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OBJECTIVE The lateral parabrachial nucleus (lPBN) in the brainstem has emerged as a key area involved in feeding control that is targeted by several circulating anorexigenic hormones. Here, the objective was to determine whether the lPBN is also a relevant site for the orexigenic hormone ghrelin, inspired by studies in mice and rats showing that there is an abundance of ghrelin receptors in this area. METHODS This study first explored whether iPBN cells respond to ghrelin involving Fos mapping and electrophysiological studies in rats. Next, rats were injected acutely with ghrelin, a ghrelin receptor antagonist, or vehicle into the lPBN to investigate feeding-linked behaviors. RESULTS Curiously, ghrelin injection (intracerebroventricular or intravenous) increased Fos protein expression in the lPBN yet the predominant electrophysiological response was inhibitory. Intra-lPBN ghrelin injection increased chow or high-fat diet intake, whereas the antagonist decreased chow intake only. In a choice paradigm, intra-lPBN ghrelin increased intake of chow but not lard or sucrose. Intra-lPBN ghrelin did not alter progressive ratio lever pressing for sucrose or conditioned place preference for chocolate. CONCLUSIONS The lPBN is a novel locus from which ghrelin can alter consummatory behaviors (food intake and choice) but not appetitive behaviors (food reward and motivation).

Objective Intermittent (INT) access to a high-fat diet (HFD) can induce excessive-intake phenotypes in rodents. This study hypothesized that impaired satiation responses contribute to elevated intake in an INT-HFD access model. Methods First, this study characterized the intake and meal patterns of female rats that were subjected to an INT HFD in which a 45% HFD was presented for 20 hours every fourth day. To examine nutrient-induced satiation, rats received intragastric infusions of saline or Ensure Plus prior to darkness-onset food access. A similar design was used to examine sensitivity to the satiating effect of amylin. This study then examined whether an INT HFD influences amylin-induced c-Fos in feeding-relevant brain areas. Results Upon INT HFD access, rats consumed meals of larger size. The anorexic response to intragastric Ensure infusion and exogenous amylin treatment was blunted in INT rats on both chow-only and INT-HFD days of the diet regimen, compared with chow-maintained and continuous-HFD rats. An INT HFD did not influence amylin-induced c-Fos in the area postrema, nucleus of the solitary tract, and lateral parabrachial nucleus. Conclusions Impaired satiation responses, mediated in part by reduced sensitivity to amylin, may explain the elevated intake observed upon INT HFD access and may play a role in disorders of INT overconsumption, including binge eating.

Both warm- and cold-sensitive neurons are found in the lateral parabrachial nucleus (LPB), a crucial relay for skin temperature information from the spinal cord to the preoptic area. The aims of this study were to investigate the electrophysiological properties of temperature-sensitive and -insensitive neurons in brain slices, and elucidate the basic mechanisms underlying the thermosensitivity of rat LPB neurons. In warm-sensitive neurons, temperature exerted no significant effects on resting membrane potential (RMP), threshold potential, and amplitude of the afterhyperpolarizing potential. However, warming significantly increased the prepotential rates of depolarization and the inactivation rates of potassium A current (IA) in warm-sensitive neurons, which in turn shortened their interspike interval and elevated the firing rate. In contrast, temperature had no significant effects on the depolarizing prepotentials and inactivation rate of IA in temperature-insensitive neurons. Besides, in cold-sensitive neurons, cooling and warming produced membrane depolarization and hyperpolarization, respectively, and there was a strong correlation between firing rate and membrane potential thermosensitivity. Nevertheless, temperature exhibited no significant effect on the depolarizing prepotential of cold-sensitive neurons. These results suggest that LPB neuronal warm sensitivity may reside in the temperature-dependent prepotentials and IA, while neuronal cold sensitivity might be mainly due to heat-induced changes in RMP.

The neural circuitry mechanism that underlies dopaminergic (DA) control of innate feeding behavior is largely uncharacterized. Here, we identified a subpopulation of DA neurons situated in the caudal ventral tegmental area (cVTA) directly innervating DRD1-expressing neurons within the lateral parabrachial nucleus (LPBN). This neural circuit potently suppresses food intake via enhanced satiation response. Notably, this cohort of DAcVTA neurons is activated immediately before the cessation of each feeding bout. Acute inhibition of these DA neurons before bout termination substantially suppresses satiety and prolongs the consummatory feeding. Activation of postsynaptic DRD1LPBN neurons inhibits feeding, whereas genetic deletion of Drd1 within the LPBN causes robust increase in food intake and subsequent weight gain. Furthermore, the DRD1LPBN signaling manifests the central mechanism in methylphenidate-induced hypophagia. In conclusion, our study illuminates a hindbrain DAergic circuit that controls feeding through dynamic regulation in satiety response and meal structure.

Lateral parabrachial nucleus (LPB) is a critical region in the integration and transmission of peripheral nociceptive information. The parabrachio‐amygdaloid (P‐Amy) pathway and parabrachio‐ventral tegmental area (P‐VTA) pathway is thought to be significant in regulation of pain‐related negative emotions. In present study, retrograde tract tracers Fluoro‐gold (FG) and tetramethylrhodramine‐dextran (TMR) were stereotaxically injected into the right central amygdaloid nucleus (CeA) and right VTA, respectively. Then, part of these rats were performed with the spare nerve injury (SNI) in the controlateral side of FG and TMR injection. Afterwards, double‐ or triple‐immunofluorescent histochemistry was used to examine FG/TMR double‐ and FG/TMR/FOS or FG/TMR/CGRP triple‐labeled neurons in the LPB. The results showed that all of FG, TMR single‐ and FG/TMR double‐labeled neurons were distributed in the LPB bilaterally with an ipsilateral predominance. The proportion of FG/TMR double‐labeled neurons to the total number of FG‐ and TMR‐labeled neurons was 10.78% and 13.07%, respectively. Nearly all of the FG/TMR double‐labeled neurons (92.67%) showed calcitonin gene‐related peptide (CGRP) immunopositive. On the other hand, in the SNI rats, about 89.49% and 77.87% of FG‐ and TMR‐labeled neurons were FG/FOS‐ and TMR/FOS‐positive neurons; about 93.33% of the FG/TMR double‐labeled neurons were FOS‐LI. Our results suggest that the part of CGRP immunopositive neurons in the LPB send projection fibers to both the CeA and VTA by the way of axon collaterals, which are activated by the nociceptive stimulation in the SNI condition, and may play an important role in the transmission of peripheral nociceptive information. Anat Rec, 302:1178–1186, 2019. © 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Growing evidence implicates neurons that project from the lateral parabrachial nucleus (LPBN) to the hypothalamic ventromedial nucleus (VMN) in a neurocircuit that drives counterregulatory responses to hypoglycemia, including increased glucagon secretion. Among LPBN neurons in this circuit is a subset that expresses cholecystokinin (LPBN(CCK) neurons) and is tonically inhibited by leptin. Because uncontrolled diabetes is associated with both leptin deficiency and hyperglucagonemia, and because intracerebroventricular (ICV) leptin administration reverses both hyperglycemia and hyperglucagonemia in this setting, we hypothesized that deficient leptin inhibition of LPBN(CCK) neurons drives activation of this LPBN→VMN circuit and thereby results in hyperglucagonemia. Here, we report that although bilateral microinjection of leptin into the LPBN does not ameliorate hyperglycemia in rats with streptozotocin-induced diabetes mellitus (STZ-DM), it does attenuate the associated hyperglucagonemia and ketosis. To determine if LPBN leptin signaling is required for the antidiabetic effect of ICV leptin in STZ-DM, we studied mice in which the leptin receptor was selectively deleted from LPBN(CCK) neurons. Our findings show that although leptin signaling in these neurons is not required for the potent antidiabetic effect of ICV leptin, it is required for leptin-mediated suppression of diabetic hyperglucagonemia. Taken together, these findings suggest that leptin-mediated effects in animals with uncontrolled diabetes occur through actions involving multiple brain areas, including the LPBN, where leptin acts specifically to inhibit glucagon secretion and associated ketosis.

