The neuromedin U system: Pharmacological implications for the treatment of obesity and binge eating behavior.

Neuromedin U (NMU) is a bioactive peptide produced in the gut and in the brain, with a role in multiple physiological processes. NMU acts by binding and activating two G protein coupled receptors (GPCR), the NMU receptor 1 (NMU-R1), which is predominantly expressed in the periphery, and the NMU receptor 2 (NMU-R2), mainly expressed in the central nervous system (CNS). In the brain, NMU and NMU-R2 are consistently present in the hypothalamus, commonly recognized as the main "feeding center". Considering its distribution pattern, NMU revealed to be an important neuropeptide involved in the regulation of food intake, with a powerful anorexigenic ability. This has been observed through direct administration of NMU and by studies using genetically modified animals, which revealed an obesity phenotype when the NMU gene is deleted. Thus, the development of NMU analogs or NMU-R2 agonists might represent a promising pharmacological strategy to treat obese individuals. Furthermore, NMU has been demonstrated to influence the non-homeostatic aspect of food intake, playing a potential role in binge eating behavior. This review aims to discuss and summarize the current literature linking the NMU system with obesity and binge eating behavior, focusing on the influence of NMU on food intake and the neuronal mechanisms underlying its anti-obesity properties. Pharmacological strategies to improve the pharmacokinetic profile of NMU will also be reported.

Postnatal ontogeny of Neuromedin S and its receptors NMUR1 and NMUR2 expression in mouse testis.

Neuromedin S (NMS) is a well-known anorexigenic neuropeptide. Despite some reports of the presence of its transcript and precursor protein in testis, the expression and localization of NMS and its receptors during the postnatal development of mammalian testis remains elusive. We investigated the expression patterns and testicular localization of NMS and its receptors NMUR1 and NMUR2, during 5, 10, 20, 30, and 90 days of postnatal development, using real time PCR, immunoblot analysis and immunohistochemistry in mice. NMS and its receptors are present at all age groups at transcript level in mouse testis. At the protein level, NMS and NMUR2 are present in all age groups, whereas NMUR1 is present primarily in 30- and 90-day testis. Immunolocalization study showed that NMS and NMUR2 are expressed in spermatogonia, spermatocytes, Sertoli cells, and Leydig cells, in contrast to NMUR1 which is expressed exclusively in the Leydig cells of 30- and 90-day testis. The results also confirm the intranuclear localization of NMS in spermatogonia and spermatocytes. Although NMS-NMUR2 is expressed in Sertoli cells at all stages of the spermatogenic cycle, they showed a stage-specific expression pattern in spermatogonia and primary spermatocytes. In conclusion, NMS and its receptors NMUR1 and NMUR2 are expressed in the testis and may regulate spermatogenesis, possibly by modulating steroidogenesis and Sertoli cell function in the testis.

Female fertility does not require Bmal1 in suprachiasmatic nucleus neurons expressing arginine vasopressin, vasoactive intestinal peptide, or neuromedin-S.

Disruptions to the circadian system alter reproductive capacity, particularly in females. Mice lacking the core circadian clock gene, Bmal1, are infertile and have evidence of neuroendocrine disruption including the absence of the preovulatory luteinizing hormone (LH) surge and enhanced responsiveness to exogenous kisspeptin. Here, we explore the role of Bmal1 in suprachiasmatic nucleus (SCN) neuron populations known to project to the neuroendocrine axis. We generated four mouse lines using Cre/Lox technology to create conditional deletion of Bmal1 in arginine vasopressin (Bmal1fl/fl:Avpcre ), vasoactive intestinal peptide (Bmal1fl/fl:Vipcre ), both (Bmal1fl/fl:Avpcre+Vipcre ), and neuromedin-s (Bmal1fl/fl:Nmscre ) neurons. We demonstrate that the loss of Bmal1 in these populations has substantial effects on home-cage circadian activity and temperature rhythms. Despite this, we found that female mice from these lines demonstrated normal estrus cycles, fecundity, kisspeptin responsiveness, and inducible LH surge. We found no evidence of reproductive disruption in constant darkness. Overall, our results indicate that while conditional Bmal1 knockout in AVP, VIP, or NMS neurons is sufficient to disrupted locomotor activity, this disruption is insufficient to recapitulate the neuroendocrine reproductive effects of the whole-body Bmal1 knockout.

