Distribution of orexin-A-like immunoreactivity (ORX-LI) in rat brains was investigated with the use of a rabbit polyclonal antibody against the full length peptide orexin A. Virtually all the ORX-LI cell bodies were observed in the lateral hypothalamus at the level of median eminence. The large majority of ORX-LI neurons appeared spherical or fusiform, 20-30 μm in diameter and issued two to five cell processes with few secondary branchings. Numerous ORX-LI fibers were observed in subregions of the hypothalamus. ORX- LI cell processes were sparsely distributed in the cortex, hippocampus and thalamus. Many varicose ORX-LI cell processes were situated close to the 3rd and lateral ventricles, some of which appeared to be protruding into the lumen. As a corollary, orexin A may be released into the ventricles and interact with neurons in distant targets, in addition to influencing the activity of neurons with which ORX-LI axons make synaptic contacts.

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.

Retrograde tract tracing combined with immunohistochemical techniques were used to identify the origin of orexin A-immunoreactive (OrA-ir) fibers in the rat medulla. One to 5 days following injection of the fluorescent dye Fluorogold into the dorsal medulla, labeled neurons were found in the lateral half of the lateral hypothalamus, paraventricular, perifornical, dorsomedial, dorsal and posterior hypothalamic nuclei. Labeling the same sections with OrA antisera revealed a concentration of OrA-ir neurons in the perifornical and dorsomedial regions of the tuberal hypothalamus. A maximum of 10% of Fluorogold-labeled hypothalamic neurons were OrA-ir and 15% of OrA-ir hypothalamic neurons contained Fluorogold. Our results demonstrate that a fraction of OrA-ir neurons in the tuberal hypothalamus project to areas of the medulla that are involved in autonomic functions.

Relações entre machos de roedores muitas vezes se dão com a manifestação de comportamentos agressivos, em um embate em que se define um perdedor e um ganhador. O macho perdedor, de acordo com experimentos realizados no laboratório, apresenta comportamentos de defesa (avaliação de risco) quando exposto ao contexto da derrota social. Na expressão da defesa condicionada, estruturas do hipotálamo lateral devem ser importantes, que são também mobilizadas durante o confronto. No hipotálamo lateral destacamos a a parte justadorsomedial do hipotálamo lateral (LHAjd) que tem conexões com o sistema septo-hipocampal e projeta-se liga ao prémamilar dorsal (PMD), crítico para a expressão de comportamentos de defesa. Por outro lado, o PMD, que é uma estrutura altamente mobilizada durante o confronto social, tem conexões com o núcleo anteromedial do tálamo (AMv), estrutura já conhecida pela sua importância na aquisição da memória contextual e espacial. Assim, no presente trabalho, investigamos o papel do LHAjd na expressão e do AMv na aquisição da defesa condicionada na derrota social. Nos animais com lesão do LHAjd, observamos diminuição nos comportamentos de avaliação de risco durante exposição ao contexto. Nos animais com lesão do AMv o mesmo efeito. Dado os efeitos das lesões no AMv, elaboramos um paradigma para estudo em camundongos, a fim de se realizar inativações pontuais com farmacogenética nos neurônios glutamatérgicos em animais transgênicos durante a derrota social. Foi observado o mesmo padrão em camundongos transgênicos vGlut2-cre inativados com farmacogenética, não havendo influência nos comportamentos durante a derrota social. Inativações antes do contexto não causaram efeito na defesa condicionada. As inativações durante a derrota, no entanto, não tiveram efeito quando o residente agressivo está na exposição ao contexto. Os dados sugerem que o LHAjd tem papel na expressão da defesa condicionada, enquanto que o AMv tem papel na aquisição da defesa condicionada ao contexto, mas não no reconhecimento do residente agressivo. / Male rodents may interact aggressively, and from the agonistic encounter, it results a winner and a defeated animal. Accordingingly, the defeated male shows defensive behaviors (risk assessment) to the social defeat-related