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.

Chronic pain alters sensory responses and carries a strong emotional component. Persistent pain can heighten pain experiences, resulting in hyperalgesia and allodynia. Further, patients suffering from chronic pain are more prone to experience a range of affective disorders including depression, sleep dysregulation, panic disorders, anxiety abnormalities and stress-related disorders including post-traumatic stress disorder (PTSD). Hence while pain serves a protective function to prevent additional physiological harm by driving behavioral and cognitive responses, chronic or persistent pain can lead to maladaptive nociceptive responses and exacerbate psychopathologies. Among brain regions, the amygdala is centrally situated to integrate the many descending and ascending signals to modulate the sensory and emotional components of pain. The amygdala is well studied for its role in fear and stress-related behavioral processes. The central nucleus of the amygdala (CeA), and in particular the lateral capsular subdivision of the CeA (CeLC), receives prominent ascending pain neurotransmission via the spino- parabrachioamygdaloid tract. In this pathway, peripheral nociceptive signals carried via primary sensory Aδ- and C-fibers terminate in the dorsal horn where second order neurons send projections via the spino-parabrachial pathway to the lateral parabrachial nucleus (LPBn). Thus, the LPBn collects cutaneous (mechanical and thermal), deep (muscular and articular) and visceral nociceptive signals and relays the information in a highly organized manner principally to the CeLC for nociceptive processing. In pain, the CeA and the LPBn-CeLC projections have been shown to undergo plasticity in the forms of enhanced synaptic transmission and alterations in neurotransmitter and receptor expression. Accordingly, the neurocircuit intersections in the CeA can modulate the sensory and emotional responses to pain. Yet despite these associations, the mediators and mechanisms underlying the emotional consequences of pain are poorly understood. Pituitary adenylate cyclase activating polypeptide (PACAP) is a neural and endocrine pleiotropic peptide important in the development and homeostatic regulation of many physiological systems. Recently, the expression of PACAP and its cognate PAC1 receptor has been shown to be upregulated in specific limbic regions by chronic stress. PACAP infusions into several limbic regions is anxiogenic, and altered blood PACAP levels and PAC1 receptor polymorphism have been associated with PTSD and other stress-related disorders. Here, we establish that CeLC PACAP originates from the LPBn as part of the spino-parabrachoamygdaloid pathway. Chronic pain enhanced PACAP expression along LPBn-CeLC projections, indicating it may be a component of pain- related plasticity. CeA PACAP signaling was sufficient to induce nociceptive hypersensitivity and anxiety-like behaviors. In a chronic neuropathic pain model, CeA PACAP signaling was found to contribute to heightened anxiety-like behaviors and nociceptive responses. Further, we characterized one prominent intracellular signaling mechanism through which CeA PACAP signaling influences these behaviors. In these experiments we provide evidence that CeA PACAP signaling plays an important role in the emotional components of pain and that alterations in CeA PACAP signaling are part of pain-related plasticity. This work establishes novel molecular mechanisms that underlie the emotional component of pain and may contribute to the development of chronic pain and associated affective disorders.

No presente trabalho, avaliamos o envolvimento da região Kölliker Fuse (KF) e núcleo parabraquial lateral (NPBL) nas respostas cardiorrespiratórias induzidas pela ativação dos quimiorreceptores centrais e periféricos em ratos não anestesiados. A injeção bilateral de muscimol (200 pmol/100 nl) no KF reduziu a ventilação basal (978 ± 100, vs. salina: 1436 ± 155 ml/kg/min). Injeção de muscimol no KF reduziu a hiperventilação (1827 ± 61, vs. salina: 3179 ± 325 ml/kg/min) e a taquicardia (380 ± 9, vs. salina: 423 ± 12 bpm), produzidos pela hipóxia (8% O2 - 10 min). Muscimol no KF reduziu a hiperventilação (1488 ± 277, vs. salina: 3539 ± 374 ml/kg/min) produzida por hipercapnia (7% CO2 - 10 min). Injeção de muscimol no NPBL promoveu um aumento de PAM (D = 119 ± 2, vs. salina: 104 ± 2 mmHg), mas não foi capaz de alterar a hiperventilação produzida por hipóxia e hipercapnia. Nossos experimentos mostram a participação da região KF, e não do NPBL, no controle do respiratório durante a ativação do quimiorreflexo central e periférico. / Here we evaluated the involvement of Kölliker-Fuse region (KF) and lateral parabrachial nucleus (LPBN) in the cardiorespiratory responses elicited by chemoreceptor activation in conscious rats. Bilateral injection of muscimol (200 pmol/100 nl) into the KF decreased resting ventilation (978 ± 100, vs. saline: 1436 ± 155 ml/kg/min). Muscimol injection into the KF reduced the increase in ventilation (1827 ± 61, vs. saline: 3179 ± 325 ml/kg/min) produced by hypoxia (8% O2 - 10 min) or hypercapnia (7% CO2 - 10 min) (1488 ± 277, vs. saline: 3539 ± 374 ml/kg/min). The injection of muscimol into the LPBN increased resting MAP (D =119 ± 2, vs. saline: 104 ± 2 mmHg). Muscimol into the LPBN did not change the increase in ventilation elicited by hypoxia or hypercapnia in unrestrained rats. The results of the present study suggest that KF region, but not LPBN, have mechanisms to control the ventilation in resting, hypoxic or hypercapnic conditions in conscious rats.