The aim of the study was to understand the effects of suckling on the brain of the pups by mapping their brain activation pattern in response to suckling.The c-fos method was applied to identify activated neurons. Fasted rat pups were returned to their mothers for suckling and sacrificed 2 hours later for Fos immunohistochemistry. Double labeling was also performed to characterize some of the activated neurons. For comparison, another group of fasted pups were given dry food before Fos mapping.After suckling, we found an increase in the number of Fos-immunoreactive neurons in the insular and somatosensory cortices, central amygdaloid nucleus (CAm), paraventricular (PVN) and supraoptic hypothalamic nuclei, lateral parabrachial nucleus (LPB), nucleus of the solitary tract (NTS), and the area postrema. Double labeling experiments demonstrated the activation of calcitonin gene-related peptide-ir (CGRP-ir) neurons in the LPB, corticotropin-releasing hormone-ir (CRH-ir) but not oxytocin-ir neurons in the PVN, and noradrenergic neurons in the NTS. In the CAm, Fos-ir neurons did not contain CRH but were apposed to CGRP-ir fiber terminals. Refeeding with dry food-induced Fos activation in all brain areas activated by suckling. The degree of activation was higher following dry food consumption than suckling in the insular cortex, and lower in the supraoptic nucleus and the NTS. Furthermore, the accumbens, arcuate, and dorsomedial hypothalamic nuclei, and the lateral hypothalamic area, which were not activated by suckling, showed activation by dry food.Neurons in a number of brain areas are activated during suckling, and may participate in the signaling of satiety, taste perception, reward, food, and salt balance regulation.

Semaglutide, a glucagon-like peptide 1 (GLP-1) analog, induces weight loss, lowers glucose levels, and reduces cardiovascular risk in patients with diabetes. Mechanistic preclinical studies suggest weight loss is mediated through GLP-1 receptors (GLP-1Rs) in the brain. The findings presented here show that semaglutide modulated food preference, reduced food intake, and caused weight loss without decreasing energy expenditure. Semaglutide directly accessed the brainstem, septal nucleus, and hypothalamus but did not cross the blood-brain barrier; it interacted with the brain through the circumventricular organs and several select sites adjacent to the ventricles. Semaglutide induced central c-Fos activation in 10 brain areas, including hindbrain areas directly targeted by semaglutide, and secondary areas without direct GLP-1R interaction, such as the lateral parabrachial nucleus. Automated analysis of semaglutide access, c-Fos activity, GLP-1R distribution, and brain connectivity revealed that activation may involve meal termination controlled by neurons in the lateral parabrachial nucleus. Transcriptomic analysis of microdissected brain areas from semaglutide-treated rats showed upregulation of prolactin-releasing hormone and tyrosine hydroxylase in the area postrema. We suggest semaglutide lowers body weight by direct interaction with diverse GLP-1R populations and by directly and indirectly affecting the activity of neural pathways involved in food intake, reward, and energy expenditure.

Orexin (ORX) neurons in the hypothalamus send their axons to arousal-promoting areas. We have previously shown that glutamatergic neurons in the lateral parabrachial nucleus (LPB) innervate ORX neurons. In this study, we examined potential pathways from the LPB to ORX neurons projecting to arousal-promoting areas in the brainstem by a combination of tract-tracing techniques in male Wistar rats. We injected the anterograde tracer biotinylated dextranamine (BDA) into the LPB and the retrograde tracer cholera toxin B subunit (CTb) into the ventral tegmental area, dorsal raphe nucleus, pedunculopontine tegmental nucleus, laterodorsal tegmental area, or locus coeruleus (LC). We then analyzed the BDA-labeled fibers and ORX-immunoreactive neurons in the hypothalamus. We found that double-labeled ORX and CTb neurons were the most abundant after CTb was injected into the LC. We also observed prominently overlapping distribution of BDA-labeled fibers, arising from neurons located in the lateral-most part of the dorsomedial nucleus and adjacent dorsal perifornical area. In these areas, we confirmed by confocal microscopy that BDA-labeled synaptophysin-immunoreactive axon terminals were in contiguity with cell bodies and dendrites of CTb-labeled ORX-immunoreactive neurons. These results suggest that the LPB innervates arousal-promoting areas via ORX neurons and is likely to promote arousal responses to stimuli.

The impairment of cold-evoked activation of brown adipose tissue (BAT) in rats fed a high-fat diet (HFD) requires the activity of a vagal afferent to the medial nucleus of the solitary tract (mNTS). We determined the role of transient receptor potential vanilloid 1 (TRPV1) activation in the mNTS, and of a dynorphin input to the median preoptic nucleus (MnPO) in the impaired BAT thermogenic response to cold in HFD-fed rats. The levels of some linoleic acid (LA) metabolites, which can act as endogenous TRPV1 agonists, were elevated in the NTS of HFD rats compared with chow-fed rats. In HFD rats, nanoinjections of the TRPV1 antagonist, capsazepine (CPZ) in the NTS rescued the impaired BAT sympathetic nerve activity (BAT SNA) and thermogenic responses to cold. In contrast, in chow-fed rats, cold-evoked BAT SNA and BAT thermogenesis were not changed by nanoinjections of CPZ into the NTS. Axon terminals of NTS neurons that project to the dorsal lateral parabrachial nucleus (LPBd) were closely apposed to LPBd neurons that project to the MnPO. Many of the neurons in the LPBd that expressed c-fos during cold challenge were dynorphinergic. In HFD rats, nanoinjections of the κ opioid receptor (KOR) antagonist, nor-binaltorphimine (nor-BNI), in the MnPO rescued the impaired BAT SNA and thermogenic responses to cold. These data suggest that HFD increases the content of endogenous ligands of TRPV1 in the NTS, which increases the drive to LPBd neurons that in turn release dynorphin in the MnPO to impair activation of BAT.

The external lateral parabrachial nucleus (elPBN) within the pons and rostral ventrolateral medulla (rVLM) contributes to central processing of excitatory cardiovascular reflexes during stimulation of cardiac sympathetic afferent nerves (CSAN). However, the importance of elPBN cardiovascular neurons in regulation of rVLM activity during CSAN activation remains unclear. We hypothesized that CSAN stimulation excites the elPBN cardiovascular neurons and, in turn, increases rVLM activity through elPBN-rVLM projections. Compared with controls, in rats subjected to microinjection of retrograde tracer into the rVLM, the numbers of elPBN neurons double-labeled with c-Fos (an immediate early gene) and the tracer were increased after CSAN stimulation ( P < 0.05). The majority of these elPBN neurons contain vesicular glutamate transporter 3. In cats, epicardial bradykinin and electrical stimulation of CSAN increased the activity of elPBN cardiovascular neurons, which was attenuated ( n = 6, P < 0.05) after blockade of glutamate receptors with iontophoresis of kynurenic acid (Kyn, 25 mM). In separate cats, microinjection of Kyn (1.25 nmol/50 nl) into the elPBN reduced rVLM activity evoked by both bradykinin and electrical stimulation ( n = 5, P < 0.05). Excitation of the elPBN with microinjection of dl-homocysteic acid (2 nmol/50 nl) significantly increased basal and CSAN-evoked rVLM activity. However, the enhanced rVLM activity induced by dl-homocysteic acid injected into the elPBN was reversed following iontophoresis of Kyn into the rVLM ( n = 7, P < 0.05). These data suggest that cardiac sympathetic afferent stimulation activates cardiovascular neurons in the elPBN and rVLM sequentially through a monosynaptic (glutamatergic) excitatory elPBN-rVLM pathway.

Combined the retrograde double tracing with immunofluorescence histochemical staining, we examined the neurons in the lateral parabrachial nucleus (LPB) sent collateral projections to the paraventricular thalamic nucleus (PVT) and central amygdaloid nucleus (CeA) and their roles in the nociceptive transmission in the rat. After the injection of Fluoro-gold (FG) into the PVT and tetramethylrhodamine-dextran (TMR) into the CeA, respectively, FG/TMR double-labeled neurons were observed in the LPB. The percentages of FG/TMR double-labeled neurons to the total number of FG- or TMR-labeled neurons were 6.18% and 9.09%, respectively. Almost all of the FG/TMR double-labeled neurons (95%) exhibited calcitonin gene-related peptide (CGRP) immunoreactivity. In the condition of neuropathic pain, 94% of these neurons showed FOS immunoreactivity. The present data indicates that some of CGRP-expressing neurons in the LPB may transmit nociceptive information toward the PVT and CeA by way of axon collaterals.