Nmu/Nms/Gpr176 Triple-Deficient Mice Show Enhanced Light-Resetting of Circadian Locomotor Activity.

The suprachiasmatic nucleus (SCN) is the master circadian clock in mammals and is properly entrained by environmental light cycle. However, the molecular mechanism(s) determining the magnitude of phase shift by light is still not fully understood. The orphan G-protein-coupled receptor Gpr176 is enriched in the SCN, controls the pace (period) of the circadian rhythm in behavior but is not apparently involved in the light entrainment; Gpr176-/- animals display a shortened circadian period in constant darkness but their phase-resetting responses to light are normal. Here, we performed microarray analysis and identified enhanced mRNA expression of neuromedin U (Nmu) and neuromedin S (Nms) in the SCN of Gpr176-/- mice. By generating C57BL/6J-backcrossed Nmu/Nms/Gpr176 triple knockout mice, we noted that the mutant mice had a greater magnitude of phase shift in response to early subjective night light than wildtype mice, while Nmu/Nms double knockout mice as well as Gpr176 knockout mice are normal in the phase shifts induced by light. At the molecular level, Nmu-/-Nms-/-Gpr176-/- mice had a reduced induction of Per1 and cFos mRNA expression in the SCN by light and mildly upregulated circadian expression of Per2, Prok2, Rgs16, and Rasl11b. These expressional changes may underlie the phenotype of the Nmu/Nms/Gpr176 knockout mice. Our data argue that there is a mechanism requiring Nmu, Nms, and Gpr176 for the proper modulation of light-induced phase shift in mice. Simultaneous modulation of Nmu/Nms/Gpr176 may provide a potential target option for modulating the circadian clock.

Possible effects of the central adrenergic and dopaminergic receptors on hypophagia induced by neuromedin S in neonatal layer-type chicks.

The current study was aimed to determine the possible effects of the central adrenergic and dopaminergic receptors in neuromedin S (NMS)-induced hypophagia in neonatal layer-type chickens. In the first experiment, control solution, and NMS (0.25, 0.5, and 1 nmol), were injected (intracerebroventricular (ICV)) in chickens. In the second experiment, birds were injected with a control solution,SCH23390 (D1receptor antagonist, 5 nmol), NMS (1 nmol), and a combination of the SCH23390 + NMS. Experiments 3-11 were similar to experiment 2, except that chickens were injected withAMI-193 (D2receptor antagonist, 5 nmol), NGB2904(D3receptor antagonist, 6.4 nmol), L-741,742(D4receptor antagonist, 6 nmol), 6-OHDA(6-hydroxydopamine, 2.5 nmol),Prazosin(α1receptor antagonist, 10 nmol),Yohimbine(α2receptor antagonist, 13 nmol),Metoprolol(ß1receptor antagonist receptor, 24 nmol),ICI 118,551 (ß2receptor antagonist, 5 nmol),SR 59230R (ß3 receptor antagonist, 20 nmol) instead ofSCH23390. Then, cumulative food intake was recorded at 30, 60, and 120 min following the injection. According to the results, food intake was significantly decreased after ICV injection of NMS in a dose -dependent manner (P < 0.05). Also, the co-injection of the SCH23390 + NMS significantly attenuated NMS-induced hypophagia (P < 0.05). The co-administration of AMI-193 + NMS significantly reduced NMS- induced hypophagia (P < 0.05). In addition, the co-injection of ICI 118,551 + NMS and 6-OHDA + NMS considerably decreased NMS-induced food consumption (P < 0.05). However, NGB2904, L-741742, Prazosin, Yohimbine, Metoprolol and SR 59230R had no effect on hypophagia induced by NMS (P > 0.05). These results demonstrated thatNMS- induced hypophagia might be mediated by D1/D2 dopaminergic andß2adrenergic receptors in neonatal layer-type chickens.

Neuromedins NMU and NMS: An Updated Overview of Their Functions.

