOCS3 in LepR expressing cells has proved essential to fully cause hyperphagia due to pregnancy and lactation that, as a consequence, reduces weight gain and fat accumulation during these periods, ensuring a lower weight retention postpartum. Finally, the conditional deletion of SOCS3 significantly improves glucose homeostasis during pregnant females protecting against the development of gestational diabetes.

Orientador: José de Anchieta de Castro e Horta Junior / Coorientador: Jackson Cioni Bittencourt / Banca: Luiz Fernando Takase / Banca: Luciana Pinato / Resumo: O hormônio concentrador de melanina (MCH) do rato é um nonadecapeptídeo presente em neurônios localizados, principalmente, na área hipotalâmica lateral (LHA) que se projetam para diversas regiões do neuro-eixo. O MCH está envolvido em diversas funções, tais como: reprodução, certos aspectos do comportamento motivado, atividade locomotora, percepção sensorial, controle de temperatura, memória, aprendizagem, ansiedade, ciclo sono-vigília e comportamento alimentar. Nesse, o MCH atua como neuropeptídeo orexígeno. Durante o envelhecimento, ocorre a diminuição no consumo de energia associada à idade. Nesse trabalho, analisamos em diferentes faixas etárias, as variações da expressão do MCH na área hipotalâmica lateral, empregando métodos esterológicos (para estimar o número de neurônios MCH-ir, área, volume e densidade neuronal), reconstrução tridimensional (para estudo da distribuição dos neurônios MCH-ir) e densitometria óptica de tecido hibridizado para reconhecimento do RNAm do ppMCH (para mensuração da área hibridizada, densidade óptica média e densidade óptica integrada [DOI]) na LHA e em suas regiões. Foram utilizados 35 ratos Sprague-Dawley machos, divididos em 7 grupos experimentais de diferentes idades: 14 (lactente), 28 (pré-púbere), 50 (púbere), 90 (adulto jovem), 210 (adulto maduro), 540 (adulto senescente) e 750 (adulto senil) dias pós-natais. Os animais foram submetidos à perfusão transcardíaca, seus encéfalos coletados e processados para análise da expressão do MCH na área hipotalâmica lateral, segundo protocolos de imuno-histoquímica e hibridização in situ. A LHA apresentou um aumento significativo no número de neurônios apenas entre os grupos de 14 e 28 dias e sua área e volume foram significativamente menores apenas no grupo de 14 dias em relação a todos os grupos, com exceção do grupo de 750 dias. No entanto, com relação a densidade neuronal não foram observadas... / Abstract: The Melanin-Concentrating Hormone (MCH) is a nonadecapeptide located mainly in neurons in the lateral hypothalamic area (LHA), which innervate several regions of neuraxis. MCH is involved in many functions as reproduction, aspects of motived behaviors, motor activity, sensorial information, temperature control, memory, learning, anxiety, sleep-wake cycle and feeding behavior in which the MCH plays an orexigenic role. The energy consumption in aging is reduced as well as, the expression of MCH presents alterations associated with age. Therefore, we analyzed the variations in the expression of MCH at different ages in the lateral hypothalamic area, using stereology (to estimate the number of MCH-ir neurons, area, volume and neuronal density), 3D reconstruction (to study the distribution of MCH-ir neurons) and optical density after in situ hybridization protocol for ppMCH RNAm (to measure the hibridizated area, the mean optical density and the integrated optical density [IOD]) in the LHA and its three regions. In this study, 35 animals were divided in 7 experimental groups of 14 (neonate), 28 (prepubescent), 50 (pubescent), 90 (young adult), 210 (middle aged adult), 540 (senescent adult) and 750 (elderly adult) postnatal days. All the animals were perfused via the ascending aorta, the brains were collected and processed by immunohistochemistry and in situ hybridization protocols to analyze the expression of MCH in the lateral hypothalamic area. LHA neurons number increased only between groups of 14 and 28 days, and its area and volume significantly smaller in 14-days group when in respect to all other groups but the 750-days one. However, there weren't differences in neuronal density among groups. MCH-ir neurons distribution in LHA and contiguous regions was similar among all groups as well. Hypothalamic MCH-ir neurons has shown uniform rates in and out of LHA, but after analyzing its distribution in mamillary, tuberal and anterior ... / Mestre