Activation of the immune system by e.g. bacteria induces the acute-phase-response and sickness behaviour. The latter encompasses among other things fever, lethargy, anorexia and hyperalgesia. An often used model to study sickness behaviour is the intravenous injection of the gram negative bacterial endotoxin lipopolysaccharide (LPS). LPS induces the production of inflammatory mediators, such as cytokines and prostaglandins, which in turn can interact with the central nervous system (CNS) to affect behaviour. The CNS also memorises substances that have made us sick in the past to avoid future harm, a phenomenon called conditioned taste aversion (CTA). An often used model to study CTA is the intraperitoneal injection of LiCl. The pontine parabrachial nucleus (PB) is an autonomic relay nucleus situated in the rostral brain stem that integrates afferent somatosensory and interoceptive information and forwards this information to the hypothalamus and limbic structures. PB is crucial for the acquisition of CTA and PB neurons are activated by many anorexigenic substances. Further, PB neurons express neuropeptides, among those calcitonin gene related peptide (CGRP) and enkephalin, both of which have been implicated in immune signalling, nociception, food intake, and aversion. By using a dual-labelling immunohistochemical/in situ hybridization technique we investigated if enkephalinergic neurons in PB are activated by systemic immune challenge. While there were many neurons in the external lateral parabrachial subnucleus (PBel) that expressed the immediate early gene fos after intravenous injection of LPS and while a large proportion of the PBel neurons expressed preproenkephalin, there were very few double-labelled cells. The fos-expressing cells were predominantly located to the outer part of the PBel (PBelo), whereas the preproenkephalin-expressing PBel neurons were located closest to the peduncle. Thus we conclude that although enkephalin has been implicated in autonomic and immune signalling, enkephalinergic neurons in PB do not seem to be activated by immune stimulation (paper I). To further characterise the PBelo neurons activated by immune challenge we investigated if these neurons expressed CGRP. Dual-labelling in situ hybridisation showed that PBelo neurons that expressed fos after intravenous injection of LPS to a large extent co-expressed CGRP mRNA, indicating that CGRP may be involved in the regulation of the sickness response in immune challenge (paper II). Using dual-labelling immunohistochemistry we examined if PBel neurons activated by an immune stimulus projected to the amygdala, a limbic structure implicated in the affective response to homeostatic challenge. Animals were injected with the retrograde tracer substance cholera toxin b (CTb) into the amygdala and subsequently subjected to immune challenge. We found that approximately a third of the neurons that expressed fos after the intravenous injection of LPS also were labelled with CTb. Thus PBel neurons activated by immune challenge project to the amygdala. The PBel-amygdala pathway has earlier been suggested to be important in nociceptive signalling. To investigate if amygdala-projecting PBel neurons are activated by nociceptive stimuli we again injected animals with CTb into the amygdala. After recovery the animals were injected with formalin into a hindpaw. Dual-labelling immunohistochemistry against fos and CTb showed that very few noxiously activated PB neurons projected to the amygdala. Thus, the PBel-amygdala projection seems to be important in immune challenge but not in nociceptive signalling (paper III). Many PBel neurons express fos after intraperitoneal injection of LiCl. Melanocortins are neuropeptides that recently have been implicated in metabolism, food intake and aversive mechanisms. The PB is known to express melanocortin receptor-4 (MC4-R) mRNA. Using dual-labelling in situ hybridization we investigated if PB neurons activated by intravenous injection of LPS or intraperitoneal injection of LiCl expressed MC4-R mRNA. We found that many PBelo neurons were activated by either LPS or LiCl and that a large proportion of such activated neurons expressed MC4-R mRNA. Further, using dual-labelling in situ hybridization against MC4-R mRNA and CGRP mRNA, we found that a large proportion of the CGRP positive PBelo neurons also expressed MC4-R mRNA. In summary, this thesis shows that CGRP-expressing neurons in the PBel are activated by peripheral immune challenge, that lipopolysaccharide-activated PBel neurons project to the amygdala, that the amygdala-projecting neurons in the PBel are CGRP-positive, and that PBel neurons activated by immune or aversive challenge express MC4-R. Taken together, these data suggest the presence of a melanocortin-regulated CGRP-positive pathway from the PBel to the amygdala that relays information of importance to certain aspects of sickness behaviour. / On the day of the defence date the title of article II was: Feeding-related immune responsive brain stem neurons: association with CGRP. Article II: Erratum for in Neuroreport 2001;12(16):inside back cover. Neuroreport 2001;12(13):inside back cover. Article III: Erratum in: J Comp Neurol. 2005; 483:489-90.