More than 35 years have passed since the identification of neuromedin U (NMU). Dozens of publications have been devoted to its physiological role in the organism, which have provided insight into its occurrence in the body, its synthesis and mechanism of action at the cellular level. Two G protein-coupled receptors (GPCRs) have been identified, with NMUR1 distributed mainly peripherally and NMUR2 predominantly centrally. Recognition of the role of NMU in the control of energy homeostasis of the body has greatly increased interest in this neuromedin. In 2005 a second, structurally related peptide, neuromedin S (NMS) was identified. The expression of NMS is more restricted, it is predominantly found in the central nervous system. In recent years, further peptides related to NMU and NMS have been identified. These are neuromedin U precursor related peptide (NURP) and neuromedin S precursor related peptide (NSRP), which also exert biological effects without acting via NMUR1, or NMUR2. This observation suggests the presence of another, as yet unrecognized receptor. Another unresolved issue within the NMU/NMS system is the differences in the effects of various NMU isoforms on diverse cell lines. It seems that development of highly specific NMUR1 and NMUR2 receptor antagonists would allow for a more detailed understanding of the mechanisms of action of NMU/NMS and related peptides in the body. They could form the basis for attempts to use such compounds in the treatment of disorders, for example, metabolic disorders, circadian rhythm, stress, etc.

Comparison of physiological functions between neuromedin U-related peptide and neuromedin S-related peptide in the rat central nervous system.

Two novel peptides, neuromedin U precursor-related peptide (NURP) and neuromedin S precursor-related peptide (NSRP), are produced from neuromedin U (NMU) and neuromedin S (NMS) precursors, respectively, as these precursors have multiple consensus sequences for proteolytic processing. Our group has shown previously that one of these two novel peptides, NURP, stimulates body temperature and locomotor activity, but not food intake. However, the physiological function of the other peptide, NSRP, has remained unclear. Therefore, the aim of this study was to characterize differences in the regions of the rat brain targeted by the NMU/NMS peptide family, including NURP and NSRP, and their physiological functions. First, we explored the regions of c-Fos expression after intracerebroventricular (i.c.v.) injection of NURP and NSRP and found that these were fewer than after i.c.v. injection of NMU and NMS in the hypothalamus, possibly because NURP and NSRP cannot activate NMU/NMS receptors. In the ventral subiculum, which is one region of the hippocampus, c-Fos expression was evident only after i.c.v. injection of NURP. We also examined the effects of NSRP on food intake, body temperature and locomotor activity. Like NURP, NSRP increased both body temperature and locomotor activity, but not food intake, indicating that NSRP is also a functional peptide. However, these effects of NSRP were distinctly weaker than those of NURP. These findings suggest differences in the affinity of NURP and/or NSRP for specific receptors, or in their respective biological activities.

Neuromedin U suppresses prolactin secretion via dopamine neurons of the arcuate nucleus.

Neuromedin U (NMU) has a precursor that contains one additional peptide consisting of 33 or 36 amino acid residues. Recently, we identified this second peptide from rat brain and designated it neuromedin U precursor-related peptide (NURP), showing it to stimulate prolactin release from the pituitary when injected via the intracerebroventricular (icv) route. Here, we examined whether NMU, like NURP, also stimulates prolactin release. Unlike NURP, icv injection of NMU significantly decreased the secretion of prolactin from the pituitary. This suppression of prolactin release by NMU was observed in hyper-prolactin states such as lactation, stress, pseudopregnancy, domperidone (dopamine antagonist) administration, and icv injection of NURP. Immunohistochemical analysis revealed that icv injection of NMU induced cFos expression in dopaminergic neurons of the arcuate nucleus, but not the substantia nigra. Mice with double knockout of NMU and neuromedin S (NMS), the latter also binding to NMU receptors, showed a significant increase of the plasma prolactin level after domperidone treatment relative to wild-type mice. These results suggest that NMU and NURP may play important reciprocal roles in physiological prolactin secretion.

Comparison of glucose tolerance between wild-type mice and mice with double knockout of neuromedin U and neuromedin S.