Made available in DSpace on 2018-12-11T17:27:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-07-01 The activation of GABAergic receptors, GABAA and GABAB, in the lateral parabrachial nucleus (LPBN) increases water and sodium intake in satiated and fluid-depleted rats. The present study investigated the presence of the GABAC receptor in the LPBN, its involvement in water and sodium intake, and its effects on cardiovascular parameters during the acute fluid depletion induced by furosemide combined with captopril (Furo/Cap). One group of male Wistar rats (290-300 g) with bilateral stainless steel LPBN cannulas was used to test the effects of a GABAC receptor agonist and antagonist on the fluid intake and cardiovascular parameters. We investigated the effects of bilateral LPBN injections of trans-4-aminocrotonic acid (TACA) on the intake of water and 0.3 M NaCl induced by acute fluid depletion (subcutaneous injection of Furo/Cap). c-Fos expression increased (P 0.05). Treatment with the GABAC receptor antagonist (Z)-3-[(aminoiminomethyl)thio]prop-2-enoic acid sulfate (ZAPA, 10 nmol) or with ZAPA (10 nmol) plus TACA (160 nmol) did not change the sodium or water intake compared with that for vehicle (saline) (P > 0.05). Bilateral injections of the GABAC agonist in the LPBN of Furo/Cap-treated rats did not affect the mean arterial pressure (MAP) or heart rate (HR). The GABAC receptor expression in the LPBN was confirmed by the presence of a 50 kDa band. Although LPBN neurons might express GABAC receptors, their activation produced no change in water and sodium intake or in the cardiovascular parameters in the acute fluid depletion rats. Therefore, the GABAC receptors in the LPBN might not interfere with fluid and blood pressure regulation. Department of Physiology School of Medicine of Ribeirão Preto University of São Paulo Department of Basic Sciences School of Dentistry UNESP - Universidade Estadual Paulista Rodovia Department of Basic Sciences School of Dentistry UNESP - Universidade Estadual Paulista Rodovia

Spinal afferents such as nociceptive afferents and group III-IV muscle afferents are known to exert an acute excitatory effect on breathing when activated. Here, we report the surprising existence of latent spinal afferents which exerted tonic inhibitory influence on breathing subliminally in anesthetized rats, an effect which was reversed upon activation of serotonin-1A receptors (5-HT1ARs) in lumbar spinal cord, lesion of pontine lateral parabrachial nucleus or suppression of the adjacent Kolliker-Fuse nucleus with NMDA receptor blockade. Small-interfering RNA knockdown of 5-HT1ARs in lumbar spinal cord unequivocally localized the site of 5-HT1AR-mediated gating of these respiratory-inhibiting interoceptive afferents to relay neurons in the spinal superficial dorsal horn at the lumbar level and not cervical spinal or supraspinal levels. Our results reveal a novel somatosensory/viscerosensory mechanism which exerts tonic inhibitory influence on homeostatic regulation of breathing independent from the classical chemoreflex excitatory pathways, and suggest a hitherto unrecognized therapeutic target in spinal dorsal horn for 5-HT1AR-based treatment of a variety of respiratory abnormalities.

Fasted rats place their tails underneath their body trunks in the cold (tail-hiding behavior), which is a thermoregulatory behavior. The aim of the present study was to investigate the effect of fasting and des-acyl ghrelin, a hormone related to fasting, on tail-hiding behavior and neural activity in the cold. Wistar rats were divided into 'fed', '42-h fasting' and des-acyl ghrelin groups. The rats received an intraperitoneal saline or 30-μg des-acyl ghrelin injection, and were then exposed to 27 °C or 15 °C for 2-h with continuous body temperature (Tb), tail skin temperature (Ttail), and tail-hiding behavior measurements. cFos immunoreactive (cFos-IR) cells in the insula, secondary somatosensory cortex, medial preoptic nucleus, parastrial nucleus, amygdala, and lateral parabrachial nucleus were counted in four segments: seg1, 2, 3, and 4 (bregma -0.36, -1.44, -2.64, and -9.00 mm), respectively. At 15 °C, Tb and Ttail were lower in the 42-h fasting group than in the fed and des-acyl ghrelin groups, and the duration of tail-hiding behavior was longer in the 42-h fasting and des-acyl ghrelin groups than in the fed group. The onset of tail-hiding behavior more advanced in the des-acyl ghrelin group than in the fed group at 15 °C. Only at the insula in seg3 at 15 °C, the number of cFos-IR cells was greater in the 42-h fasting group than in the fed group. Both the 42-h fasting and des-acyl ghrelin groups might modulate the tail-hiding behavior of rats in a cold, and a part of the insula might be involved this response during fasting.

The pancreatic hormone amylin and its agonist salmon calcitonin (sCT) act via the area postrema (AP) and the lateral parabrachial nucleus (PBN) to reduce food intake. Investigations of amylin and sCT signaling in the ventral tegmental area (VTA) and nucleus accumbens (NAc) suggest that the eating inhibitory effect of amylin is, in part, mediated through the mesolimbic 'reward' pathway. Indeed, administration of the sCT directly to the VTA decreased phasic dopamine release (DA) in the NAc. However, it is not known if peripheral amylin modulates the mesolimbic system directly or whether this occurs via the AP and PBN. To determine whether and how peripheral amylin or sCT affect mesolimbic reward circuitry we utilized fast scan cyclic voltammetry under anesthesia to measure phasic DA release in the NAc evoked by electrical stimulation of the VTA in intact, AP lesioned and bilaterally PBN lesioned rats. Amylin (50μg/kg i.p.) did not change phasic DA responses compared to saline control rats. However, sCT (50μg/kg i.p.) decreased evoked DA release to VTA-stimulation over 1h compared to saline treated control rats. Further investigations determined that AP and bilateral PBN lesions abolished the ability of sCT to suppress evoked phasic DA responses to VTA-stimulation. These findings implicate the AP and the PBN as important sites for peripheral sCT to decrease evoked DA release in the NAc and suggest that these nuclei may influence hedonic and motivational processes to modulate food intake.

Thermoregulatory behaviour, such as migration to a comfortable thermal environment, is a representative innate animal behaviour and facilitates effective autonomic regulation of body temperature with a reduced cost of resources. Here we determine the central thermosensory ascending pathway that transmits information on environmental temperature from cutaneous thermoreceptors to elicit thermoregulatory behaviour. To examine the contribution of the spinothalamocortical pathway, which is known to mediate thermosensory transmission for perception of skin temperature, we lesioned thalamic regions mediating this pathway in rats. Thalamic-lesioned rats showed compromised electroencephalographic responses in the primary somatosensory cortex to changes in skin temperature, indicating functional ablation of the spinothalamocortical pathway. However, these lesioned rats subjected to a two-floor innocuous thermal plate preference test displayed intact heat- and cold-avoidance thermoregulatory behaviours. We then examined the involvement of the lateral parabrachial nucleus (LPB), which mediates cutaneous thermosensory signaling to the thermoregulatory center for autonomic thermoregulation. Inactivation of neurons in the LPB eliminated both heat- and cold-avoidance thermoregulatory behaviours and ablated heat defense. These results demonstrate that the LPB, but not the thalamus, mediates the cutaneous thermosensory neural signaling required for behavioural thermoregulation, contributing to understanding of the central circuit that generates thermal comfort and discomfort underlying thermoregulatory behaviours.

Made available in DSpace on 2018-12-11T17:33:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-08-30 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Angiotensin II (ANG II) is a typical facilitatory stimulus for sodium appetite. Surprisingly, hyperosmolarity and central cholinergic stimulation, two classical antinatriorexigenic stimuli, also facilitate NaCl intake when they are combined with injections of the α2-adrenoceptor/imidazoline agonist moxonidine into the lateral parabrachial nucleus (LPBN). In the present study, we tested the relative importance of central angiotensinergic and cholinergic mechanisms for the control of water and NaCl intake by combining different dipsogenic or natriorexigenic stimuli with moxonidine injection into the LPBN. Adult male Holtzman rats (n = 9–10/group) with stainless steel cannulas implanted in the lateral ventricle and LPBN were used. Bilateral injections of moxonidine (0.5 nmol) into the LPBN increased water and 0.3 M NaCl intake in rats that received furosemide + captopril injected subcutaneously, ANG II (50 ng) or carbachol (cholinergic agonist, 4 nmol) injected intracerebroventricularly (icv) or 2 M NaCl infused intragastrically (2 ml/rat). Losartan (AT1 antagonist, 100 μg) or atropine (muscarinic antagonist, 20 nmol) injected icv abolished the effects on water and 0.3 M NaCl of moxonidine combined to either 2 M NaCl intragastrically or carbachol icv. However, atropine icv did not change 0.3 M NaCl intake produced by direct central action of ANG II like that induced by ANG II icv or furosemide + captopril combined with moxonidine into the LPBN. The results suggest that different stimuli, including hyperosmolarity and central cholinergic stimulation, share central angiotensinergic activation as a common mechanism to facilitate sodium intake, particularly when they are combined with deactivation of the LPBN inhibitory mechanisms. Department of Physiology and Pathology School of Dentistry São Paulo State University UNESP Department of Physiology and Pharmacology School of Medicine Federal University of Ceará Department of Physiology and Pathology School of Dentistry São Paulo State University UNESP