context. Contextual responses are known to rely on hippocampal processing, and one of the main targets of the hippocampal system is the justodorsomedial part of lateral hypothalamus (LHAjd), which projects to dorsal premamillary nucleus (PMD), known to be involved in the expression of social defensive behaviors. Notably, PMD, a hypothalamic site highly responsive to the social defeat, in turn, projects to the ventral part of thalamic anteromedial nucleus (AMv), previously shown to be involved in the acquistion of spatial and contextual memory to predatory threats. Thus, in present study, we investigated the role of LHAjd in expression and of AMv in acquisition of social defeat conditioned defensive behaviors. In LHAjd lesioned animals, we observed a decrease in risk assessment behaviors during exposute to the social defeat associated context , but not during the social defeat itself, suggesting a role in the expression of contextual but not in the innate social defeat. In AMv lesioned animals, we observed that the animals lost contextual defensive response, suggesting a role in the acquisition and/or expression of contextual responses. Next, using pharmacogetic inhibition, we investigated in vGlut2-cre transgenic mice the role of the AMV in the acquisition and expression of contextual defensive behavior. We have found that AMV inactivation prior to the social defeaf, but not prior to the exposure to the social defeat related context, was able to decrease contextual responses in animals tested withot the presence of the male aggressor, but not in the situation where the male aggressor was present. Overall, our results suggest that the LHAjd has a role in the expression of conditioned defense, and that the AMV is involved in the acquisision of contextual fear responses, but not in social recognition of aggressive male.

Der laterale Hypothalamus (LH) reguliert angeborene Verhaltensweisen. Ob und wie die Koordination von hypothalamischen Neuronengruppen Verhaltensübergänge reguliert, blieb jedoch unbekannt. In dieser Arbeit wurde Optogenetik mit neuronalen Ableitungen in verhaltenden Mäusen kombiniert. LHVgat Neurone erhöhten ihre Aktivitätsrate während Übergängen vom NREM-Schlaf zum Wachzustand. LHVgat Zellen projizieren zum Nucleus reticularis des Thalamus (RTN). Optogenetische Aktivierung von Vgat Ausgängen im RTN führte eine starke, frequenzabhängige Inhibierung von RTN Zellen herbei und replizierte Verhaltenszustands-abhängige Aktivitätsraten in RTN Neuronen. Ableitungen von LH Neuronen während Umgebungserkundung ergaben, dass 65% der LH Neurone ihre Aktivitätsrate erhöhten, wenn das Tier began sich fortzubewegen. 'Top-down’ Vorderhirn Innervation des LH erfolgt größtenteils durch Signale ausgehend vom lateralen Septums (LS). Während spontaner Umgebungserkundung und freiem Zugang zu Futter wiesen der LH und das LS Gamma-Oszillationen (30-90 Hz) auf, welche neuronale Aktivität innerhalb und zwischen diesen beiden Gehirnregionen synchronisierten. Optogenetische Stimulation von Somatostatin-positiven GABAergen Projektionen zum LH mit Gamma-Frequenz förderte die Nahrungssuche und erhöhte die Wahrscheinlichkeit des Betretens der Nahrungszone. Inhibitorische Signale des LS bewirkten eine Unterteilung der LH Neurone: entsprechend ihrer Aktivität im Bezug zur Nahrungsstelle wurden sie während bestimmter Phasen der Gamma-Oszillation aktiviert. Dabei führte optogenetische Stimulation von LS-LH Neuronen mit Gamma-Frequenz keine Veränderung bei der Nahrungsaufnahme selbst herbei. Insgesamt liefert diese Arbeit neue Einsichten über die Funktion der neuronalen Netzwerke des LH, welche durch Signalgebung mit unterschiedlichen Zeitskalen über die Koordination mit vor- und nachgeschalteten neuronalen Netzwerken Übergange zwischen verschiedenen angeborenen Verhaltensweisen regeln. / Lateral hypothalamus (LH) is crucial for regulation of innate behaviors. However, it remained unknown whether and how temporal coordination of hypothalamic neuronal populations regulates behavioral transitions. This work combined optogenetics with neuronal recordings in behaving mice. LHVgat cells were optogenetically identified. LHVgat neurons