Area Postrema Neurons Research Articles

7753 Anoctamin 4 in the Area Postrema Regulates Glucose and Energy Homeostasis

Abstract Disclosure: L. Tu: None. Y. Xu: None. Located on the floor of the 4th ventricle within the dorsal surface of the medulla oblongata, the area postrema (AP) is a circumventricular organ where a proper blood-brain barrier is missing. Numerous studies reveal that neurons in the AP are implicated into suppression of food intake (e.g., glucagon-like peptide 1 (GLP-1)-mediated anorexia), but the role of AP neurons in whole-body glucose homeostasis is less investigated. Here we identified and fully characterized the physiological functions of anoctamin-4 (Ano4, a calcium activated chloride channel)1 in the AP. CRISPR-mediated deletion of Ano4 in the AP caused a lower body weight and reduced blood glucose, concurrent with an improved glucose tolerance and increased insulin sensitivity. Chemogenetic activation of Ano4-expressing neurons in the AP (APAno4) increased food intake and blood glucose, and did not induce conditioned flavor avoidance. Both Ano4 neurons and non-Ano4 neurons (i.e., neurons do not express Ano4) sent projections to the lateral parabrachial nucleus (LPBN). Optogenetic stimulation of APAno4 ◊ LPBN circuit promoted feeding behavior and increased blood glucose, whereas stimulation of APnon-Ano4 ◊ LPBN circuit inhibited food intake and did not influence glucose homeostasis. Finally, RNAscope studies reveal that 45.7% of APAno4 neurons are glutamatergic neurons, and 41.8% of them are GABAergic neurons. Furthermore, APAno4 neurons showed only a 2.6% overlap with Glp-1r-expressing neurons in the AP, but had a 60.8% co-localization with protein tyrosine phosphatase receptor δ2, which serves as orexigenic asprosin receptor. Together, our results indicate that Ano4 neurons in the AP are the first reported orexigenic neurons, and represent potential therapeutic targets for human diseases with glucose imbalance and abnormal feeding behavior. Reference: (1) Tu, L. et al. Anoctamin 4 channel currents activate glucose-inhibited neurons in the mouse ventromedial hypothalamus during hypoglycemia. J Clin Invest 133,14. (2023). (2) Mishra, I. et al. Protein tyrosine phosphatase receptor δ serves as the orexigenic asprosin receptor. Cell Metab 34, 549-563. (2022). Presentation: 6/2/2024

9207 Anoctamin 4 in the Area Postrema Regulates Glucose and Energy Homeostasis

Abstract Disclosure: L. Tu: None. Y. Xu: None. Located on the floor of the 4th ventricle within the dorsal surface of the medulla oblongata, the area postrema (AP) is a circumventricular organ where a proper blood-brain barrier is missing. Numerous studies reveal that neurons in the AP are implicated into suppression of food intake (e.g., glucagon-like peptide 1 (GLP-1)-mediated anorexia), but the role of AP neurons in whole-body glucose homeostasis is less investigated. Here we identified and fully characterized the physiological functions of anoctamin-4 (Ano4, a calcium activated chloride channel)1 in the AP. CRISPR-mediated deletion of Ano4 in the AP caused a lower body weight and reduced blood glucose, concurrent with an improved glucose tolerance and increased insulin sensitivity. Chemogenetic activation of Ano4-expressing neurons in the AP (APAno4) increased food intake and blood glucose, and did not induce conditioned flavor avoidance. Both Ano4 neurons and non-Ano4 neurons (i.e., neurons do not express Ano4) sent projections to the lateral parabrachial nucleus (LPBN). Optogenetic stimulation of APAno4 ◊ LPBN circuit promoted feeding behavior and increased blood glucose, whereas stimulation of APnon-Ano4 ◊ LPBN circuit inhibited food intake and did not influence glucose homeostasis. Finally, RNAscope studies reveal that 45.7% of APAno4 neurons are glutamatergic neurons, and 41.8% of them are GABAergic neurons. Furthermore, APAno4 neurons showed only a 2.6% overlap with Glp-1r-expressing neurons in the AP, but had a 60.8% co-localization with protein tyrosine phosphatase receptor δ2, which serves as orexigenic asprosin receptor. Together, our results indicate that Ano4 neurons in the AP are the first reported orexigenic neurons, and represent potential therapeutic targets for human diseases with glucose imbalance and abnormal feeding behavior. Reference: (1) Tu, L. et al. Anoctamin 4 channel currents activate glucose-inhibited neurons in the mouse ventromedial hypothalamus during hypoglycemia. J Clin Invest 133,14. (2023). (2) Mishra, I. et al. Protein tyrosine phosphatase receptor δ serves as the orexigenic asprosin receptor. Cell Metab 34, 549-563. (2022). Presentation: 6/2/2024