No presente trabalho, avaliamos o envolvimento da região Kölliker Fuse (KF) e núcleo parabraquial lateral (NPBL) nas respostas cardiorrespiratórias induzidas pela ativação dos quimiorreceptores centrais e periféricos em ratos não anestesiados. A injeção bilateral de muscimol (200 pmol/100 nl) no KF reduziu a ventilação basal (978 ± 100, vs. salina: 1436 ± 155 ml/kg/min). Injeção de muscimol no KF reduziu a hiperventilação (1827 ± 61, vs. salina: 3179 ± 325 ml/kg/min) e a taquicardia (380 ± 9, vs. salina: 423 ± 12 bpm), produzidos pela hipóxia (8% O2 - 10 min). Muscimol no KF reduziu a hiperventilação (1488 ± 277, vs. salina: 3539 ± 374 ml/kg/min) produzida por hipercapnia (7% CO2 - 10 min). Injeção de muscimol no NPBL promoveu um aumento de PAM (D = 119 ± 2, vs. salina: 104 ± 2 mmHg), mas não foi capaz de alterar a hiperventilação produzida por hipóxia e hipercapnia. Nossos experimentos mostram a participação da região KF, e não do NPBL, no controle do respiratório durante a ativação do quimiorreflexo central e periférico. / Here we evaluated the involvement of Kölliker-Fuse region (KF) and lateral parabrachial nucleus (LPBN) in the cardiorespiratory responses elicited by chemoreceptor activation in conscious rats. Bilateral injection of muscimol (200 pmol/100 nl) into the KF decreased resting ventilation (978 ± 100, vs. saline: 1436 ± 155 ml/kg/min). Muscimol injection into the KF reduced the increase in ventilation (1827 ± 61, vs. saline: 3179 ± 325 ml/kg/min) produced by hypoxia (8% O2 - 10 min) or hypercapnia (7% CO2 - 10 min) (1488 ± 277, vs. saline: 3539 ± 374 ml/kg/min). The injection of muscimol into the LPBN increased resting MAP (D =119 ± 2, vs. saline: 104 ± 2 mmHg). Muscimol into the LPBN did not change the increase in ventilation elicited by hypoxia or hypercapnia in unrestrained rats. The results of the present study suggest that KF region, but not LPBN, have mechanisms to control the ventilation in resting, hypoxic or hypercapnic conditions in conscious rats.

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Orientador: Patricia Maria de Paula / Banca: Luciane Helena Gargaglioni Batalhão / Banca: Lisandra Brandino de Oliveira / Resumo: Estudos recentes demonstram que os receptores purinérgicos estão presentes no núcleo parabraquial lateral (NPBL), uma estrutura pontina envolvida no controle da ingestão de sódio. No presente estudo, investigamos os efeitos das injeções do, -methyleneadenosine 5 -triphosphate (, -metileno ATP, agonista dos receptores P2X) sozinho ou combinado com o ácido piridoxalfosfato-6-azofenil-2',4'-disulfônico (PPADS, antagonista dos receptores P2X) ou suramin (antagonista não seletivo dos receptores P2) no NPBL sobre a ingestão de NaCl 1,8% induzida por depleção de sódio. Também investigamos os efeitos da injeção de, -metileno ATP sozinho ou combinado com o PPADS no NPBL sobre a pressão arterial média (PAM) e freqüência cardíaca (FC) em ratos saciados e depletados de sódio. Foram utilizados ratos Holtzman com implante de cânulas implantadas bilateralmente em direção ao NPBL. A depleção de sódio foi induzida pelo tratamento com o diurético furosemida (20 mg/kg do peso corporal) acompanhado de uma dieta deficiente em sódio por 24 horas. As injeções bilaterais de, -metileno ATP (2,0 e 4,0 nmol/0,2 μL) no NPBL aumentaram a ingestão de NaCl 1,8% induzida por depleção de sódio (25,3 ± 0,8 e 26,5 ± 0,9 mL/2 h, respectivamente, vs. salina: 15,2 ± 1,3 mL/2 h). O pré-tratamento com o suramin (2,0 nmol/0,2 μL) ou com o PPADS (4,0 nmol/0,2 μL) no NPBL aboliu os efeitos do, -metileno-ATP na ingestão de NaCl 1,8% (15,2 ± 1,2 e 16,9 ± 0,9 mL/2 h, respectivamente). As injeções de PPADS sozinho no NPBL não alteraram a ingestão de NaCl 1,8% (14,6 ± 0,8 mL/2 h vs. salina: 18,3 ± 1,8 mL/2 h). No entanto, as injeções de suramin sozinho no NPBL quase aboliram a ingestão de NaCl 1,8% (5,7 ± 1,9 mL/120 min, vs. salina: 15,5 ± 1,1 mL/120 min) e aumentaram a ingestão de sacarose 2% somente no tempo de 90 minutos (7,1 ± 1,3 vs. salina: 5,3 ± 0,8 mL/90 min) sem alterar...