Made available in DSpace on 2018-12-11T17:09:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-03-27 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Chronic infusion of aldosterone into the 4th ventricle (4th V) induces robust daily sodium intake, whereas acute injection of aldosterone into the 4th V produces no sodium intake. The inhibitory mechanism of the lateral parabrachial nucleus (LPBN) restrains sodium intake induced by different natriorexigenic stimuli and might affect the acute response to aldosterone into the 4th V. In the present study, 1.8% NaCl and water intake was tested in rats treated with acute injections of aldosterone into the 4th V combined with the blockade of the inhibitory mechanisms with injections of moxonidine (α2 adrenergic/imidazoline agonist) or methysergide (a serotonergic antagonist) into the LPBN. Male Holtzman rats with stainless steel cannulas implanted in the 4th V and bilaterally in the LPBN were used. Aldosterone (250 or 500 ng) into the 4th V combined with vehicle into the LPBN induced no 1.8% NaClintake compared to control (1.5 ± 1.1 and 1.1 ± 0.4, respectively, vs. vehicle into 4th V: 1.0 ± 0.5 ml/2 h). However, aldosterone (250 or 500 ng) into the 4th V combined with moxonidine (0.5 nmol) into the LPBN induced strong ingestion of 1.8% NaCl (12.7 ± 4.6 and 17.6 ± 3.7 ml/2 h, respectively). Aldosterone (250 ng) into the 4th V combined with methysergide (4 μg) into the LPBN also induced 1.8% NaCl intake (17.6 ± 5.4 ml/2 h). These data suggest that the inhibitory mechanisms of the LPBN counteract the facilitation of sodium intake produced by aldosterone injected into the 4th, restraining sodium intake in this condition. Department of Physiology and Pathology School of Dentistry of Araraquara São Paulo State University UNESP Department of Physiology and Pathology School of Dentistry of Araraquara São Paulo State University UNESP FAPESP: 2015/23467-7 FAPESP: FAPESP 2011/50770-1 FAPESP: FAPESP 2013/00026-0

Rats place their tails underneath their bodies in the cold (tail-hiding behavior), which is a behavioral indicator of thermoregulation. The aim of the present study was to elucidate the effect of estradiol (E2) on tail-hiding behavior and neural activity assessed by immunohistochemistry. Ovariectomized rats were implanted with a silastic tube with or without E2 underneath the dorsal skin (E2(-) and E2(+) groups), and exposed to 27°C, 16°C, and 10°C for 2h with continuous body temperature (Tb), tail skin temperature (Ttail), and behavioral measurements. cFos immunoreactive (cFos-IR) cells in the insula, secondary somatosensory cortex, medial preoptic nucleus, parastrial nucleus, amygdala, and lateral parabrachial nucleus were counted. Tb and Ttail were not different between the E2(-) and E2(+) groups. At 16°C, the duration and the onset of tail-hiding behavior in the E2(+) group were greater than that in the E2(-) group. The number of cFos-IR cells in the insula of the E2(-) group was greater than that of the E2(+) group in rats kept at 16°C. E2 might modulate tail-hiding behavior of female rats at 16°C, and the insula may be involved in the response.

Cisplatin chemotherapy is commonly used to treat cancer despite severe energy balance side effects. In rats, cisplatin activates nucleus tractus solitarius (NTS) projections to the lateral parabrachial nucleus (lPBN) and calcitonin-gene related peptide (CGRP) projections from the lPBN to the central nucleus of the amygdala (CeA). We demonstrated previously that CeA glutamate receptor signaling mediates cisplatin-induced anorexia and body weight loss. Here, we used neuroanatomical tracing, immunofluorescence, and confocal imaging to demonstrate that virtually all NTS→lPBN and lPBN→CeA CGRP projections coexpress vesicular glutamate transporter 2 (VGLUT2), providing evidence that excitatory projections mediate cisplatin-induced energy balance dysregulation. To test whether lPBN→CeA projection neurons are required for cisplatin-induced anorexia and weight loss, we inhibited these neurons chemogenetically using a retrograde Cre-recombinase-expressing canine adenovirus-2 in combination with Cre-dependent inhibitory Designer Receptors Exclusive Activated by Designer Drugs (DREADDs) before cisplatin treatment. Inhibition of lPBN→CeA neurons attenuated cisplatin-induced anorexia and body weight loss significantly. Using a similar approach, we additionally demonstrated that inhibition of NTS→lPBN neurons attenuated cisplatin-induced anorexia and body weight loss significantly. Together, our data support the view that excitatory hindbrain–forebrain projections are necessary for cisplatin's untoward effects on energy intake, elucidating a key neuroanatomical circuit driving pathological anorexia and weight loss that accompanies chemotherapy treatment.SIGNIFICANCE STATEMENTChemotherapy treatments are commonly used to treat cancers despite accompanying anorexia and weight loss that may limit treatment adherence and reduce patient quality of life. Strikingly, we lack a neural understanding of, and effective treatments for, chemotherapy-induced anorexia and weight loss. The current data characterize the excitatory nature of neural projections activated by cisplatin in rats and reveal the necessity of specific hindbrain–forebrain projections for cisplatin-induced anorexia and weight loss. Together, these findings help to characterize the neural mechanisms mediating cisplatin-induced anorexia, advancing opportunities to develop better-tolerated chemotherapies and adjuvant therapies to prevent anorexia and concurrent nutritional deficiencies during cancer treatment.

Pontine parabrachial nucleus (PBN) neurons integrate visceral, oral, and other sensory information, playing an integral role in the neural control of feeding. Current experiments probed whether lateral PBN (lPBN) leptin receptor (LepRb) signaling contributes to this function. Intra-lPBN leptin microinjection significantly reduced cumulative chow intake, average meal size, and body weight in rats, independent of effects on locomotor activity or gastric emptying. In contrast to the effects observed following LepRb activation in other nuclei, lPBN LepRb stimulation did not affect progressive ratio responding for sucrose reward or conditioned place preference for a palatable food. Collectively, results suggest that lPBN LepRb activation reduces food intake by modulating the neural processing of meal size/satiation signaling, and highlight the lPBN as a novel site of action for leptin-mediated food intake control.

The parabrachial nucleus (PBN) is a key nucleus for the regulation of feeding behavior. Inhibitory inputs from the hypothalamus to the PBN play a crucial role in the normal maintenance of feeding behavior, because their loss leads to starvation. Viscerosensory stimuli result in neuronal activation of the PBN. However, the origin and neurochemical identity of the excitatory neuronal input to the PBN remain largely unexplored. Here, we hypothesize that hindbrain glucagon-like peptide 1 (GLP-1) neurons provide excitatory inputs to the PBN, activation of which may lead to a reduction in feeding behavior. Our data, obtained from mice expressing the yellow fluorescent protein in GLP-1-producing neurons, revealed that hindbrain GLP-1-producing neurons project to the lateral PBN (lPBN). Stimulation of lPBN GLP-1 receptors (GLP-1Rs) reduced the intake of chow and palatable food and decreased body weight in rats. It also activated lPBN neurons, reflected by an increase in the number of c-Fos-positive cells in this region. Further support for an excitatory role of GLP-1 in the PBN is provided by electrophysiological studies showing a remarkable increase in firing of lPBN neurons after Exendin-4 application. We show that within the PBN, GLP-1R activation increased gene expression of 2 energy balance regulating peptides, calcitonin gene-related peptide (CGRP) and IL-6. Moreover, nearly 70% of the lPBN GLP-1 fibers innervated lPBN CGRP neurons. Direct intra-lPBN CGRP application resulted in anorexia. Collectively, our molecular, anatomical, electrophysiological, pharmacological, and behavioral data provide evidence for a functional role of the GLP-1R for feeding control in the PBN.

Serotonin is widely distributed throughout the brain and is involved in a multiplicity of visceral, cognitive and behavioral responses. It has been previously shown that injections of different doses of ondansetron, a 5-HT3 receptor antagonist, into the medial septum/vertical limb of the diagonal band complex (MS/vDB) induce a hypertensive response in rats. On the other hand, administration of m-CPBG, a 5-HT3 agonist, into the MS/vDB inhibits the increase of blood pressure during restraint stress. However, it is unclear which neuronal circuitry is involved in these responses. The present study investigated Fos immunoreactive nuclei (Fos-IR) in different brain areas following the blockade of 5-HT3 receptors located in the MS/vDB in sham and in sinoaortic denervated (SAD) rats. Ondansetron injection into the MS/vDB increases Fos-IR in different brain areas including the limbic system (central amygdala and ventral part of the bed nucleus of the stria terminalis), hypothalamus (medial parvocellular parts of the paraventricular nucleus, anterodorsal preoptic area, dorsomedial hypothalamic nucleus), mesencephalon (ventrolateral periaqueductal gray region) and rhombencephalon (lateral parabrachial nucleus) in sham rats. Barodenervation results in higher Fos expression at the parvocellular and magnocellular part of the paraventricular nucleus, the lateral parabrachial nucleus, the central nucleus of amygdala, the locus coeruleus, the medial part of the nucleus of the solitary tract, the rostral ventrolateral medulla and the caudal ventrolateral medulla following 5-HT3receptor blockade in the MS/vDB. Based on the present results and previous data showing a hypertensive response to ondansetron injected into the MS/vDB, it is reasonable to suggest that 5-HT3receptors in the MS/vDB exert an inhibitory drive that may oscillate as a functional regulatory part of the complex central neuronal network participating in the control of blood pressure.