Area postrema neurons mediate interleukin-6 function in cancer cachexia

Interleukin-6 (IL-6) has been long considered a key player in cancer cachexia. It is believed that sustained elevation of IL-6 production during cancer progression causes brain dysfunctions, which ultimately result in cachexia. However, how peripheral IL-6 influences the brain remains poorly understood. Here we show that neurons in the area postrema (AP), a circumventricular structure in the hindbrain, is a critical mediator of IL-6 function in cancer cachexia in male mice. We find that circulating IL-6 can rapidly enter the AP and activate neurons in the AP and its associated network. Peripheral tumor, known to increase circulating IL-6, leads to elevated IL-6 in the AP, and causes potentiated excitatory synaptic transmission onto AP neurons and AP network hyperactivity. Remarkably, neutralization of IL-6 in the brain of tumor-bearing mice with an anti-IL-6 antibody attenuates cachexia and the hyperactivity in the AP network, and markedly prolongs lifespan. Furthermore, suppression of Il6ra, the gene encoding IL-6 receptor, specifically in AP neurons with CRISPR/dCas9 interference achieves similar effects. Silencing Gfral-expressing AP neurons also attenuates cancer cachectic phenotypes and AP network hyperactivity. Our study identifies a central mechanism underlying the function of peripheral IL-6, which may serve as a target for treating cancer cachexia.

GDF15 enhances body weight and adiposity reduction in obese mice by leveraging the leptin pathway

GDF15 regulates its anorexic effects through the hindbrain area postrema (AP) and nucleus of the solitary tract (NTS) neurons where its receptor, glial-derived neurotrophic factor receptor alpha-like (GFRAL), is expressed. The actions of GDF15 may interact with other appetite regulators elevated in obesity, such as leptin. Here, we report that in mice with high-fat-diet-induced obesity (HFD), the combined infusion of GDF15 and leptin causes significantly greater weight and adiposity loss than either treatment alone, indicating potentiation between GDF15 and leptin. Furthermore, obese, leptin-deficient ob/ob mice are less responsive to GDF15, as are normal mice treated with a competitive leptin antagonist. GDF15 and leptin induce more hindbrain neuronal activation in HFD mice than either treatment alone does. We report extensive connections between GFRAL- and LepR-expressing neurons and find LepR knockdown in the NTS to reduce the GDF15-mediated activation of AP neurons. Overall, these findings suggest that leptin signaling pathways in the hindbrain increase GDF15's metabolic actions.

Neither GLP-1 receptors nor GFRAL neurons are required for aversive or anorectic response to DON (vomitoxin).

Deoxynivalenol (DON), a type B trichothecene mycotoxin contaminating grains, promotes nausea, emesis and anorexia. With DON exposure, circulating levels of intestinally derived satiation hormones, including glucagon-like peptide 1 (GLP-1) are elevated. To directly test whether GLP-1 signaling mediates the effects of DON, we examined the response of GLP-1 or GLP-1R-deficient mice to DON injection. We found comparable anorectic and conditioned taste avoidance learning responses in GLP-1/GLP-1R deficient mice compared to control littermates, suggesting that GLP-1 is not necessary for the effects of DON on food intake and visceral illness. We then used our previously published data from translating ribosome affinity purification with RNA sequencing (TRAP-seq) analysis of area postrema neurons that express the receptor for the circulating cytokine growth differentiation factor (GDF15), growth differentiation factor a-like (GFRAL). Interestingly, this analysis showed that a cell surface receptor for DON, calcium sensing receptor (CaSR), is heavily enriched in GFRAL neurons. Given that GDF15 potently reduces food intake and can cause visceral illness by signaling through GFRAL neurons, we hypothesized that DON may also signal by activating CaSR on GFRAL neurons. Indeed, circulating GDF15 levels are elevated after DON administration but both GFRAL knockout and GFRAL neuron-ablated mice exhibited similar anorectic and conditioned taste avoidance responses compared to WT littermates. Thus, GLP-1 signaling and GFRAL signaling and neurons are not required for DON-induced visceral illness or anorexia.