increased firing rates upon transitions from non-REM (NREM) sleep to wakefulness, and their optogenetic stimulation during NREM sleep induced a fast transition to wakefulness. LHVgat cells project to the reticular thalamic nucleus (RTN). Optogenetic activation of LHVgat terminals in the RTN exerted a strong frequency-dependent inhibition of RTN cells and replicated state-dependent changes in RTN neurons activity. Recordings of LH neurons during exploration revealed that 65% of LH neurons increased their activity upon the onset of locomotion. Top-down forebrain innervation of LH is provided, to a great extent, by inhibitory inputs from the lateral septum (LS). During spontaneous exploration in a free-feeding model, LS and LH displayed prominent gamma oscillations (30-90 Hz) which entrained neuronal activity within and across the two regions. Optogenetic gamma-frequency stimulation of somatostatin-positive GABAergic projections to LH facilitated food-seeking, and increased the probability of entering the food zone. LS inhibitory input enabled separate signaling by LH neurons according to their feeding-related activity, making them fire at distinct phases of the gamma oscillation. In contrast to increased food intake during optogenetic stimulation of LHVgat cells, food intake during gamma-rhythmic LS-LH stimulation was not changed. Overall this works provides new insight into the function of LH circuitry, that employs signalling at different time scales, which, in coordination with upstream and downstream circuits, regulates transitions between innate behaviors.

Made available in DSpace on 2016-06-02T19:22:09Z (GMT). No. of bitstreams: 1 5394.pdf: 2154328 bytes, checksum: 49114074adb778dc78d219f32b8ee5b4 (MD5) Previous issue date: 2013-03-22 / Sodium intake is induced by facilitatory signals, like angiotensin II and aldosterone Cell dehydration, a classical inhibitory signal for sodium intake, may also induce paradoxical sodium intake if the sodium intake inhibition by the ocitocinergic hypothalamic mechanism or by the lateral parabraquial nucleus (LPBN) is absent. Thus, the LPBN deactivation could modify the activity of hypothalamic oxytocinergic pathways or the gastric emptying control system, another inhibitory system, as well as facilitatory areas and the reward system. The aim of this study was to investigate the effect of LPBN injections of methysergide (4 μg/0.2 μl, serotonergic antagonist) in cell dehydrated animals on: activity of brain areas involved in ingestive behavior by measuring c-Fos protein immunoreactivity and tissue levels of dopamine, serotonin and metabolites or plasma hormone levels; pre-systemic satiety involving gastric emptying; selectivity of paradoxical sodium intake. The effect of disinhibition of the natriorexigenesis on the reward system was tested by repeated deactivations of the LPBN with muscimol (2 nmol/0.2 μl; GABAA agonist) and its effect on ingestive behavior sensitization and water deprivation with partial rehydration followed by sodium access (WD-PR protocol) on the lateral hypothalamus self-stimulation (LHSS). Holtzman or Sprague-Dawley rats (280-320 g), intacts or operated (femoral vein cannulation and/or guide cannulas implanted in direction to the LPBN or bipolar electrode implanted in the hypothalamus), were used in the experiments. Animals treated with methysergide and hyperosmotic by gavage of 2 M NaCl (2 ml) compared to the control treatment (vehicle) showed: (a) increase in ir-Fos in the area postrema and intermediate nucleus of the solitary tract, subfornical organ and non-oxytocinergic neurons of the ventral portion of the paraventricular nucleus of the hypothalamus; (b) increase in tissue levels of dopamine in the amygdala, but not in the accumbens; (c) unchanged activity of oxytocinergic system (ir-Fos in oxytocinergic neurons and oxytocin plasma levels similar to control group). Hyperosmotic rats (iv infusion of 2 ml of 2 M NaCl) treated with methysergide and a gavage of 0.3 M NaCl (3 ml) showed a hypertonic gastric and intestinal content similar to the control group (vehicle) after gavage. In hydrated animals with a history of two previous treatments of