The central nervous system (CNS) has a crucial role in the maintenance of energy homeostasis by orchestrating a plethora of signals from peripheral organs about the state of energy stores and the current energy intake needed to match energy expenditure. These signals converge into the hypothalamic regions and its complex local circuitry. CNS-derived cholecystokinin (CCK) is acting at central level to modulate energy balance by regulating the neuronal activity of hypothalamic neuronal populations that regulate food intake, energy storage and consumption. Moreover, our recent published work identifies CCK neurons as key integrators of the neuroendocrine negative feedback of glucocorticoids to the PVN. Glucose sensing neurons of the Ventromedial Hypothalamus (VMH) are integrating energy signals and are essential for mounting a counter-regulatory response and glucose homeostasis. VMH is also important in energy expenditure by regulating body weight and thermogenesis. CCK neurons are present in high density in the VMH.The source of endogenous CCK that acts on distinct neuronal components has not been elucidated. The research so far does not address the purpose of CCK neurons in the hypothalamus and their potential role in the network dynamics regarding energy homeostasis. In this study, we untangle the role of CCK neurons in the VMH nucleus by employing stereotactic intracranial delivery of adeno-associated viruses that result in cell-type specific chemogenetic inhibition or ablation of these neurons. Acute silencing of their neurotransmission with the cre-dependent AAV expression of the chemogenetic tool of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) increases their daily food intake due to increased meal numbers and eating frequency without meal size or meal duration being affected. CCK ablation by a newly generated double-recombinase-mediated Diphtheria Toxin Receptor (DTR) mouse line or AAV-DTA-mediated ablation resulted in hyperphagia, obesity and hyperglycaemia. We conclude that CCK^VMH neurons are implicated in the regulation of food intake, body weight and glucose homeostasis in the adult brain.

Na realização do tratamento ortodôntico, é descrito que a dor ocorre imediatamente após a aplicação das forças ortodônticas e desaparece após alguns dias. Com relação às vias neurais envolvidas na mediação sensorial da movimentação ortodôntica (MO) poucos são os estudos que abordam esse tema, e nas últimas décadas achados consistentes demonstram o envolvimento do sistema trigeminal, bem como de suas áreas de projeção nessa mediação. Nesse contexto, achados demonstram que além de áreas do tronco encefálico, regiões da substância cinzenta periaquedutal, o núcleo espinhal do trigêmio, a área parabraquial e áreas límbicas apresentam aumento da imunorreatividade à proteína FOS (IR-FOS) 24hs após o início da movimentação dentária de molares de ratos. É possível que esta ativação límbica ocorra devido à liberação local de prostaglandinas. Assim, o presente trabalho avaliou temporalmente (3, 6, 24 e 48hs) a ativação do núcleo central da amígdala (CEA) e do hipotálamo lateral (HL) após MO. Em adição, verificamos se a pré-administração de diclofenaco de sódio (analgésico e antiinflamatório) ou de sulfato de morfina (analgésico de ação central) altera a IR-FOS após a aplicação de força para movimentação dentária de incisivos superiores de ratos, nas áreas analisadas. Nossos resultados mostram que a MO aumentou a IR-FOS no CEA e no HL após 3, 6 e 24hs. Após a MO por 48hs, a IR-FOS foi semelhante ao controle. A análise histológica do periodonto desses animais evidenciou processo ativo de remodelação óssea, sugerindo aposição de matriz colágena e a análise radiográfica demonstrou que a separação dos incisivos superiores dos animais ocorreu devido ao movimento ortodôntico e não ortopédico, já que não houve abertura da sutura palatina mediana. O pré-tratamento com sulfato de morfina (2mg/kg) inibiu a IR-FOS em ambas as áreas estudadas, sugerindo que a expressão de FOS após MO possa ser devido à transmissão da informação nociceptiva. Ainda, o pré-tratamento com diclofenaco de sódio (5mg/kg) reduziu a IR-FOS no CEA e no HL nos animais que receberam o aparelho ortodôntico por 6hs / In the accomplishment of the orthodontic treatment, it is described that the pain happens immediately after the application of orthodontic forces and disappears after few days. Regarding the neural pathways involved in the sensorial mediation of the orthodontic movement (OM) few are the studies that approach this theme, and in the last decades solid founds demonstrate the involvement of the trigeminal system as well as its projection areas in this mediation. In this context, discoveries demonstrate that besides brainstem areas, such as periaquedutal gray matter, trigeminal spinal nucleus, and parabrachial area, limbic areas can present increase in FOS immunorreactivity(IR-FOS) 24 hours after the beginning of the tooth movement of rat molars. It is possible that this limbic activation happens due to the local liberation of prostaglandins. In this way, this work evaluated the activation of the central amygdala (CEA) and the lateral hypothalamus (LH) after OM. In addition, we verified if the previous administration of sodium diclofenac (painkiller and anti-inflammatory) or of morphine sulfate (painkiller of central action) alters IR-FOS after the application of force for tooth movement of rat superior incisors, in the analyzed limbic areas. Our results show that the OM increased the IR-FOS in CEA and in HL after 3, 6 and 24hs. After the OM for 48hs, the IR-FOS was similar to the control. The histological periodontal analysis of those animals evidenced an active process of bone remodeling, suggesting the deposition of osteoid material and the radiographic analysis demonstrated that the separation of the superior incisors of the animals happened due to the orthodontic movement and not orthopedic movement, since the interpremaxillary suture was fused. The pre-treatment with morphine sulfate (2mg/kg) inhibited the IR-FOS in both studied areas, suggesting that the expression of FOS after OM can be due to the transmission of nociceptive information. In addition, pre-treatment with sodium diclofenac (5mg/kg) reduced IR-FOS in CEA and in HL in the animals that received the orthodontic apparel for 6hs.