Selective PDE4 subtype inhibition provides new opportunities to intervene in neuroinflammatory versus myelin damaging hallmarks of multiple sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal inflammatory lesions and prominent demyelination. Even though the currently available therapies are effective in treating the initial stages of disease, they are unable to halt or reverse disease progression into the chronic progressive stage. Thus far, no repair-inducing treatments are available for progressive MS patients. Hence, there is an urgent need for the development of new therapeutic strategies either targeting the destructive immunological demyelination or boosting endogenous repair mechanisms. Using in vitro, ex vivo, and in vivo models, we demonstrate that selective inhibition of phosphodiesterase 4 (PDE4), a family of enzymes that hydrolyzes and inactivates cyclic adenosine monophosphate (cAMP), reduces inflammation and promotes myelin repair. More specifically, we segregated the myelination-promoting and anti-inflammatory effects into a PDE4D- and PDE4B-dependent process respectively. We show that inhibition of PDE4D boosts oligodendrocyte progenitor cells (OPC) differentiation and enhances (re)myelination of both murine OPCs and human iPSC-derived OPCs. In addition, PDE4D inhibition promotes in vivo remyelination in the cuprizone model, which is accompanied by improved spatial memory and reduced visual evoked potential latency times. We further identified that PDE4B-specific inhibition exerts anti-inflammatory effects since it lowers in vitro monocytic nitric oxide (NO) production and improves in vivo neurological scores during the early phase of experimental autoimmune encephalomyelitis (EAE). In contrast to the pan PDE4 inhibitor roflumilast, the therapeutic dose of both the PDE4B-specific inhibitor A33 and the PDE4D-specific inhibitor Gebr32a did not trigger emesis-like side effects in rodents. Finally, we report distinct PDE4D isoform expression patterns in human area postrema neurons and human oligodendroglia lineage cells. Using the CRISPR-Cas9 system, we confirmed that pde4d1/2 and pde4d6 are the key targets to induce OPC differentiation. Collectively, these data demonstrate that gene specific PDE4 inhibitors have potential as novel therapeutic agents for targeting the distinct disease processes of MS.

A brainstem circuit for nausea suppression.

SUMMARYNausea is a discomforting sensation of gut malaise that remains a major clinical challenge. Several visceral poisons induce nausea through the area postrema, a sensory circumventricular organ that detects blood-borne factors. Here, we use genetic approaches based on an area postrema cell atlas to reveal inhibitory neurons that counteract nausea-associated poison responses. The gut hormone glucose insulinotropic peptide (GIP) activates area postrema inhibitory neurons that project locally and elicit inhibitory currents in nausea-promoting excitatory neurons through γ-aminobutyric acid (GABA) receptors. Moreover, GIP blocks behavioral responses to poisons in wild-type mice, with protection eliminated by targeted area postrema neuron ablation. These findings provide insights into the basic organization of nausea-associated brainstem circuits and reveal that area postrema inhibitory neurons are an effective pharmacological target for nausea intervention.