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Recent studies have shown that purinergic receptors are present in the lateral parabrachial nucleus (LPBN), a pontine structure involved in the control of sodium intake. In the present study, we investigated the effects of, -methyleneadenosine 5 -triphosphate (, - methylene ATP, selective P2X purinergic agonist) alone or combined with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, P2X purinergic antagonist) or suramin (non-selective P2 purinergic antagonist) injected into the LPBN on sodium depletion-induced by 1.8% NaCl intake. We also investigated the effects of, -methylene ATP alone or combined with PPADS injected into the LPBN on mean arterial pressure (MAP) and heart rate (HR) on replete and sodium depleted rats. Male Holtzman rats with stainless steel cannulas implanted into the LPBN were used. Sodium depletion was induced by treating rats with the diuretic furosemide (20 mg/kg of body weight) followed by 24 h of sodium-deficient diet. Bilateral injections of, -methylene ATP (2.0 and 4.0 nmol/0.2 μL) into the LPBN increased sodium depletion-induced 1.8% NaCl intake (25.3 ± 0.8 and 26.5 ± 0.9 mL/2 h, respectively, vs. saline: 15.2 ± 1.3 mL/2 h). Pre-treatment with suramin (2.0 nmol/0.2 μL) or PPADS (4 nmol/0.2 μL) into the LPBN abolished the effects of, - methylene-ATP on 1.8% NaCl intake (15.2 ± 1.2 and 16.9 ± 0.9 mL/2 h, respectively). Injections of PPADS alone into the LPBN did not change 1.8% NaCl intake (14.6 ± 0.8 ml/2 h vs. saline: 18.3 ± 1.8 mL/2 h). However, injections of suramin alone into the LPBN strongly reduced 1.8% NaCl intake (5.7 ± 1.9 mL/120 min, vs. saline: 15.5 ± 1.1 mL/2 h) and increased the 2% sucrose intake only at 90 min (7.1 ± 1.3 vs. saline: 5.3 ± 0.8 mL/90 min), without changing 24h water deprivation-induced water intake (16.7 ± 1.8 mL/2 h vs. saline: 15.0 ± 2.1 mL/2 h)... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2016-06-02T19:22:13Z (GMT). No. of bitstreams: 1 6779.pdf: 4081865 bytes, checksum: f8c80484ba3b15da8905a6d3499ac8f2 (MD5) Previous issue date: 2015-02-26 / Universidade Federal de Minas Gerais / The lateral parabrachial nucleus (LPBN) is an important area of the hindbrain circuitry involved in cardiorespiratory control. Adenosine triphosphate (ATP) is considered an important central neurotransmitter and purinergic receptors are present in the LPBN. The involvement of purinergic mechanisms of the LPBN in the cardiorespiratory control during hypoxia is still unknown. In the present study, we investigated the effects of alpha, beta-me ATP (P2X purinergic agonist) alone or combined with PPADS (P2 purinergic receptor antagonist) injected into the LPBN on cardiorespiratory responses induced by acute hypoxia (7% O2 for 60 min) in unanesthetized rats and chronic intermittent hypoxia (CIH) (10% O2, 8 hours/7 days) in anesthetized rats. Additionally, in another unanesthetized rats group, we investigated the effect of acute hypoxia (7% O2 for 60 minutes) on the activity of LPBN neurons and also the effect of alpha, beta-me ATP injected into the LPBN on Fos immunoreactivity at NTS induced by acute hypoxia (7% O2 for 60 min). In unanesthetized group, we used male Holtzman rats (290-310 g, n=8/group) with stainless steel cannulas implanted bilaterally into the LPBN. A polyethylene tubing was inserted into abdominal aorta through femoral artery for recording mean arterial pressure (MAP) and heart rate (HR). Respiratory frequency (fR), tidal volume (VT) and ventilation (VE) were recorded by whole-body plethysmography. The unanesthetized rats received bilateral injections of PPADS (4 nmol/0.2 &#956;L) into the LPBN 10 minutes before injections of alpha, beta-me ATP (2 nmol/0.2 &#956;L) or saline into the LPBN. Ten minutes after the LPBN injections, a hypoxic gas mixture (7% O2) was ventilated in the chamber for 60 minutes. In anesthetized group, we used Sprague Dawley rats (300-400 g, n=7) were exposed for 7 days to CIH (alternating 6 min periods of 10% O2 and 4 min of 21% O2 from 8 am to 4 pm; and continuous exposition to normoxic at 21% O2 from 4 pm to 8 am). Mean arterial pressure (MAP), heart rate (HR), renal sympathetic nerve discharge (RSND) and amplitude and phrequency of phrenic nerve activity (PNA) were recorded in rats anesthetized with urethane and alpha chloralose, vagotomized and mechanically ventilated. The anesthetized rats received a unilateral injections of alpha, beta-me ATP (2.0 nmol/50 nL) before, 10 and 30 minutes after PPADS (0.125 nmol/50 nL) into the LPBN. For immunohistochemistry group we used male Holtzman rats, we studied the expression of Fos in the LPBN and medial parabrachial nucleus (MPBN) of unanesthetized rats exposed to 1 h of acute hypoxia, on another group, the animals received a bilateral injections of alpha, beta-me ATP into the LPBN or saline 10 minutes (n= 5/group) before the acute hypoxia for 1 h. After this period, the rats were deeply anesthetized