Kisspeptin (encoded by Kiss1) is a hypothalamic neuropeptide that is directly regulated by sex steroids and directly stimulates gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin cell models were established in order to facilitate future molecular analysis of kisspeptin. mHypoA-51 and mHypoA-63 cell lines were found to express kisspeptin, estrogen receptor α and β, substance P, but not tyrosine hydroxyase. Furthermore, estrogen decreased Kiss1 expression in both cell lines. Based on these results, it was concluded that mHypoA-51 and mHypoA-63 are representative of arcuate kisspeptin neurons. Accumulating evidence also indicates that kisspeptin indirectly stimulates GnRH neurons through afferent neurons. Kisspeptin receptor expression was detected in native neuropeptide Y (NPY) neurons. Using the mHypoE-38 cell line, kisspeptin was found to directly regulate NPY mRNA expression and secretion via the ERK1/2 and p38 MAPK pathways. This is the first evidence that kisspeptin directly stimulates NPY neurons to potentially exert indirect effects on GnRH neurons.

The naked mole-rat (Heterocephalus glaber) demonstrates a strict social and reproductive hierarchy. Oxytocin (OXT) is a peptide hormone that acts both peripherally and centrally in the regulation of a number of sexual and social behaviours. The main area of central production of this peptide is the paraventricular nucleus of the hypothalamus (PVN). The present study characterized differences that exist in OXT neurons in this region. Breeders and subordinates from established colonies were sacrificed and brains were processed for OXT-immunoreactivity. Four further groups of paired animals underwent various social and hormonal manipulations (opposite-sex paired, same sex-paired, opposite-sex/gonadectomised paired, opposite-sex/vasectomized paired) and were also used for analysis. Results showed that subordinate naked mole-rats had significantly more OXT-immunoreactive neurons in the PVN than either breeders or paired animals that had been gonadectomised. However, no differences were found on measures of OXT cell volume. Possible functional significance of these differences is discussed.

Kisspeptin (encoded by Kiss1) is a hypothalamic neuropeptide that is directly regulated by sex steroids and directly stimulates gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin cell models were established in order to facilitate future molecular analysis of kisspeptin. mHypoA-51 and mHypoA-63 cell lines were found to express kisspeptin, estrogen receptor α and β, substance P, but not tyrosine hydroxyase. Furthermore, estrogen decreased Kiss1 expression in both cell lines. Based on these results, it was concluded that mHypoA-51 and mHypoA-63 are representative of arcuate kisspeptin neurons. Accumulating evidence also indicates that kisspeptin indirectly stimulates GnRH neurons through afferent neurons. Kisspeptin receptor expression was detected in native neuropeptide Y (NPY) neurons. Using the mHypoE-38 cell line, kisspeptin was found to directly regulate NPY mRNA expression and secretion via the ERK1/2 and p38 MAPK pathways. This is the first evidence that kisspeptin directly stimulates NPY neurons to potentially exert indirect effects on GnRH neurons.

The naked mole-rat (Heterocephalus glaber) demonstrates a strict social and reproductive hierarchy. Oxytocin (OXT) is a peptide hormone that acts both peripherally and centrally in the regulation of a number of sexual and social behaviours. The main area of central production of this peptide is the paraventricular nucleus of the hypothalamus (PVN). The present study characterized differences that exist in OXT neurons in this region. Breeders and subordinates from established colonies were sacrificed and brains were processed for OXT-immunoreactivity. Four further groups of paired animals underwent various social and hormonal manipulations (opposite-sex paired, same sex-paired, opposite-sex/gonadectomised paired, opposite-sex/vasectomized paired) and were also used for analysis. Results showed that subordinate naked mole-rats had significantly more OXT-immunoreactive neurons in the PVN than either breeders or paired animals that had been gonadectomised. However, no differences were found on measures of OXT cell volume. Possible functional significance of these differences is discussed.