muscimol into the LPBN, the hypertonic NaCl intake induced by muscimol was higher than the control animals pretreated with vehicle. The LHSS was not altered at any stage of WD-PR protocol, as well as in cell dehydrated animals in comparison with hydrated control group. The results demonstrate that the deactivation of the LPBN enhances specifically the intake of solutions containing sodium and suggest the involvement of the brain stem and the amygdala during the appetitive phase of the paradoxical sodium intake, while the deactivation of other inhibitory mechanisms (oxytocin and gastric retention) seems not to be essential. Furthermore, the repetition of LPBN deactivation sensitizes hydrated animals for sodium intake. Removing the inhibition of sodium appetite by partial rehydration in WD-PR does not change the LHSS reward. / A ingestão de sódio é induzida por sinais facilitadores, como a angiotensina II e a aldosterona. Apesar de classicamente considerada antinatriorexigênica, a desidratação celular também se mostra facilitadora da ingestão paradoxal de sódio quando a inibição da ingestão de sódio pelo mecanismo ocitocinérgico do hipotálamo ou pelo núcleo parabraquial lateral (NPBL) está desativada. A desativação do NPBL poderia, então, atuar sobre o mecanismo ocitocinérgico ou de controle do esvaziamento gástrico, ambos inibidores, ou ainda modificar a atividade de áreas facilitadoras ou envolvidas na recompensa. O objetivo deste estudo foi investigar o efeito da injeção de metisergida (4 μg/0,2 μl, antagonista serotonérgico) no NPBL em animais com desidratação celular sobre a: atividade de áreas encefálicas envolvidas no comportamento ingestivo, medindo-se imunorreatividade para proteína c-Fos e dosagem tecidual de dopamina, serotonina e metabólitos em áreas de interesse ou dosagem hormonal; saciedade pré-sistêmica envolvendo esvaziamento gástrico; seletividade da ingestão paradoxal de sódio. Também foi avaliado o possível efeito da desinibição da natriorexigênese sobre o sistema de recompensa através da sensibilização por desativações repetidas do NPBL pelo muscimol (2 nmol/0,2 μl; agonista GABAA) e na privação hídrica com reidratação parcial e posterior acesso ao sódio (modelo PH-RP) sobre a autoestimulação elétrica do hipotálamo lateral (AEHL). Ratos (280-320 g) Holtzman ou Sprague-Dawley, intactos ou operados (canulação da veia femoral e/ou cânulas-guia direcionadas ao NPBL ou eletrodo bipolar no hipotálamo), foram utilizados nos experimentos. Animais tratados com metisergida e hiperosmóticos por gavagem de NaCl 2 M (2 ml) apresentaram, em relação ao tratamento controle (veículo): (a) aumento da ir-Fos na área postrema e núcleo do trato solitário intermediário, no órgão subfornical e em neurônios não ocitocinérgicos da porção ventral do núcleo paraventricular do hipotálamo; (b) aumento da concentração tecidual de dopamina na amígdala, mas não no accumbens; (c) atividade do sistema ocitocinérgico inalterada (ir-Fos em neurônios ocitocinérgicos e ocitocina plasmática semelhantes ao controle). Ratos tratados com metisergida e hiperosmóticos por infusão iv de NaCl 2 M (1,5 ml), que receberam uma gavagem de NaCl 0,3 M (3 ml) apresentaram conteúdo gástrico e intestinal hipertônico semelhantes ao controle (veículo) após a gavagem. Em animais hidratados com histórico de dois tratamentos prévios de muscimol no NPBL, a ingestão de NaCl hipertônico induzida por muscimol foi superior aos animais controle tratados previamente com veículo. A AEHL não foi alterada em nenhuma fase do protocolo PH-RP, assim como em animais com desidratação celular, em relação aos controles hidratados. Os resultados demonstram que a desativação do NPBL potencializa especificamente a ingestão de solução contendo sódio e sugerem a participação do tronco encefálico e da amígdala na fase apetitiva da ingestão paradoxal de sódio, enquanto a desativação de outros mecanismos inibidores (ocitocina e retenção gástrica) parece não ser essencial. Ainda, a repetição da desativação do NPBL sensibiliza a ingestão de sódio em animais hidratados. A remoção da inibição ao apetite ao sódio pela reidratação parcial na PHRP não altera a recompensa na AEHL.