and perfused to remove the brains and carrying out immunohistochemical procedures. In unanesthetized rats, bilateral injections of alpha,beta-me ATP into the LPBN potentiated acute hypoxia-induced increase in VT (&#61508;&#61472;= 4.0±0.3 mL/kg, vs. saline 2.2±0.2 mL/kg, or 81% of increase, p= 0.005) and VE (&#61508;&#61472;= 871±55 mL/kg/min, vs. saline: 598±60 ml/kg/min, or 45% of increase, p= 0.009), without changing hypoxia-induced tachypnea (&#61508;&#61472;fR = 49±5 cpm, vs. saline: 48±5 cpm). The pre-treatment with PPADS into the LPBN abolished the responses produced by alpha, beta-me ATP. Bilateral injections of alpha,beta-me ATP into the LPBN did not affect the hypotension, and tachycardia induced by acute hypoxia. In normoxic anesthetized rats, unilateral injections of alpha, beta-me ATP (2.0 nmol/50 nL) into the LPBN increased MAP (&#916; = 10±2 mmHg, vs. saline: 0±1 mmHg, p<0.05), RSND (&#916; = 40±12%, vs. saline: 1±1%) and phrequency PNA (&#916; = 17±5 cpm, vs. saline: 0±1 cpm, p<0.05), without changing HR and amplitude PNA. Unilateral injection of PPADS into the LPBN abolished the increase in MAP (&#916; = 0±1 mmHg), RSND (&#916; = 3±3.1%) and phrequency PNA (&#916; = 1±1 cpm) produced by alpha, beta-me ATP injected into LPBN. In anesthetized CIH rats, the injection of alpha, beta-me ATP into the LPBN increased even more MAP (&#916; = 20±2, vs. saline: -1±1 mmHg), HR (&#916; = 25±5 bpm, vs. saline: -1±1 bpm) and amplitude PNA (&#916; = 87±31%, vs. saline: 2±1%), in addition to have increased also frequency PNA (&#916; = 17±5 cpm, vs. saline: -1±1 cpm). In immunohistochemistry group the acute hypoxia produces activation of the LPBN neurons (93 ± 8, vs. normoxic 22 ± 8 cells), while the injection of alpha, beta-me ATP into the LPBN potentiated the Fos expression in caudal NTS (NTSc; 88 ± 4, vs. saline: 42 ± 8 cells) and rostral NTS (NTSr, 62 ± 8, vs. saline: 38 ± 4 cells). In conclusion, our data suggest that the P2 receptors into the LPBN are involved in the cardiorespiratory responses induced by acute hypoxia and chronic intermittent hypoxia and these responses activate neurons in the NTS, suggesting possible direct or indirect projections between the LPBN and the NTS. / O núcleo parabraquial lateral (NPBL) é uma importante área da circuitaria do tronco encefálico envolvida no controle cardiorrespiratório. A adenosina trifosfato (ATP) é considerada um importante neurotransmissor e os receptores purinérgicos estão presentes no NPBL. O envolvimento de mecanismos purinérgicos do NPBL no controle cardiorrespiratório durante a hipóxia ainda é desconhecido. No presente estudo, investigamos os efeitos do alfa, beta-metileno ATP (alfa, beta-me ATP, agonista purinérgico P2X) sozinho ou combinado com PPADS (antagonista dos receptores purinérgicos P2) injetado no NPBL sobre as respostas cardiorrespiratórias induzidas por hipóxia aguda (7% O2 por 60 min) em ratos não anestesiados e hipóxia crônica intermitente (HCI, 10% de O2, 8 horas/7 dias) em ratos anestesiados. Além disso, em um outro grupo de ratos não anestesiados, investigamos o efeito da hipóxia aguda (7% de O2, durante 60 min) sobre a atividade dos neurônios do NPBL e também o efeito da injeção de alfa, beta-me ATP no NPBL sobre a imunorreatividade à proteína Fos no NTS induzido por hipóxia aguda. No grupo de ratos não anestesiados, foram utilizados ratos Holtzman (290-310 g, n = 8/ grupo) com cânulas de aço inoxidável implantadas bilateralmente no NPBL. Um tubo de polietileno foi inserido na aorta abdominal através da artéria femoral para registro da pressão arterial média (PAM) e frequência cardíaca (FC). A frequência respiratória (fR), volume corrente (VC) e ventilação (VE) foram registrados através da pletismografia de corpo inteiro. Os ratos não anestesiados receberam injeções bilaterais de PPADS (4 nmol/0,2 &#956;L) no NPBL 10 minutos antes da injeção de alfa, beta-me ATP (2 nmol/0,2 &#956;L) ou salina no NPBL. Dez minutos após as injeções no NPBL, uma mistura de gás hipóxico (7% de O2) foi ventilado na câmara, durante 60 minutos. No grupo de ratos anestesiado, foram utilizados ratos Sprague Dawley (300-400 g, n = 7) que foram expostos por 7 dias à HCI (alternando períodos de 6 minutos de 10% de O2 e 4 min de 21% O2 entre as 08:00- 16:00 hs; e exposição contínua à normóxia em 21% O2 entre as 08:00-16:00 hs). PAM, FC, atividade nervosa simpática renal (ANSR) e atividade do nervo frênico (ANF) foram registradas em ratos anestesiados com uretana e alfa cloralose, vagotomizados