Bilateral injections of the GABA(A) agonist muscimol into the lateral parabrachial nucleus (LPBN) induce 0.3 M NaCl and water intake in satiated and normovolemic rats, a response reduced by intracerebroventricular (icv) administration of losartan or atropine (angiotensinergic type 1 (AT₁) and cholinergic muscarinic receptor antagonists, respectively). In the present study, we investigated the effects of the injections of losartan or atropine into the subfornical organ (SFO) on 0.3M NaCl and water intake induced by injections of muscimol into the LPBN. In addition, using intracellular calcium measurement, we also tested the sensitivity of SFO-cultured cells to angiotensin II (ANG II) and carbachol (cholinergic agonist). In male Holtzman rats with cannulas implanted bilaterally into the LPBN and into the SFO, injections of losartan (1 μg/0.1 μl) or atropine (2 nmol/0.1 μl) into the SFO almost abolished 0.3M NaCl and water intake induced by muscimol (0.5 nmol/0.2 μl) injected into the LPBN. In about 30% of the cultured cells of the SFO, carbachol and ANG II increased intracellular calcium concentration ([Ca²⁺](i)). Three distinct cell populations were found in the SFO, i.e., cells activated by either ANG II (25%) or carbachol (2.6%) or by both stimuli (2.3%). The results suggest that the activation of angiotensinergic and cholinergic mechanisms in the SFO is important for NaCl and water intake induced by the deactivation of LPBN inhibitory mechanisms with muscimol injections. They also show that there are cells in the SFO activated by both angiotensinergic and cholinergic stimuli, perhaps those involved in the responses to muscimol into the LPBN.

Made available in DSpace on 2014-09-30T18:18:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-01-10Bitstream added on 2014-10-01T14:03:57Z : No. of bitstreams: 1 S0100-879X2014000100011.pdf: 258243 bytes, checksum: 492697b800bc76cd5a8b1a12c4ab45f3 (MD5) Made available in DSpace on 2016-01-28T16:55:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2014. Added 1 bitstream(s) on 2016-01-28T17:02:22Z : No. of bitstreams: 1 ISSN1414-431X-2014-47-01-11-18.pdf: 258243 bytes, checksum: 492697b800bc76cd5a8b1a12c4ab45f3 (MD5) Item merged in doublecheck by Luana Priscila Costa (luana@reitoria.unesp.br) on 2018-05-17T19:05:59Z Item was identical to item(s): 108564, 132734 at handle(s): http://hdl.handle.net/11449/109481, http://hdl.handle.net/11449/133605 Central α2-adrenoceptors and the pontine lateral parabrachial nucleus (LPBN) are involved in the control of sodium and water intake. Bilateral injections of moxonidine (α2-adrenergic/imidazoline receptor agonist) or noradrenaline into the LPBN strongly increases 0.3 M NaCl intake induced by a combined treatment of furosemide plus captopril. Injection of moxonidine into the LPBN also increases hypertonic NaCl and water intake and reduces oxytocin secretion, urinary sodium, and water excreted by cell-dehydrated rats, causing a positive sodium and water balance, which suggests that moxonidine injected into the LPBN deactivates mechanisms that restrain body fluid volume expansion. Pretreatment with specific α2-adrenoceptor antagonists injected into the LPBN abolishes the behavioral and renal effects of moxonidine or noradrenaline injected into the same area, suggesting that these effects depend on activation of LPBN α2-adrenoceptors. In fluid-depleted rats, the palatability of sodium is reduced by ingestion of hypertonic NaCl, limiting intake. However, in rats treated with moxonidine injected into the LPBN, the NaCl palatability remains high, even after ingestion of significant amounts of 0.3 M NaCl. The changes in behavioral and renal responses produced by activation of α2-adrenoceptors in the LPBN are probably a consequence of reduction of oxytocin secretion and blockade of inhibitory signals that affect sodium palatability. In this review, a model is proposed to show how activation of α2-adrenoceptors in the LPBN may affect palatability and, consequently, ingestion of sodium as well as renal sodium excretion. Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Fisiologia e Patologia, Araraquara, Rua Humaitá, 1680, Centro, CEP 14801903, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Fisiologia e Patologia, Araraquara, Rua Humaitá, 1680, Centro, CEP 14801903, SP, Brasil

Numerous studies have demonstrated that both the hypothalamic paraventricular nuclei (PVN) and ventromedial nuclei (VMN) regulate energy homeostasis through behavioral and metabolic mechanisms. Receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) are abundantly expressed in these nuclei, suggesting PACAP may be critical for the regulation of feeding behavior and body weight. To characterize the unique behavioral and physiological responses attributed to select hypothalamic cell groups, PACAP was site-specifically injected into the PVN or VMN. Overall food intake was significantly reduced by PACAP at both sites; however, meal pattern analysis revealed that only injections into the PVN produced significant reductions in meal size, duration, and total time spent eating. PACAP-mediated hypophagia in both the PVN and VMN was abolished by PAC1R antagonism, whereas pretreatment with a VPACR antagonist had no effect. PACAP injections into the VMN produced unique changes in metabolic parameters, including significant increases in core body temperature and spontaneous locomotor activity that was PAC1R dependent whereas, PVN injections of PACAP had no effect. Finally, PACAP-containing afferents were identified using the neuronal tracer cholera toxin subunit B (CTB) injected unilaterally into the PVN or VMN. CTB signal from PVN injections was colocalized with PACAP mRNA in the medial anterior bed nucleus of the stria terminalis, VMN, and lateral parabrachial nucleus (LPB), whereas CTB signal from VMN injections was highly colocalized with PACAP mRNA in the medial amygdala and LPB. These brain regions are known to influence energy homeostasis perhaps, in part, through PACAP projections to the PVN and VMN.

This study examined which brain regions are influenced by an inhibitory lateral parabrachial nucleus (LPBN) mechanism that affects water intake. Controls and rats with bilateral LPBN lesions were administered angiotensin II (AngII) (0.5mg/kg subcutaneous - SC), drinking responses measured, and brains processed for Fos-immunohistochemistry. A separate group of LPBN-lesioned and non-lesioned animals were denied water for 90 min prior to perfusion to remove any confounding factor of water intake. LPBN-lesioned rats drank a cumulative volume of 9 mL compared with

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:29:46Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:29:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-20 Objective: Gamma-aminobutyric acid A (GABAA) receptor activation with muscimol in the lateral parabrachial nucleus (LPBN) induces water and 0.3 M NaCl intake. The purpose of this study was to investigate whether a local inflammatory event, such as periodontal disease (PD), is able to alter the effects of muscimol on water and 0.3 M NaCl intake in fluid-replete rats and in rats treated with furosemide (FURO) combined with captopril (CAP) injected subcutaneously. Design: Male Wistar rats were divided into two groups: with PD and those without PD (control condition). Fifteen days after PD, both groups had cannulas implanted bilaterally into the LPBN. Results: In fluid-replete rats without PD, injections of muscimol (0.5 nmol/0.2 μl) into the LPBN induced 0.3 M NaCl and water intake and a pressor response. In fluid-replete rats with PD, a decrease was observed in water intake and pressor response but not in 0.3 M NaCl intake. In control rats with FURO + CAP treatment, injections of muscimol into the LPBN increased 0.3 M NaCl and water intake. In PD rats with FURO + CAP treatment, a decrease was observed in 0.3 M NaCl and water intake after muscimol in the LPBN. Alveolar bone loss and interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) plasmatic concentration were higher in PD rats in comparison with controls. Conclusion: These results suggest that PD is able to reduce the pressor response and the dipsogenic and natriorexigenic effects induced by the activation of GABAA receptors in the LPBN, probably due to the elevation of the plasmatic concentration of pro-inflammatory cytokines IL-6 and TNF-α. © 2013 Elsevier Ltd. All rights reserved.