Minocycline Activates the Nucleus of the Solitary Tract-Associated Network to Alleviate Lipopolysaccharide-Induced Neuroinflammation

ObjectiveTo examine the neuroanatomical substrates underlying the effects of minocycline in alleviating lipopolysaccharide (LPS)-induced neuroinflammation. MethodsForty C57BL/6 male mice were randomly and equally divided into eight groups. Over three consecutive days, saline was administered to four groups of mice and minocycline to the other four groups. Immediately after the administration of saline or minocycline on the third day, two groups of mice were additionally injected with saline and the other two groups were injected with LPS. Six or 24 hours after the last injection, mice were sacrificed and the brains were removed. Immunohistochemical staining across the whole brain was performed to detect microglia activation via Iba1 and neuronal activation via c-Fos. Morphology of microglia and the number of c-Fo-positive neurons were analyzed by Image-Pro Premier 3D. One-way ANOVA and Fisher's least-significant differences were employed for statistical analyses. ResultsMinocycline alleviated LPS-induced neuroinflammation as evidenced by reduced activation of microglia in multiple brain regions, including the shell part of the nucleus accumbens (Acbs), paraventricular nucleus (PVN) of the hypothalamus, central nucleus of the amygdala (CeA), locus coeruleus (LC), and nucleus tractus solitarius (NTS). Minocycline significantly increased the number of c-Fo-positive neurons in NTS and area postrema (AP) after LPS treatment. Furthermore, in NTS-associated brain areas, including LC, lateral parabrachial nucleus (LPB), periaqueductal gray (PAG), dorsal raphe nucleus (DR), amygdala, PVN, and bed nucleus of the stria terminali (BNST), minocycline also significantly increased the number of c-Fo-positive neurons after LPS administration. ConclusionMinocycline alleviates LPS-induced neuroinflammation in multiple brain regions, possibly due to increased activation of neurons in the NTS-associated network.

Fibroblast Growth Factor-1 Activates Neurons in the Arcuate Nucleus and Dorsal Vagal Complex

Central administration of fibroblast growth factor-1 (FGF1) results in long-lasting resolution of hyperglycemia in various rodent models, but the pre- and postsynaptic mechanisms mediating the central effects of FGF1 are unknown. Here we utilize electrophysiology recordings from neuronal populations in the arcuate nucleus of the hypothalamus (ARH), nucleus of the solitary tract (NTS), and area postrema (AP) to investigate the mechanisms underlying FGF1 actions. While FGF1 did not alter membrane potential in ARH-NPY-GFP neurons, it reversibly depolarized 83% of ARH-POMC-EGFP neurons and decreased the frequency of inhibitory inputs onto ARH-POMC-EGFP neurons. This depolarizing effect persisted in the presence of FGF receptor (R) blocker FIIN1, but was blocked by pretreatment with the voltage-gated sodium channel (VGSC) blocker tetrodotoxin (TTX). Non-FGF1 subfamilies can activate vascular endothelial growth factor receptors (VEGFR). Surprisingly, the VEGFR inhibitors axitinib and BMS605541 blocked FGF1 effects on ARH-POMC-EGFP neurons. We also demonstrate that FGF1 induces c-Fos in the dorsal vagal complex, activates NTS-NPY-GFP neurons through a FGFR mediated pathway, and requires VGSCs to activate AP neurons. We conclude that FGF1 acts in multiple brain regions independent of FGFRs. These studies present anatomical and mechanistic pathways for the future investigation of the pharmacological and physiological role of FGF1 in metabolic processes.

Fibroblast Growth Factor 19 Increases the Excitability of Pre-Motor Glutamatergic Dorsal Vagal Complex Neurons From Hyperglycemic Mice.

Intracerebroventricular administration of the protein hormone fibroblast growth factor 19 (FGF19) to the hindbrain produces potent antidiabetic effects in hyperglycemic mice that are likely mediated through a vagal parasympathetic mechanism. FGF19 increases the synaptic excitability of parasympathetic motor neurons in the dorsal motor nucleus of the vagus (DMV) from hyperglycemic, but not normoglycemic, mice but the source of this synaptic input is unknown. Neurons in the area postrema (AP) and nucleus tractus solitarius (NTS) express high levels of FGF receptors and exert glutamatergic control over the DMV. This study tested the hypothesis that FGF19 increases glutamate release in the DMV by increasing the activity of glutamatergic AP and NTS neurons in hyperglycemic mice. Glutamate photoactivation experiments confirmed that FGF19 increases synaptic glutamate release from AP and NTS neurons that connect to the DMV in hyperglycemic, but not normoglycemic mice. Contrary to expectations, FGF19 produced a mixed effect on intrinsic membrane properties in the NTS with a trend towards inhibition, suggesting that another mechanism was responsible for the observed effects on glutamate release in the DMV. Consistent with the hypothesis, FGF19 increased action potential-dependent glutamate release in the NTS in hyperglycemic mice only. Finally, glutamate photoactivation experiments confirmed that FGF19 increases the activity of glutamatergic AP neurons that project to the NTS in hyperglycemic mice. Together, these results support the hypothesis that FGF19 increases glutamate release from AP and NTS neurons that project to the DMV in hyperglycemic mice. FGF19 therefore modifies the local vago-vagal reflex circuitry at several points. Additionally, since the AP and NTS communicate with several other metabolic regulatory nuclei in the brain, FGF19 in the hindbrain may alter neuroendocrine and behavioral aspects of metabolism, in addition to changes in parasympathetic output.