e ventilados mecanicamente. Os animais anestesiados receberam injeções unilaterais de alfa, beta-me ATP (2,0 nmol/50 nL) antes, 10 e 30 minutos após PPADS (0,125 nmol/50 nL) no NPBL. Para o grupo de imunohistoquímica, foram utilizados ratos Holtzman, onde estudamos a expressão da proteína Fos no NBPL e núcleo parabraquial medial (NPBM) de ratos não anestesiados submetidos a 1 h de hipóxia aguda. Em um outro grupo, os animais receberam injeções bilaterais no NPBL de alfa, beta-me ATP ou salina (n = 5/grupo) 10 minutos antes da hipóxia aguda durante 1 h. Após este período, os ratos foram anestesiados profundamente e perfundidos para a remoção dos encéfalos e realização de procedimentos de imunohistoquímica. Nos ratos não anestesiados as injeções bilaterais de alfa, beta-me ATP no NPBL potencializou o aumento no VC (&#61508;= 4,0 ± 0,3 ml/kg, vs. salina: 2,2 ± 0,2 mL/kg, ou 81% de aumento, p = 0,005) e VE (&#61508;= 871 ± 55 mL/kg/min, vs. salina: 598 ± 60 ml/kg/min, ou seja 45% de aumento, p = 0,009), sem alterar taquipneia (&#61508;fR = 49 ± 5 cpm, vs. salina: 48 ± 5 cpm) induzida por hipóxia aguda. O pré-tratamento com PPADS no NPBL aboliu as respostas de alfa, beta-me ATP. Injeções bilaterais de alfa, beta-me ATP no NPBL não afetou a hipotensão e taquicardia induzidos pela hipóxia aguda. Em ratos anestesiados expostos a normóxia por 7 dias, as injeções unilaterais de alfa, beta-me ATP (2,0 nmol/50 nL) aumentou a PAM (&#916; = 10 ± 2 mmHg, vs. salina: 0 ± 1 mmHg, p <0,05), ANSR (&#916; = 40 ± 12%, vs. salina: 1 ± 1%) e frequência da ANF (&#916; = 17 ± 5 cpm, vs. salina: 0 cpm ± 1cpm), sem alterar FC e a amplitude da ANF. O pré-tratamento com PPADS (0,125 nmol/50 nL) no NPBL aboliu o aumento da PAM (&#916; = 0 ± 1 mmHg), ANSR (&#916; = 3 ± 3,1%) e a frequência da ANF (&#916; = 1 ± 1 cpm) produzidos pela injeção de alfa, beta-me ATP. Em ratos anestesiados expostos à HCI, a injeção de alfa, beta-me ATP no NPBL aumentou ainda mais PAM (&#916; = 20 ± 2, vs. salina: -1 ± 1 mmHg), FC (&#916; = 25 ± 5 bpm, vs. salina: -1 ± 1 bpm) e a amplitude da ANF (&#916; = 87 ± 31%, vs. salina: 2 ± 1%), além de ter aumentado também a freqüência da ANF (&#916; = 17 ± 5 cpm, vs. salina: -1 ± 1 cpm, p <0,05). No grupo imunohistoquímica, a hipóxia aguda produziu a ativação dos neurônios do NPBL (93 ± 8, normóxia, vs. 22 ± 8 células), enquanto que a injeção de alfa, beta-me ATP no NPBL potencializou a expressão de Fos no NTS caudal (NTSc; 88 ± 4, vs. salina: 42 ± 8 células) e rostral (NTSr, 62 ± 8, vs. salina: 38 ± 4 células). Concluindo, nossos resultados sugerem que os receptores P2 do NPBL estão envolvidos nas respostas cardiorrespiratórias induzidas por hipóxia aguda e hipóxia crônica intermitente e essas respostas ativam neurônios no NTS, sugerindo possíveis projeções diretas ou indiretas entre o NPBL e o NTS.

Made available in DSpace on 2014-06-11T19:23:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-30Bitstream added on 2014-06-13T20:30:21Z : No. of bitstreams: 1 menezes_mf_me_arafo.pdf: 831526 bytes, checksum: 44cecb40bcf8a2490778eb7d99c815ee (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Estudos recentes demonstram que os receptores purinérgicos estão presentes no núcleo parabraquial lateral (NPBL), uma estrutura pontina envolvida no controle da ingestão de sódio. No presente estudo, investigamos os efeitos das injeções do , -methyleneadenosine 5 -triphosphate ( , -metileno ATP, agonista dos receptores P2X) sozinho ou combinado com o ácido piridoxalfosfato-6-azofenil-2',4'-disulfônico (PPADS, antagonista dos receptores P2X) ou suramin (antagonista não seletivo dos receptores P2) no NPBL sobre a ingestão de NaCl 1,8% induzida por depleção de sódio. Também investigamos os efeitos da injeção de , -metileno ATP sozinho ou combinado com o PPADS no NPBL sobre a pressão arterial média (PAM) e freqüência cardíaca (FC) em ratos saciados e depletados de sódio. Foram utilizados ratos Holtzman com implante de cânulas implantadas bilateralmente em direção ao NPBL. A depleção de sódio foi induzida pelo tratamento com o diurético furosemida (20 mg/kg do peso corporal) acompanhado de uma dieta deficiente em sódio por 24 horas. As injeções bilaterais de , -metileno ATP (2,0 e 4,0 nmol/0,2 μL) no NPBL aumentaram a ingestão de NaCl 1,8% induzida por depleção de sódio (25,3 ± 0,8 e 26,5 ± 0,9 mL/2 h, respectivamente, vs. salina: 15,2 ± 1,3 mL/2 h). O pré-tratamento com o suramin (2,0 nmol/0,2 μL) ou com o PPADS (4,0 nmol/0,2 μL) no NPBL aboliu os efeitos