Recently, it has been proposed that neuromedin U (NMU) is "decretin", which suppresses insulin secretion from the pancreas in vitro. Here we examined the possible involvement of NMU in insulin secretion in vivo by comparing the plasma glucose and insulin levels of wild-type mice with those of double knockout (D-KO) of the NMU and neuromedin S (NMS) genes, as NMS binds to the neuromedin U receptor. If NMU is, in fact, "decretin", which inhibits insulin secretion from the pancreas, then NMU-deficient mice might result in higher plasma insulin levels than is the case in wild-type mice, or injection of NMU lead to suppression of plasma insulin level. In this study, we found that the fasting plasma level of insulin was not increased in D-KO mice. Glucose tolerance tests revealed no significant difference in plasma insulin levels between wild-type mice and D-KO mice under non-fasting conditions. After peripheral injection of NMU, plasma glucose and insulin levels did not show any significant changes in either wild-type or D-KO mice. Glucose tolerance testing after 3 weeks of high fat feeding revealed no significant difference in plasma insulin levels during 60 min after glucose injection between wild-type and D-KO mice. These results suggest that even if NMU is a decretin candidate, its physiological involvement in suppression of insulin secretion may be very minor in vivo.

Characterization of neuromedin U (NMU), neuromedin S (NMS) and their receptors (NMUR1, NMUR2) in chickens.

Neuromedin U (NMU) and its structurally-related peptide, neuromedin S (NMS), are reported to regulate many physiological processes and their actions are mediated by two NMU receptors (NMUR1, NMUR2) in mammals. However, the information regarding NMU, NMS, and their receptors is limited in birds. In this study, we examined the structure, functionality, and expression of NMS, NMU, NMUR1 and NMUR2 in chickens. The results showed that: 1) chicken (c-) NMU cDNA encodes a 181-amino acid precursor, which may generate two forms of NMU peptide with 9 (cNMU-9) and 25 amino acids (cNMU-25), respectively. 2) Interestingly, two cNMS transcripts encoding two cNMS precursors of different lengths were identified from chicken pituitary, and both cNMS precursors may produce a mature cNMS peptide of 9 amino acids (cNMS-9). 3) cNMU-9, cNMU-25 and cNMS-9 could activate cNMUR1 expressed in HEK293 cells potently, as monitored by three cell-based luciferase reporter systems, indicating that cNMUR1 can act as a receptor common for cNMU and cNMS peptides, whereas cNMUR2 could be potently activated by cNMS-9, but not by cNMU-9/cNMU-25. 4) cNMU and cNMUR1 are widely expressed in chicken tissues with abundant expression noted in the gastrointestinal tract, as detected by quantitative real-time PCR, whereas cNMUR2 expression is mainly restricted to the brain and anterior pituitary, and cNMS is widely expressed in chicken tissues. Collectively, our data helps to elucidate the physiological roles of NMU/NMS peptides in birds and reveal the functional conservation and changes of NMU/NMS-NMUR axis across vertebrates.

Postnatal developmental of Neuromedin S and its receptor in the male Xiaomeishan pig reproductive axis.

Neuromedin S (NMS) has been identified as an endogenous ligand for FM-3/GPR66 and FM-4/TGR-1 which are NMU receptors NMUR1 and NMUR2, respectively. The NMS molecule is present in some peripheral tissues and the central nervous system (CNS), and it had been documented that NMS has fundamental and important roles in multiple physiological functions and processes such as circadian rhythm, energy balance, feeding behavior, stress responses and reproduction. The possible role of NMS in sexual development postnatally, however, is still obscure. This study aims to determine the change of NMS and its receptor gene expression in the reproductive axis of male Xiaomeishan pigs, postnatally. Firstly, the cDNA of the NMS and its receptors was cloned and sequenced. The results showed that there was a lack of 12 amino acids in the C-terminal of the male Xiaomeishan pig NMS amino-acid sequences compared with other animal species, but the main protein structure of prepro-NMS was high in homology. In addition, the nucleotide sequence and amino acids of the male Xiaomeishan pig's NMUR1 and NMUR2 had high homology. The NMS and NMUR2 mRNA in the male Xiaomeishan pig was detected in the reproductive axis at postnatal development stages, including postnatal day 3, 30, 60, 90 and 120, using real-time PCR and immunohistochemistry. The data showed that there were developmental changes in NMS and NMUR2 in the reproductive axis of the male Xiaomeishan pigs, postnatally, which suggested that NMS and NMUR2 might have a role in the development of the boar reproductive axis, but its regulatory mechanism remains to be elucidated.

PEGylated neuromedin U-8 shows long-lasting anorectic activity and anti-obesity effect in mice by peripheral administration.