The calcitonin receptor is the main mediator of LAAMA's body weight lowering effects in male mice

The anorectic action of the pancreatic hormone amylin is mainly mediated through the area postrema (AP). Amylin activates AP neurons using a heterodimeric receptor (AMY) composed of the calcitonin receptor (CTR) and the receptor activity modifying protein (RAMP 1, 2 or 3). The aim of the following experiments is to test the effects of the long acting amylin analogue (LAAMA) in RAMP1/3 knock-out (KO) male mice and in neuronal CTR KO Nestin-CreCTR male mice. In vitro, LAAMA exerted an equipotent effect on CTR and AMYs that was maintained across species. Following one week of 45% high fat diet, WT, RAMP1/3 KO and Nestin-CreCTR mice were injected daily for one week with vehicle or LAAMA. LAAMA decreased body weight gain in WT and in RAMP1/3 KO mice suggesting that RAMP1/3 are not necessary for LAAMA-induced effects. However, LAAMA was not able to produce any body lowering and anorectic effects in Nestin-CreCTR mice. This was accompanied by the absence of any c-Fos signal in the AP opposite to WT control mice. Together, these results suggest that LAAMA's effects are mainly mediated through CTR rather than specific AMY. The study of LAAMA or any amylin receptor agonist in different receptor KO mouse models helps disentangle the underlying mechanisms used by these molecules.

Hypoglycemia attenuates acute amylin-induced reduction of food intake in male rats

The ability of amylin to inhibit gastric emptying and glucagon secretion in rats is reduced under hypoglycemic conditions. These effects are considered part of a fail-safe mechanism that prevents amylin from further decreasing nutrient supply when blood glucose levels are low. Because these actions and amylin-induced satiation are mediated by the area postrema (AP), it is plausible that these phenomena are based on the co-sensitivity of AP neurons to amylin and glucose. Using hyperinsulinemic glucose clamps in unrestrained and freely-feeding rats, we investigated whether amylin's ability to inhibit food intake is also reduced by hypoglycemia (HYPO). Following an 18 h fast, rats were infused with insulin and glucose for 45 min to clamp blood glucose at baseline levels (between 90 and 100 mg/dL). HYPO (approximately 55 mg/dL) was induced between 45 and 60 min and then maintained for the remainder of the clamp. Rats were injected with amylin (20 µg/kg) or saline and offered normal chow at 85 min. Food intake was measured at 30 and 60 min after amylin. Control hyperinsulinemic/euglycemic (EU) rats were maintained at approximately 150 mg/dL (which is a physiological periprandial glucose level) before and after amylin injection. Terminal experiments tested the effect of amylin to induce the phosphorylation of ERK, a marker of amylin action in the AP, in EU and HYPO conditions. Amylin significantly reduced 30- and 60-min food intake in EU rats, but the effect at 60-min was attenuated in HYPO rats. Interestingly, glucose infusion rate had to be dramatically reduced at meal onset in saline-treated, but not in amylin-treated, EU or HYPO rats; this suggests that meal-related glucose appearance in the blood was inhibited by amylin under both EU and HYPO. Finally, amylin induced a similar pERK response in the AP in EU and HYPO rats. We conclude that amylin's action to decrease eating is blunted in hypoglycemia, and this effect seems to be downstream from amylin-induced pERK in AP neurons. These data allow us to extend the idea of a hypoglycemic brake on amylin's actions to its food intake-reducing effect, but also demonstrate that amylin can buffer meal-induced glucose appearance at EU and HYPO levels.