do , -metileno-ATP na ingestão de NaCl 1,8% (15,2 ± 1,2 e 16,9 ± 0,9 mL/2 h, respectivamente). As injeções de PPADS sozinho no NPBL não alteraram a ingestão de NaCl 1,8% (14,6 ± 0,8 mL/2 h vs. salina: 18,3 ± 1,8 mL/2 h). No entanto, as injeções de suramin sozinho no NPBL quase aboliram a ingestão de NaCl 1,8% (5,7 ± 1,9 mL/120 min, vs. salina: 15,5 ± 1,1 mL/120 min) e aumentaram a ingestão de sacarose 2% somente no tempo de 90 minutos (7,1 ± 1,3 vs. salina: 5,3 ± 0,8 mL/90 min) sem alterar... / Recent studies have shown that purinergic receptors are present in the lateral parabrachial nucleus (LPBN), a pontine structure involved in the control of sodium intake. In the present study, we investigated the effects of , -methyleneadenosine 5 -triphosphate ( , - methylene ATP, selective P2X purinergic agonist) alone or combined with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, P2X purinergic antagonist) or suramin (non-selective P2 purinergic antagonist) injected into the LPBN on sodium depletion-induced by 1.8% NaCl intake. We also investigated the effects of , -methylene ATP alone or combined with PPADS injected into the LPBN on mean arterial pressure (MAP) and heart rate (HR) on replete and sodium depleted rats. Male Holtzman rats with stainless steel cannulas implanted into the LPBN were used. Sodium depletion was induced by treating rats with the diuretic furosemide (20 mg/kg of body weight) followed by 24 h of sodium-deficient diet. Bilateral injections of , -methylene ATP (2.0 and 4.0 nmol/0.2 μL) into the LPBN increased sodium depletion-induced 1.8% NaCl intake (25.3 ± 0.8 and 26.5 ± 0.9 mL/2 h, respectively, vs. saline: 15.2 ± 1.3 mL/2 h). Pre-treatment with suramin (2.0 nmol/0.2 μL) or PPADS (4 nmol/0.2 μL) into the LPBN abolished the effects of , - methylene-ATP on 1.8% NaCl intake (15.2 ± 1.2 and 16.9 ± 0.9 mL/2 h, respectively). Injections of PPADS alone into the LPBN did not change 1.8% NaCl intake (14.6 ± 0.8 ml/2 h vs. saline: 18.3 ± 1.8 mL/2 h). However, injections of suramin alone into the LPBN strongly reduced 1.8% NaCl intake (5.7 ± 1.9 mL/120 min, vs. saline: 15.5 ± 1.1 mL/2 h) and increased the 2% sucrose intake only at 90 min (7.1 ± 1.3 vs. saline: 5.3 ± 0.8 mL/90 min), without changing 24h water deprivation-induced water intake (16.7 ± 1.8 mL/2 h vs. saline: 15.0 ± 2.1 mL/2 h)... (Complete abstract click electronic access below)

Made available in DSpace on 2016-06-02T19:22:11Z (GMT). No. of bitstreams: 1 6151.pdf: 1975366 bytes, checksum: 23b842452d75ee4bd80f5408eccc025c (MD5) Previous issue date: 2014-08-26 / Universidade Federal de Minas Gerais / Sodium intake is induced by facilitatory signals, such as angiotensin II (ANG II) and aldosterone. Hyperosmolarity and central cholinergic activation, classic antinatriorexigenic stimuli, also induce NaCl intake when the inhibitory mechanisms of the lateral parabrachial nucleus (LPBN) are deactivated. In the present study, we investigated the possible interaction between osmoreceptors and cholinergic and angiotensinergic mechanisms in the control of water and NaCl intake induced by different dipsogenic and/or natriorexigenic stimuli combined with the blockade of LPBN inhibitory mechanisms. Rats with stainless steel cannulas implanted in the lateral ventricle (LV) or subfornical organ (SFO) and bilaterally into the LPBN were used to study the effects of injections of atropine (muscarinic cholinergic antagonist), losartan or ZD 7155 (AT1 receptor antagonists) into the LV or SFO on water and 0.3 M NaCl intake induced by bilateral injections of moxonidine (&#945;2- adrenoceptor/imidazoline agonist) into the LPBN combined with a) plasma hyperosmolarity induced by intragastric (ig) 2 M NaCl; b) injections of carbachol (cholinergic agonist) into the LV or SFO; c) subcutaneous injections of furosemide (FURO) and captopril (CAP); d) injection of ANG II into the LV. Additionally, we also investigated whether acute application of osmotic, angiotensinergic and cholinergic stimuli would activate cultured SFO dissociated cells and if the same cell would be activated by different stimuli. In rats treated with ig 2 M NaCl, injections of moxonidine (0.5 nmol/0.2 &#956;l) into the LPBN increased water and 0.3 M NaCl intake. Injections into the LV or SFO of atropine (20 nmol/1.0 &#956;l and 2 nmol/0.1 &#956;l, respectively) or losartan (100 &#956;g/1.0 &#956;l and 1 &#956;g/0.1 &#956;l, respectively) abolished water and 0.3 M NaCl intake in rats treated with ig 2 M NaCl combined