Classically, opioids produce their effects by activating Gi-proteins that inhibit adenylate cyclase activity. Previous studies proposed that mu-opioid receptors can also stimulate adenylate cyclase due to an initial transient coupling to Gs-proteins. Treatment with ultra-low doses of the nonselective opioid antagonist (–)-naloxone or its inactive enantiomer (+)-naloxone blocks this excitatory effect and enhances Gi-coupling. Previously we reported that infusion of the mu-opioid receptor agonist [D-Ala2, N-Me-Phe4, Glycinol5]-Enkephalin (DAMGO) into the mu-opioid receptor expressing lateral parabrachial nucleus increases feeding. Pretreatment with (–)-naloxone blocks this effect. We used this parabrachial circuit as a model to assess cellular actions of ultra-low doses of (–)-naloxone and (+)-naloxone in modifying the effects of DAMGO. Our results showed that an ultra-low concentration of (–)-naloxone (0.001 nM) and several concentrations of (+)-naloxone (0.01–10 nM) enhanced DAMGO-stimulated guanosine-5′-0-(γ-thio)-triphosphate incorporation in parabrachial sections in vitro. Further, we analyzed the relevance of these effects in vivo. In the present study, we show that (+)-naloxone can potentiate DAMGO-induced feeding at doses at which (–)-naloxone was an antagonist. These results implicated (+)-naloxone as a novel tool for studying mu-opioid receptor functions and suggest that (+)-naloxone may have therapeutic value to enhance clinical actions of opiate drugs.

The present study investigated the effects of a microinjection of GABA A receptor agonist (muscimol) and antagonist (bicuculline) into the central nucleus of the amygdala (CeA) in sodium-depleted rats. We measured the sodium intake and identified the neuronal activation in the brainstem induced by activating the GABA A receptors in the CeA using Fos immunohistochemistry. Muscimol (0.20, 0.35 or 0.50 nmol, in 0.2 μl) that was injected bilaterally into the CeA decreased the 0.3 M NaCl and water intake in a dose-dependent manner. Microinjection of 0.02 nmol/0.2 μl muscimol also decreased the NaCl intake, but had no effect on the water intake. The inhibitory effect of muscimol (0.20 nmol) on the sodium and water intake could be blocked by pretreatment with bicuculline intra-CeA microinjection (0.4 nmol). However, bilateral injections of bicuculline alone into the CeA did not affect the NaCl or water intake. Furthermore, microinjection of muscimol (0.20 nmol) into the CeA increased the number of Fos-like immunoreactive (FLI) neurons in the caudal and intermediate parts of the nucleus of the tractus solitarius (cNTS and iNTS) and the lateral parabrachial nucleus (LPBN). These results suggest that GABA A receptors within the CeA may be involved in mediating the sodium intake in the sodium-depleted rat, and the cNTS, iNTS and LPBN were probably involved in this mechanism.

Multiple lines of evidence document a role for glutamatergic input to the hypothalamic paraventricular nucleus (PVH) in stress-induced activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. However, the neuro-anatomical origins of the glutamatergic input have yet to be definitively determined. We have previously shown that vesicular glutamate transporter 2 (VGLUT2) is the predominant VGLUT isoform expressed in the basal forebrain and brainstem, including PVH-projecting regions, and that the PVH is preferentially innervated by VGLUT2-immunoreactive terminals/boutons. The present study employed a dual-labeling approach, combining immunolabeling for a retrograde tract tracer, Fluoro-Gold (FG), with in situ hybridization for VGLUT2 mRNA, to map the brainstem and caudal forebrain distribution of glutamatergic PVH-projecting neurons. The present report presents evidence for substantial dual labeling in the periaqueductal gray, caudal portions of the zona incerta and subparafascicular nucleus, and the lateral parabrachial nucleus. The current data also suggest that relatively few PVH-projecting neurons in ascending raphe nuclei, nucleus of the solitary tract, or ventrolateral medulla are VGLUT2 positive. The data reveal multiple brainstem origins of glutamatergic input to PVH that are positioned to play a role in transducing a diverse range of stressful stimuli.

The deactivation of the inhibitory mechanisms with injections of moxonidine (α2-adrenoceptor/imidazoline receptor agonist) into the lateral parabrachial nucleus (LPBN) increases hypertonic NaCl intake by intra- or extracellular dehydrated rats. In the present study, we investigated the changes in the urinary sodium and volume, sodium balance, and plasma vasopressin and oxytocin in rats treated with intragastric (i.g.) 2 M NaCl load (2 ml/rat) combined with injections of moxonidine into the LPBN. Male Holtzman rats (n=5-12/group) with stainless steel cannulas implanted bilaterally into LPBN were used. Bilateral injections of moxonidine (0.5 nmol/0.2 μl) into the LPBN decreased i.g. 2 M NaCl-induced diuresis (4.6±0.7 vs. vehicle: 7.4±0.6 ml/120 min) and natriuresis (1.65±0.29 vs. vehicle: 2.53±0.17 mEq/120 min), whereas the previous injection of the α2-adrenoceptor antagonist RX 821002 (10 nmol/0.2 μl) into the LPBN abolished the effects of moxonidine. Moxonidine injected into the LPBN reduced i.g. 2 M NaCl-induced increase in plasma oxytocin and vasopressin (14.6±2.8 and 2.2±0.3 vs. vehicle: 25.7±7 and 4.3±0.7 pg/ml, respectively). Moxonidine injected into the LPBN combined with i.g. 2 M NaCl also increased 0.3 M NaCl intake (7.5±1.7 vs. vehicle: 0.5±0.2 mEq/2 h) and produced positive sodium balance (2.3±1.4 vs. vehicle: -1.2±0.4 mEq/2 h) in rats that had access to water and NaCl. The present results show that LPBN α2-adrenoceptor activation reduces renal and hormonal responses to intracellular dehydration and increases sodium and water intake, which facilitates sodium retention and body fluid volume expansion.

Airway obstruction in animals leads to compensation and avoidance behavior and elicits respiratory mechanosensation. The pattern of respiratory load compensation and neural activation in response to intrinsic, transient, tracheal occlusions (ITTO) via an inflatable tracheal cuff are unknown. We hypothesized that ITTO would cause increased diaphragm activity, decreased breathing frequency, and activation of neurons within the medullary and pontine respiratory centers without changing airway compliance. Obstructions were performed for 2–3 breaths followed by a minimum of 15 unobstructed breaths with an inflatable cuff sutured around the trachea in rats. The obstruction procedure was repeated for 10 min. The brains of obstructed and control animals were removed, fixed, sectioned, and stained for c-Fos. Respiratory pattern was measured from esophageal pressure (Pes) and diaphragm electromyography (EMGdia). The obstructed breaths resulted in a prolonged inspiratory and expiratory time, an increase in EMGdia amplitude, and a more negative Pes compared with control breaths. Neurons labeled with c-Fos were found in brain stem and suprapontine nuclei, with a significant increase in c-Fos expression for the occluded experimental group compared with the control groups in the nucleus ambiguus, nucleus of the solitary tract, lateral parabrachial nucleus, and periaqueductal gray matter. The results of this study demonstrate tracheal occlusion-elicited activation of neurons in brain stem respiratory nuclei and neural areas involved in stress responses and defensive behaviors, suggesting that these neurons mediate the load compensation breathing pattern response and may be part of the neural pathway for respiratory mechanosensation.

Lesions of the area postrema (AP) block many of the behavioral and physiological effects of lithium chloride (LiCl) in rats, including formation of conditioned taste aversions (CTAs). Systemic administration of LiCl induces c-Fos immunoreactivity in several brain regions, including the AP, nucleus of the solitary tract (NTS), lateral parabrachial nucleus (latPBN), supraoptic nucleus (SON), paraventricular nucleus (PVN), and central nucleus of the amygdala (CeA). To determine which of these brain regions may be activated in parallel with the acquisition of LiCl-induced CTAs, we disrupted CTA learning in rats by ablating the AP and then quantified c-Fos-positive cells in these brain regions in sham- and AP-lesioned rats 1 h following LiCl or saline injection. Significant c-Fos induction after LiCl was observed in the CeA and SON of AP-lesioned rats, demonstrating activation independent of an intact AP. LiCl-induced c-Fos was significantly attenuated in the NTS, latPBN, PVN and CeA of AP-lesioned rats, suggesting that these regions are dependent on AP activation. Almost all of the lesioned rats showed some damage to the subpostremal NTS, and some rats also had damage to the dorsal motor nucleus of the vagus; this collateral damage in the brainstem may have contributed to the deficits in c-Fos response. Because c-Fos induction in several regions was correlated with magnitude of CTA acquisition, these regions are implicated in the central mediation of lithium effects during CTA learning.