GFRAL-expressing neurons suppress food intake via aversive pathways

The TGFβ cytokine family member, GDF-15, reduces food intake and body weight and represents a potential treatment for obesity. Because the brainstem-restricted expression pattern of its receptor, GDNF Family Receptor α-like (GFRAL), presents an exciting opportunity to understand mechanisms of action for area postrema neurons in food intake; we generated GfralCre and conditional GfralCreERT mice to visualize and manipulate GFRAL neurons. We found infection or pathophysiologic states (rather than meal ingestion) stimulate GFRAL neurons. TRAP-Seq analysis of GFRAL neurons revealed their expression of a wide range of neurotransmitters and neuropeptides. Artificially activating GfralCre -expressing neurons inhibited feeding, decreased gastric emptying, and promoted a conditioned taste aversion (CTA). GFRAL neurons most strongly innervate the parabrachial nucleus (PBN), where they target CGRP-expressing (CGRPPBN) neurons. Silencing CGRPPBN neurons abrogated the aversive and anorexic effects of GDF-15. These findings suggest that GFRAL neurons link non-meal-associated pathophysiologic signals to suppress nutrient uptake and absorption.

Effects of methyl methacrylate on the excitability of the area postrema neurons in rats

ObjectivesThe aim of the present study is to demonstrate the effects of inhaled methyl methacrylate (MMA) on the excitability of neurons in the area postrema (AP). We also investigated the relation between vagal afferent inputs and responding cells in the AP. MethodsWe set up two groups of experimental animals, such as rats inhaling MMA and rats inhaling room air. To visualize the changes of AP neuron excitability after inhalation of MMA for 90 min, c-Fos protein expression was identified and quantified by immunohistochemical analysis. Some rats receiving ventral gastric branch vagotomy were also subjected to the abovementioned experiment. ResultsThe number of c-Fos-immunoreactive (Fos-ir) cells in the MMA group was more than six times greater than that of the control group (statistically significant, p < 0.01). In vagotomized rats inhaling MMA, markedly smaller number of Fos-ir cells was identified in the AP compared to that of rats inhaling MMA without vagotomy. ConclusionsThese results indicate that inhalation of MMA increases neuronal excitability in the AP, suggesting that vagal afferent inputs are involved in the induction mechanism of Fos-ir cells by MMA.

Area postrema: fetal maturation, tumours, vomiting centre, somatic growth and role in neuromyelitis optica

The area postrema (AP) in the caudal 4th ventricular floor is unique, highly vascular without blood/brain or /CSF barrier. In addition to its function as the vomiting centre, several other important functions are: part of the circumventricular organs for vasomotor and angiotensin II regulation; a role in neuromyelitis optica related to aquaporin-4; contributor to fetal and postnatal somatic growth. Functions are immature at birth.The purpose of this study was to demonstrate AP neuronal/synaptic/glial maturation in normal fetuses and 3 AP tumours. Transverse sections of the caudal 4th ventricle of 18 normal human fetal brains at autopsy, 6 to 40 weeks were examined; also 3 infants 3-18mos; 2 children. A battery of immunocytochemical neuronal and glial markers: MAP2; calretinin; synaptophysin; vimentin; nestin; GFAP; S-100β protein; were applied to paraffin sections. Two children with AP tumours and one with neurocutaneous melanocytosis, all with pernicious vomiting, were studied. In normal fetuses, AP neurons exhibited cytological maturity and well-formed synaptic circuitry by 14wk gestation. Size/volume increase was disproportionately greater than brainstem growth in 2nd and 3rd trimesters and postnatally. Astrocytes co-expressed vimentin/GFAP but glia were best demonstrated by S-100β protein. Ependyma over the AP in fetuses is simple cuboidal, adjacent to pseudostratified columnar of the 4th ventricular floor. Melanocytes infiltrated AP in the toddler with pernicious vomiting; 2 children had primary AP pilocytic astrocytomas. Though AP is cytologically mature by 14wk, growth increases and functions mature into the postnatal months. We recommend that AP neuropathology include synaptophysin and S-100β at autopsy if AP dysfunction suspected.LEARNING OBJECTIVESThis presentation will enable the learner to:1.Explain the maturation of neurons, synaptic circuits and glial elements of the AP2.List and recognize tumours that can affect the area postrema3.Describe functions of the area postrema

Isoflurane induces c-Fos expression in the area postrema of the rat.