with moxonidine into the LPBN. Moxonidine injected into the LPBN also increased water and 0.3 M NaCl intake induced by FURO + CAP, injections of ANG II (50 ng/1.0 &#956;l) and carbachol (4 nmol/1.0 &#956;l) into the LV or carbachol (0.5 nmol/0.1 &#956;l) into the SFO. The blockade of AT1 receptors with injections of losartan into the LV or ZD 7155 (1 &#956;g/0.1 &#956;l) into the SFO abolished water and 0.3 M NaCl intake in rats treated with carbachol into the LV or SFO combined with LPBN injections of moxonidine. However, atropine injected into the LV, despite reducing water intake, did not change 0.3 M NaCl intake in rats treated with FURO + CAP or injection of ANG II into the LV combined with injections of moxonidine into the LPBN. Injections of losartan into the LV reduced 0.06 M sucrose intake, but did not change food intake induced by 24 h of food deprivation. Finally, in vitro studies showed that osmotic, angiotensinergic and cholinergic stimuli activate SFO dissociated cells and that different stimuli can activate the same SFO cell. Therefore, the results of the present study suggest that different stimuli, such as hyperosmolarity and central cholinergic activation, facilitate NaCl intake through activation of central angiotensinergic mechanisms. / A ingestão de sódio é induzida por sinais facilitatórios, como angiotensina II (ANG II) e aldosterona. A hiperosmolaridade e a estimulação colinérgica central, estímulos classicamente considerados antinatriorexigênicos, também induzem ingestão de NaCl quando os mecanismos inibitórios do núcleo parabraquial lateral (NPBL) são bloqueados. No presente estudo, investigamos a possível interação entre osmorreceptores e mecanismos colinérgicos e angiotensinérgicos centrais no controle da ingestão de água e NaCl induzida por diferentes estímulos dipsogênicos e/ou natriorexigênicos combinados com bloqueio dos mecanismos inibitórios do NPBL. Em ratos com cânulas de aço inoxidável implantadas no ventrículo lateral (VL) ou órgão subfornical (OSF) e bilateralmente no NPBL, foram estudados os efeitos de injeções de atropina (antagonista colinérgico muscarínico), losartan ou ZD 7155 (antagonistas de receptores AT1) no VL ou diretamente no OSF na ingestão de água e NaCl 0,3 M induzida por injeções bilaterais de moxonidina (agonista adrenérgico &#945;2/imidazólico) no NPBL combinadas com: a) hiperosmolaridade plasmática induzida por sobrecarga intragástrica de NaCl 2 M; b) injeções de carbacol (agonista colinérgico) no VL ou OSF; c) injeções subcutâneas de furosemida (FURO) e captopril (CAP); d) injeção de ANG II no VL. Adicionalmente, também foi investigado se a aplicação aguda de estímulos osmóticos, angiotensinérgico e colinérgico ativariam neurônios dissociados do OSF mantidos em cultura e se um mesmo neurônio seria ativado por diferentes estímulos. Em ratos tratados com NaCl 2 M ig, injeções de moxonidina (0,5 nmol/0,2 &#956;l) no NPBL aumentaram a ingestão de água e NaCl 0,3 M. Injeções no VL ou OSF de atropina (20 nmol/1,0 &#956;l e 2 nmol/0,1 &#956;l, respectivamente) ou losartan (100 &#956;g/1,0 &#956;l e 1 &#956;g/0,1 &#956;l, respectivamente) aboliram a ingestão de água e NaCl em ratos tratados com NaCl 2 M ig que receberam injeções de moxonidina no NPBL. Injeções de moxonidina também aumentaram a ingestão de água e NaCl 0,3 M induzida por FURO + CAP, injeções de ANG II (50 ng/1,0 &#956;l) e carbacol (4 nmol/1,0 &#956;l) no VL ou carbacol (0,5 nmol/0,1 &#956;l) no OSF. O bloqueio de receptores AT1 com injeções de losartan no VL ou ZD 7155 (1 &#956;g/0,1 &#956;l) no OSF aboliu a ingestão de água e NaCl 0,3 M em ratos tratados com injeção de carbacol no VL ou OSF combinada com injeções de moxonidina no NPBL. No entanto, injeção de atropina no VL, apesar de reduzir a ingestão de água, não alterou a ingestão de NaCl 0,3 M em ratos tratados com FURO + CAP ou injeção de ANG II no VL combinados com injeções de moxonidina no NPBL. Injeções de losartan no VL reduziram a ingestão de sacarose 0,06 M, mas não alteraram a ingestão de ração induzida por privação alimentar por 24 h. Finalmente, os estudos in vitro mostraram que estímulos osmóticos, angiotensinérgico e colinérgico ativam as células dissociadas do OSF e que diferentes estímulos podem ativar uma mesma célula do OSF. Portanto, os resultados do presente estudo sugerem que diferentes estímulos, tais como hiperosmolaridade e ativação colinérgica central, facilitam a ingestão de NaCl através da ativação de mecanismos angiotensinérgicos centrais.