Some of the benefits of exercise appear to be mediated through modulation of neuronal excitability in central autonomic control circuits. Previously, we identified that six weeks of voluntary wheel running had a protective effect during hemorrhage (HEM), limiting both the hypotensive phase of HEM and enhancing recovery. The present study was undertaken to evaluate the role of opioid release in the lateral parabrachial nucleus (LPBN) on the response to severe HEM in chronically exercised (EX, voluntary) versus sedentary (SED) controls. Male Sprague Dawley rats were allowed either free access to running wheels (EX) or normal cage conditions (SED). After 6 weeks of “training” animals were instrumented with a bilateral cannula directed toward the dorsolateral pons and arterial catheters. After a recovery period, animals underwent central microinjection of either vehicle (VEH; n = 3/group) or the opioid receptor antagonist naloxone (NAL; n = 6/group) followed by withdrawal of 30% of their total estimated blood volume. Following VEH injection, the drop in MAP during and following HEM was significantly attenuated in the EX vs SED animals. Alternatively, NAL microinjection in the dorsolateral pons (20 μM, 200–500 nl) reversed the beneficial effect of EX on the HEM response. NAL microinjection in SED rats did not significantly alter the response to HEM. These data suggest chronic voluntary EX has a beneficial effect on the autonomic response to severe HEM which is mediated, in part, via EX-induced plasticity of the opioid system within the dorsolateral pons.

Amylin is an important control of nutrient fluxes because it reduces energy intake modulates nutrient utilization by inhibiting postprandial glucagon secretion and increases energy disposal by preventing compensatory decreases of energy expenditure in weight reduced individuals. The best investigated function of amylin which is cosecreted with insulin is to reduce eating by promoting meal ending satiation. This effect is thought to be mediated by a stimulation of specific amylin receptors in the area postrema. Secondary brain sites to mediate amylin action include the nucleus of the solitary tract and the lateral parabrachial nucleus which convey the neural signal to the lateral hypothalamic area and other hypothalamic nuclei. Amylin may also signal adiposity because plasma levels of amylin are increased in adiposity and because higher amylin concentrations in the brain result in reduced body weight gain and adiposity while amylin receptor antagonists increase body adiposity. The central mechanisms involved in amylin's effect on energy expenditure are much less known. A series of recent experiments in animals and humans indicate that amylin is a promising option for anti obesity therapy especially in combination with other hormones. The most extensive dataset is available for the combination therapy of amylin and leptin. Ongoing research focuses on the mechanisms of these interactions. © 2011 Springer Basel AG.

Place preference induction by intracerebral electrical stimulation was initially shown by Olds and Milner. It has since proven possible to induce concurrent stimulation-induced place preference (cCPP) after electrical stimulation of the lateral hypothalamus (LH) and, more recently, of the external lateral parabrachial nucleus (LPBe). The objective of this experimental study was to examine whether the rewarding effects of electrical stimulation of the LH and LPBe involve the activation of similar opioid systems in an alternative cCPP task. Administration of the opioid antagonist naloxone (4 mg/kg) blocked the conditioned place preference effect induced after LPBe but not after LH stimulation (at 4 or 10 mg/kg). These results are interpreted in relation to the presence of multiple reward systems that might anatomically and neurochemically differ with respect to the involvement of the opioid system.

GABA(A) and GABA(B) receptors are present in the lateral parabrachial nucleus (LPBN), a pontine area involved with inhibitory mechanisms related to the control of sodium appetite. Activation of GABA(A) receptors in the LPBN induces strong ingestion of 0.3 M sodium chloride (NaCl) in normonatremic and euhydrated rats. In the present study, we investigated the effects of the GABA(B) receptor agonist baclofen, injected alone or combined with GABA(A) or GABA(B) receptor antagonists into the LPBN on 0.3 M NaCl, water, 0.06 M sucrose and food intake in normonatremic and euhydrated rats. Male Holtzman rats with stainless steel cannulas implanted bilaterally in the LPBN were used. In normonatremic and euhydrated rats, bilateral injections of baclofen (0.5 nmol/0.2 μl) into the LPBN induced 0.3 M NaCl (24.0±3.1 vs. saline: 2.0±0.8 ml/240 min) and water intake (10.6±1.4 vs. saline: 3.5±0.7 ml/240 min) in a two-bottle test. Injections of GABA(B) receptor antagonists CGP 35348 (50 nmol/0.2 μl) or 2-hydroxysaclofen (5 nmol/0.2 μl) or GABA(A) receptor antagonist bicuculline (1.6 nmol/0.2 μl) into the LPBN reduced 0.3 M NaCl (14.1±4.7 ml/240 min; 9.97±2.5 ml/210 min; 8.8±5.9 ml/240 min, respectively) and water intake induced by baclofen injected into the LPBN. Baclofen (0.5 nmol/0.2 μl) injected into the LPBN also induced 0.06 M sucrose intake (21.8±5.9 vs. saline: 5.0±2.6 ml/180 min). Urinary volume and sodium excretion had a tendency to decrease after baclofen injection into the LPBN, whereas arterial pressure and food intake were not affected. The results show that baclofen injected into the LPBN, in normonatremic and euhydrated rats, produces a natriorexigenic effect dependent on GABA(A) and GABA(B) receptor activation. The natriorexigenic effect is not secondary to alterations in blood pressure or sodium urinary excretion. In addition, baclofen injected into the LPBN also induces 0.06 M sucrose intake.

Neuropeptide S (NPS) is the endogenous ligand for GPR154, now referred to as neuropeptide S receptor (NPSR). Physiologically, NPS has been characterized as a modulator of arousal and has been shown to produce anxiolytic-like effects in rodents. Neuroanatomical analysis in the rat revealed that the NPS precursor mRNA is strongly expressed in the brainstem in only three distinct regions: the locus coeruleus area, the principal sensory trigeminal nucleus, and the lateral parabrachial nucleus. NPSR mRNA expression in the rat is widely distributed, with the strongest expression in the olfactory nuclei, amygdala, subiculum, and some cortical structures, as well as various thalamic and hypothalamic regions. Here we report a comprehensive map of NPS precursor and receptor mRNA expression in the mouse brain. NPS precursor mRNA is only expressed in two regions in the mouse brainstem: the Kolliker-Fuse nucleus and the pericoerulear area. Strong NPSR mRNA expression was found in the dorsal endopiriform nucleus, the intra-midline thalamic and hypothalamic regions, the basolateral amgydala, the subiculum, and various cortical regions. In order to elucidate projections from NPS-producing nuclei in the brainstem to NPSR-expressing structures throughout the brain, we performed immunohistochemical analysis in the mouse brain by using two polyclonal anti-NPS antisera. The distribution of NPS-immunopositive fibers overlaps well with NPSR mRNA expression in thalamic and hypothalamic regions. Mismatches between NPSR expression and NPS-immunoreactive fiber staining were observed in hippocampal, olfactory, and cortical regions. These data demonstrate that the distribution pattern of the central NPS system is only partially conserved between mice and rats. J. Comp. Neurol. 519:1867–1893, 2011. © 2011 Wiley-Liss, Inc.

A part of the mediobasal hypothalamus (known as hypothalamic attack area) plays a central role in the control of aggressive behavior as its electrical stimulation reliably and rapidly elicits biting attacks in cats and rodents. The efferent connections of this brain region were described in rats, but afferent pathways were not investigated so far. We injected the retrograde tracer cholera toxin B subunit into the mediobasal hypothalamus of male Wistar rats and studied the distribution of labeled cells by immunohistochemical method. The retrograde labeling outlined three continuous, distinct afferent cell populations: (i) a telencephalic midline "plate" containing the orbitofrontal - medial prefrontal - septal regions which ends in the bed nucleus of stria terminalis; (ii) a temporal column including the medial amygdala, amigdalohippocampal area and subiculum; (iii) a diffuse column along the medial hypothalamus which ends in the posterior hypothalamic nucleus. Sparse labeling was present in brainstem nuclei, except for the lateral parabrachial nucleus that provides a significant input. The projections of the medial prefrontal cortex to the hypothalamic attack area indicate a direct, earlier undescribed pathway with marked importance in the control of aggressive behavior. Similarly, we identified several brain regions which send very significant projections to the hypothalamic attack area but their importance in the control of aggressive behavior are nearly unknown. The comparison of the present and earlier findings shows that efferent and afferent connections overlap in many regions to a significant extent, suggesting that reverberating circuits are important in the control of aggressive behavior.

We performed this study to understand the anatomical substrates of parabrachial nucleus (PBN) modulation of orexin (ORX)-containing neurons in the hypothalamus. After biotinylated dextranamine (BDA) injection into the lateral PBN and immunostaining of ORX-containing neurons in the rat, the prominent overlap of the distribution field of the BDA-labeled fibers and that of the ORX-immunoreactive (ir) neurons was found in the lateralmost part of the dorsomedial nucleus and adjacent dorsal perifornical area (this overlapping field was referred to as "suprafornical area" in the present study), and the labeled axon terminals made asymmetrical synaptic contacts with somata and dendrites of the ORX-ir neurons. We further revealed that almost all the "suprafornical area"-projecting lateral PBN neurons were positive for vesicular glutamate transporter 2 mRNA and very few of them were positive for glutamic acid decarboxylase 67 mRNA. The present data suggest that ORX-containing neurons in the "suprafornical area" may be under the excitatory influence of the glutamatergic lateral PBN neurons probably for the regulation of arousal and waking.