Volatile anesthetics are speculated to cause postoperative nausea and vomiting via stimulation of the chemoreceptor trigger zone (CTZ). However, the precise mechanism underlying the emetic action of these drugs is not well understood. In this study, we assessed whether isoflurane induced the expression of c-Fos, a neuronal activation marker, in the area postrema (AP), the locus of the CTZ, in rats, which do not have vomiting action. Male rats were exposed to 1.3% isoflurane for 0-240min, or to various concentrations of isoflurane (0, 1.3%, or 2.6%) for 120min. Finally, the rats were exposed to 1.3% isoflurane for 120min after ondansetron administration. After the treatments, immunohistochemistry of the rat AP was performed using c-Fos antibody staining. One-way analysis of variance showed that isoflurane exposure significantly increased c-Fos expression in the AP; however, the rats pretreated with 4mg/kg ondansetron showed significantly decreased c-Fos expression. Moreover, we evaluated the effect of the anesthetic on inducing pica in the rats, and found that kaolin intake was not influenced by isoflurane exposure. Overall, these results suggest that isoflurane activates AP neurons and may be involved in the emetic mechanism of isoflurane. This study further suggests the feasibility of using rats as a model for studying emetic mechanisms of drugs, despite their lack of vomit action.

In Renovascular Hypertension, TNF‐α Type‐1 Receptors in the Area Postrema Mediate Increases in Cardiac and Renal Sympathetic Nerve Activity and Blood Pressure

Neuroinflammation is a common feature in renovascular, obesity-related, and angiotensin II mediated hypertension. There is evidence that increased release of the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α) contributes to the development of the hypertension, but the underlying neural mechanisms are unclear. In rats with renovascular hypertension, area postrema neurons that expressed TNF-α type-1 receptor (TNFR1) remained constantly activated (expressed c-Fos) and injection of TNFR1 neutralizing antibody into the area postrema returned blood pressure (systolic: ~151 mmHg) to normotensive levels (systolic: ~108 mmHg). Nanoinjection of TNF-α (100 pg/50 nl) into the area postrema of anaesthetized normotensive rats increased blood pressure (~16 mmHg) and sympathetic nerve activity, predominantly to the heart (~53%), but also to the kidneys (~35%). These responses were abolished by prior injection of a TNFR1 neutralizing antibody (1 ng/50 nl) within the same site. TNFR1 were expressed in the somata of neurons activated by TNF-α that were retrogradely labelled from the rostral ventrolateral medulla. These findings indicate that in renovascular hypertension, activation of TNFR1 expressing neurons in the area postrema by TNF-α contributes to the sustained blood pressure in rats. This is mediated, in part, by projections to the rostral ventrolateral medulla and an increase in both cardiac and renal sympathetic nerve activity. Support or Funding Information This work was supported by the National Health and Medical Research Council of Australia (GNT 1079680 to STY), the High Blood Pressure Research Council of Australia, the Rebecca L Cooper Medical Foundation, the Wade Institute and Ormond College - University of Melbourne (to WSK) and the Victorian Government through the Operational Infrastructure Scheme. KE and AS are supported by the Australian Government Research Training Program Scholarships. STY is supported by an Australian Research Council Future Fellowship (FT170100363). Effects of injection of TNF-α into the AP on cardiac and renal sympathetic nerve activity. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In renovascular hypertension, TNF-α type-1 receptors in the area postrema mediate increases in cardiac and renal sympathetic nerve activity and blood pressure.