Neuromedin U (NMU) is a neuropeptide found in the brain and gastrointestinal tract. The NMU system has been shown to regulate energy homeostasis by both a central and a peripheral mechanism. Peripheral administration of human NMU-25 was recently shown to inhibit food intake in mice. We examined the possibility that other NMU-related peptides exert an anorectic activity by intraperitoneal (i.p.) administration. We found that rat NMU-23 and its structurally-related peptide rat neuromedin S (NMS) significantly reduced food intake in lean mice, whereas NMU-8, an active fragment of the octapeptide sequence conserved in porcine, human and mouse NMU, had no effect. When rat NMU-23, NMU-8, and rat NMS were covalently conjugated to polyethylene glycol (PEG) (PEGylation) at the N-terminus of these peptides, PEGylated NMU-8 showed the most long-lasting and robust anorectic activity. The exploration of the linker between NMU-8 and PEG using hetero-bifunctional chemical cross-linkers led to an identification of PEGylated NMU-8 analogs with higher affinity for NMU receptors and with more potent anorectic activity in lean mice. The PEGylated NMU-8 showed potent and robust anorectic activity and anti-obesity effect in diet-induced obesity (DIO) mice by once-daily subcutaneous (s.c.) administration. These results suggest that PEGylated NMU-8 has the therapeutic potential for treatment of obesity.

Uterotonic Neuromedin U Receptor 2 and Its Ligands Are Upregulated by Inflammation in Mice and Humans, and Elicit Preterm Birth.

Uterine labor requires the conversion of a quiescent (propregnancy) uterus into an activated (prolabor) uterus, with increased sensitivity to endogenous uterotonic molecules. This activation is induced by stressors, particularly inflammation in term and preterm labor. Neuromedin U (NmU) is a neuropeptide known for its uterocontractile effects in rodents. The objective of the study was to assess the expression and function of neuromedin U receptor 2 (NmU-R2) and its ligands NmU and the more potent neuromedin S (NmS) in gestational tissues, and the possible implication of inflammatory stressors in triggering this system. Our data show that NmU and NmS are uterotonic ex vivo in murine tissue, and they dose-dependently trigger labor by acting specifically via NmU-R2. Expression of NmU-R2, NmU, and NmS is detected in murine and human gestational tissues by immunoblot, and the expression of NmS in placenta and of NmU-R2 in uterus increases considerably with gestation age and labor, which is associated with amplified NmU-induced uterocontractile response in mice. NmU- and NmS-induced contraction is associated with increased NmU-R2-coupled Ca++ transients, and Akt and Erk activation in murine primary myometrial smooth muscle cells (mSMCs), which are potentiated with gestational age. NmU-R2 is upregulated in vitro in mSMCs and in vivo in uterus in response to proinflammatory interleukin 1beta (IL1beta), which is associated with increased NmU-induced uterocontractile response and Ca++ transients in murine and human mSMCs; additionally, placental NmS is markedly upregulated in vivo in response to IL1beta. In human placenta at term, immunohistological analysis revealed NmS expression primarily in cytotrophoblasts; furthermore, stimulation with lipopolysaccharide (LPS; Gram-negative endotoxin) markedly upregulates NmS expression in primary human cytotrophoblasts isolated from term placentas. Correspondingly, decidua of women with clinical signs of infection who delivered preterm display significantly higher expression of NmS compared with those without infection. Importantly, in vivo knockdown of NmU-R2 prevents LPS-triggered preterm birth in mice and the associated neonatal mortality. Altogether, our data suggest a critical role for NmU-R2 and its ligands NmU and NmS in preterm labor triggered by infection. We hereby identify NmU-R2 as a relevant target for preterm birth.

Involvement of endogenous neuromedin U and neuromedin S in thermoregulation.

We investigated the possible involvement of neuromedin U (NMU) and neuromedin S (NMS) in thermoregulation in rats. Intracerebroventricular (icv) injection of NMU or NMS increased the back surface temperature (BS-T) in a dose-dependent manner during both the light and dark periods. Pre-treatment with the ß3 blocker SR59230A, and the cyclooxygenase blocker indomethacin, inhibited the increase in BS-T induced by NMS. Icv injection of NMS and NMU increased the expression of mRNAs for prostaglandin E synthase and cyclooxygenase 2 (COX2) in the hypothalamus, and that of mRNA for uncoupling protein 1 (UCP1) in the brown adipose tissue. Comparison of thermogenesis in terms of body temperature under normal and cold conditions revealed that NMS-KO and double-KO mice had a significantly low BS-T during the active phase, whereas NMU-KO mice did not. Exposure to low temperature decreased the BS temperature in all KO mice, but BS-T was lower in NMS-KO and double-KO mouse than in NMU-KO mice. Calorie and oxygen consumption was also significantly lower in all KO mice than in wild-type mice during the dark period. These results suggest that NMU and NMS are involved in thermoregulation via the prostaglandin E2 and ß3 adrenergic receptors, but that endogenous NMS might play a more predominant role than NMU.