Blockade of serotonergic receptors in the lateral parabrachial nucleus (LPBN), via bilateral injections of nonselective 5-hydroxytryptamine (5-HT)(1/2)-receptor antagonist, methysergide causes a robust sodium appetite. Our aim was to elucidate which brain regions are activated when serotonergic pathways to the LPBN are blocked and combined with subcutaneous injection of isoproterenol causing a salt appetite. In the experimental group, conscious rats were administered methysergide (4 microg/0.2 microl) injected bilaterally into the LPBN. Control groups included rats administered with injections of vehicle bilaterally into the LPBN, rats administered methysergide into injection sites outside the LPB region, and rats that did not undergo surgery. Each group was treated with a subcutaneous injection of isoproterenol (30 microg/kg), a beta-adrenergic agonist, and NaCl and water intakes were measured over 2h. Bilateral injections of methysergide into the LPBN followed by subcutaneous isoproterenol induced a strong intake of 0.3M NaCl (p

The ability of the pancreatic hormone amylin to inhibit food intake relies on a direct activation of the area postrema (AP). This activation is synaptically transmitted to the nucleus of the solitary tract (NTS), the lateral parabrachial nucleus (LPB), the central amygdaloid nucleus (Ce) and the lateral bed nucleus of stria terminalis (BSTL). Interestingly, neurons of the rostro-dorsal lateral hypothalamic area (dLHA), which are activated during fasting, are inhibited by peripheral amylin, although they lack amylin receptors. Using the retrograde tracer cholera toxin-B (Ctb) we analyzed whether the dLHA receives neuronal projections from amylin-activated brain areas. The anterograde tracer biotinylated dextran-amine (BDA) was used to confirm the projections and to identify further neuronal pathways potentially involved in amylin signaling. We identified dense projections from the amylin activated neurons in the LPB and sparse projections from the NTS to the dLHA. LPB fiber efferents were found in close proximity to dLHA nuclei activated by 24h of fasting. The AP and the Ce showed no projections to the dLHA. Dense efferents were also observed from the LPB to other hypothalamic areas, namely to the ventromedial, dorsomedial, paraventricular and arcuate nuclei. This study provides neuroanatomical evidence that among the amylin activated areas, the LPB provides the strongest input to the dLHA, thus it may mediate the amylin-induced inhibition of the dLHA.

The blockade of the lateral parabrachial nucleus (LPBN) with the GABAergic receptor agonist muscimol induces strong hypertonic NaCl intake in satiated and normovolemic rats, whereas lesions of the central nucleus of the amygdala (CeA) reduce sodium intake induced by different protocols. In the present study we investigated the effects of bilateral lesions of the CeA on water and 0.3M NaCl intake induced by GABAergic receptor activation with bilateral injections of muscimol into the LPBN in satiated rats. Male Holtzman rats (n=6-10) with bilateral sham or electrolytic lesions (2mA; 10s) of the CeA and stainless steel cannulas implanted bilaterally in the LPBN were used. Bilateral injections of muscimol (0.5nmol/0.2microl) into the LPBN in satiated sham-lesioned rats induced 0.3M NaCl intake (16.1+/-5.4ml/4h, vs. saline: 1.3+/-0.5ml/4h) and water intake (8.1+/-3.5ml/4h, vs. saline: 1.6+/-0.5ml/4h). Bilateral lesions of the CeA (3days) abolished 0.3M NaCl intake (0.1+/-0.1ml/4h) and water intake (0.1+/-0.1ml/4h) induced by bilateral injections of muscimol into the LPBN in satiated rats. The present results show that water and 0.3M NaCl intake induced by the blockade of LPBN neurons with muscimol depends on the integrity of the CeA, suggesting that facilitatory mechanisms present in the CeA are essential for water and hypertonic NaCl intake that arises after the blockade of the inhibitory mechanisms of the LPBN with muscimol.

Neuropeptide W (NPW), which was recently isolated from the porcine hypothalamus, has been identified as the endogenous ligand of the orphan G protein-coupled receptors GPR7 (NPBWR1) and GPR8 (NPBWR2). Infusion of NPW increases food intake in the light phase, whereas in the dark phase, it has the opposite effect. In this study, we used RT-PCR analysis to examine the gene expression of NPW mRNA in the rat brain, and performed a detailed analysis of the distribution of NPW-positive neurons by use of immunohistochemistry at both the light and electron microscopic levels. NPW mRNA expression was demonstrated in the hypothalamic paraventricular nucleus (PVN), arcuate nucleus (ARC), ventromedial nucleus (VMH) and lateral hypothalamus (LH). At the light microscopic level, NPW-like immunoreactive (NPW-LI) cell bodies were found in the preoptic area (POA), PVN, ARC, VMH, LH, PMD (dorsal premammillary nucleus), periaqueductal gray (PAG), lateral parabrachial nucleus (LPB), and prepositus nucleus (Pr). NPW-LI axon terminals were shown in the POA, bed nucleus of the stria terminalis (BST), amygdala, PVN, ARC, VMH, LH, and PAG, LPB. In addition, at the electron microscopic level, NPW-LI cell bodies and dendritic processes were often seen to receive inputs from other unknown neurons in the ARC, PVN, VMH and amygdala. Our observations indicate that NPW-LI neurons widely distributed in the rat brain region. These finding suggest that NPW may have important roles in feeding behavior, energy homeostasis, emotional response and regulation of saliva secretion.

Modulation of salt appetite involves interactions between the circumventricular organs (CVOs) receptive areas and inhibitory hindbrain serotonergic circuits. Recent studies provide support to the idea that the serotonin action in the lateral parabrachial nucleus (LPBN) plays an important inhibitory role in the modulation of sodium appetite. The aim of the present work was to identify the specific groups of neurons projecting to the LPBN that are activated in the course of sodium appetite regulation, and to analyze the associated endocrine response, specifically oxytocin (OT) and atrial natriuretic peptide (ANP) plasma release, since both hormones have been implicated in the regulatory response to fluid reestablishment. For this purpose we combined the detection of a retrograde transported dye, Fluorogold (FG) injected into the LPBN with the analysis of the Fos immunocytochemistry brain pattern after sodium intake induced by sodium depletion. We analyzed the Fos-FG immunoreactivity after sodium ingestion induced by peritoneal dialysis (PD). We also determined OT and ANP plasma concentration by radioimmunoassay (RIE) before and after sodium intake stimulated by PD. The present study identifies specific groups of neurons along the paraventricular nucleus, central extended amygdala, insular cortex, dorsal raphe nucleus, nucleus of the solitary tract and the CVOs that are activated during the modulation of sodium appetite and have direct connections with the LPBN. It also shows that OT and ANP are released during the course of sodium satiety and fluid reestablishment. The result of this brain network activity may enable appropriate responses that re-establish the body fluid balance after induced sodium consumption.

We investigated the effects of electrolytic damage to the central nucleus of the amygdala on brain c-fos expression and 0.3 M NaCl intake of adult male rats (n = 6–12/group) submitted to a cycle of 36 h of water deprivation (WD) followed by 2 h water intake until satiety or partial rehydration (PR). The groups were divided into sham lesion (CEAs), bilateral lesion of the CEA (CEAX) and misplaced lesion with intact CEA (CEAm). The WD–PR produced a marked increase in c-fos expression in the medial parabrachial nucleus (MPBN) and some increase in the parvocelullar portion of the hypothalamic paraventricular nucleus (PVNp), compared to respective hydrated control (no water deprivation) state in CEAX, but not in CEAs or CEAm. The WD–PR induced similar c-fos expression in the lamina terminalis, supraoptic nucleus, magnocellular PVN and lateral parabrachial nucleus in both CEAX and CEAs. The CEAX showed the typical reduced daily need-free 0.3 M NaCl intake compared to CEAs. However, the 0.3 M NaCl intake of CEAX, unexpectedly, was not significantly different from CEAs or intact rats in the sodium appetite test that followed a cycle of WD–PR. The results do not allow associating the alterations in c-fos expression to the typical inhibition of sodium appetite well known in the literature to be produced by damage to the CEA. Nevertheless, the enhanced cell activation in the MPBN and PVNp suggests an inhibitory role for the CEA on the activity of these nuclei when water-deprived rats have quenched their thirst.