Neuroinflammation is a common feature in renovascular, obesity-related, and angiotensin II mediated hypertension. There is evidence that increased release of the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α) contributes to the development of the hypertension, but the underlying neural mechanisms are unclear. Here, we investigated whether TNF-α stimulates neurons in the area postrema (AP), a circumventricular organ, to elicit sympathetic excitation, and increases in blood pressure (BP). In rats with renovascular hypertension, AP neurons that expressed TNF-α type-1 receptor (TNFR1) remained constantly activated (expressed c-Fos) and injection of TNFR1 neutralizing antibody into the AP returned BP (systolic: ∼151 mmHg) to normotensive levels (systolic: ∼108 mmHg). Nanoinjection of TNF-α (100 pg/50 nL) into the AP of anaesthetized normotensive rats increased BP (∼16 mmHg) and sympathetic nerve activity, predominantly to the heart (∼53%), but also to the kidneys (∼35%). These responses were abolished by prior injection of a TNFR1 neutralizing antibody (1 ng/50 nL) within the same site. TNFR1 were expressed in the somata of neurons activated by TNF-α that were retrogradely labelled from the rostral ventrolateral medulla (RVLM). These findings indicate that in renovascular hypertension, blocking TNFR1 receptors in the AP significantly reduces BP, while activation of TNFR1 expressing neurons in the AP by TNF-α increases BP in normotensive rats. This is mediated, in part, by projections to the RVLM and an increase in both cardiac and renal sympathetic nerve activity. These findings support the notion that proinflammatory cytokines and neuroinflammation are important pathological mechanisms in the development and maintenance of hypertension.

Glucagon-like peptide-1 (GLP-1) action in the mouse area postrema neurons

Glucagon-like peptide-1 (GLP-1) is a peptide hormone and member of the incretin family. GLP-1 related drugs, such as liraglutide, are widely used to treat diabetic patients and work by stimulating pancreatic β cells to increase glucose-dependent insulin secretion. However, extrapancreatic effects, such as appetite suppression or emesis, are observed in response to GLP-1 receptor agonists. In this study we used the in vitro patch-clamp method in acute brainstem preparations of mice and demonstrated that GLP-1 acts directly on area postrema neurons. It is known that activation of the area postrema is related to the induction of homeostatic autonomic nervous systems, including nausea. Approximately,half of the neurons tested in the area postrema were excited by GLP-1 in the presence of tetrodotoxin, and is thought to be through adenylate cyclase-cAMP pathways. Excitation was not frequently observed in nucleus tractus solitaries neurons or in area postrema neurons from GLP-1 receptor knock-out mice. These results indicate that GLP-1 receptor agonists excite area postrema neurons and potentially leading to the expression of extra-pancreatic effects. This is the first study to show that GLP-1 directly activates area postrema neurons.

Role of the area postrema in the hypophagic effects of oleoylethanolamide

The satiety-promoting action of oleoylethanolamide (OEA) has been associated to the indirect activation of selected brain areas, such as the nucleus of the solitary tract (NST) in the brainstem and the tuberomammillary (TMN) and paraventricular (PVN) nuclei in the hypothalamus, where noradrenergic, histaminergic and oxytocinergic neurons play a necessary role. Visceral ascending fibers were hypothesized to mediate such effects. However, our previous findings demonstrated that the hypophagic action of peripherally administered OEA does not require intact vagal afferents and is associated to a strong activation of the area postrema (AP). Therefore, we hypothesized that OEA may exert its central effects through the direct activation of this circumventricular organ. To test this hypothesis, we subjected rats to the surgical ablation of the AP (APX rats) and evaluated the effects of OEA (10mgkg−1 i.p.) on food intake, Fos expression, hypothalamic oxytocin (OXY) immunoreactivity and on the expression of dopamine beta hydroxylase (DBH) in the brainstem and hypothalamus. We found that the AP lesion completely prevented OEA’s behavioral and neurochemical effects in the brainstem and the hypothalamus. Moreover OEA increased DBH expression in AP and NST neurons of SHAM rats while the effect in the NST was absent in APX rats, thus suggesting the possible involvement of noradrenergic AP neurons. These results support the hypothesis of a necessary role of the AP in mediating OEA’s central effects that sustain its pro-satiety action.