Structural and functional characterization of neuromedin S in the teleost fish, zebrafish (Danio rerio).

Neuromedin S (NMS) has been demonstrated to have important roles in many vertebrate physiological processes. However, the function of NMS in teleost fishes remains unclear. We explored the physiological roles of the NMS gene in the zebrafish model. An NMS cDNA was cloned from zebrafish brain tissue, and the full-length cDNA sequence was 521 bp in length and encoded a precursor of 110 amino acid residues. Interestingly, fish prepro-NMS is predicted to generate a short 34-residue peptide, designated as NMS-related peptide (NMSRP). Zebrafish prepro-NMS does not contain the NMS peptide which is found in the NMS precursors of mammals, and just retains the MNSRP peptide. A multiple-species sequence alignment showed that NMSRPs are conserved among the other sampled vertebrates. Zebrafish NMS mRNA was detected by RT-PCR revealing a tissue-specific distribution with high levels of expression in the brain, spleen, ovary, pituitary, and muscle. Furthermore, the locations of NMS-expressing cells in the zebrafish brain were detected by in situ hybridization in the parvocellular preoptic nucleus (PPa), the ventral zone of the periventricular hypothalamus (Hv), and lateral hypothalamic nucleus (LH). The levels of NMS mRNA in the hypothalamus were significantly increased after three days of food deprivation. Administration of zebrafish NMSRP by intraperitoneal injection significantly promoted the expression of neuropeptide Y (NPY) and orexin, suggesting an orexigenic role for NMSRP in zebrafish. The present study offers a new understanding of the NMS gene in vertebrates and increases our knowledge of the neuroendocrine regulation of feeding.

Neuromedins U and S involvement in the regulation of the hypothalamo-pituitary-adrenal axis.

We reviewed neuromedin U (NMU) and neuromedin S (NMS) involvement in the regulation of the hypothalamo-pituitary-adrenal (HPA) axis function. NMU and NMS are structurally related and highly conserved neuropeptides. They exert biological effects via two GPCR receptors designated as NMUR1 and NMUR2 which show differential expression. NMUR1 is expressed predominantly at the periphery, while NMUR2 in the central nervous system. Elements of the NMU/NMS and their receptors network are also expressed in the HPA axis and progress in molecular biology techniques provided new information on their actions within this system. Several lines of evidence suggest that within the HPA axis NMU and NMS act at both hypothalamic and adrenal levels. Moreover, new data suggest that NMU and NMS are involved in central and peripheral control of the stress response.

Different distribution of neuromedin S and its mRNA in the rat brain: NMS peptide is present not only in the hypothalamus as the mRNA, but also in the brainstem.

Neuromedin S (NMS) is a neuropeptide identified as another endogenous ligand for two orphan G protein-coupled receptors, FM-3/GPR66 and FM-4/TGR-1, which have also been identified as types 1 and 2 receptors for neuromedin U structurally related to NMS. Although expression of NMS mRNA is found mainly in the brain, spleen, and testis, the distribution of its peptide has not yet been investigated. Using a newly prepared antiserum, we developed a highly sensitive radioimmunoassay for rat NMS. NMS peptide was clearly detected in the rat brain at a concentration of 68.3 ± 3.4 fmol/g wet weight, but it was hardly detected in the spleen and testis. A high content of NMS peptide was found in the hypothalamus, midbrain, and pons-medulla oblongata, whereas abundant expression of NMS mRNA was detected only in the hypothalamus. These differing distributions of the mRNA and peptide suggest that nerve fibers originating from hypothalamic NMS neurons project into the midbrain, pons, or medulla oblongata. In addition, abundant expression of type 2 receptor mRNA was detected not only in the hypothalamus, but also in the midbrain and pons-medulla oblongata. These results suggest novel, unknown physiological roles of NMS within the brainstem.