GLP-1R signaling directly activates arcuate nucleus kisspeptin action in brain slices but does not rescue LH inhibition in OVX mice during negative energy balance

eNeuro

2017 Jan 05

Heppner KM, Baquero AF, Bennett CM, Lindsley SR, Kirigiti MA, Bennett B, Bosch MA, Mercer AJ, Rønnekleiv OK, True C, Grove KL, Smith MS.
PMID: - | DOI: 10.1523/ENEURO.0198-16.2016

Kisspeptin (Kiss1) neurons in the hypothalamic arcuate nucleus (ARC) are key components of the hypothalamic-pituitary-gonadal axis, as they regulate the basal pulsatile release of gonadotropin releasing hormone (GnRH). ARC Kiss1 action is dependent on energy status and unmasking metabolic factors responsible for modulating ARC Kiss1 neurons is of great importance. One possible factor is glucagon-like peptide-1 (GLP-1), an anorexigenic neuropeptide produced by brainstem preproglucagon neurons. As GLP fiber projections and the GLP-1 receptor (GLP-1R) are abundant in the ARC, we hypothesized that GLP-1R signaling could modulate ARC Kiss1 action. Using ovariectomized (OVX) mice, we found that GLP-producing fibers come in close apposition with ARC Kiss1 neurons; these neurons also contain Glp1r mRNA. Electrophysiological recordings revealed that liraglutide (a long-acting GLP-1R agonist) increased action potential firing and caused a direct membrane depolarization of ARC Kiss1 cells in brain slices. We determined that brainstem preproglucagon mRNA is decreased following a 48 h fast in mice, a negative energy state in which ARC Kiss1 expression and downstream GnRH/luteinizing hormone (LH) release are potently suppressed. However, activation of GLP-1R signaling in fasted mice with liraglutide was not sufficient to prevent LH inhibition. Furthermore, chronic central infusions of the GLP-1R antagonist, exendin (9-39) in ad libitum fed mice did not alter ARC Kiss1 mRNA or plasma LH. As a whole, these data identify a novel interaction of the GLP-1 system with ARC Kiss1 neurons but indicate that CNS GLP-1R signaling alone is not critical for the maintenance of LH during fasting or normal feeding.

Significance Statement Reproductive dysfunction is associated with metabolic imbalance, and identifying the underlying molecular mechanisms linking metabolic status with reproductive function is of great importance. Kisspeptin neurons (Kiss1) located in the arcuate nucleus of the hypothalamus (ARC) are essential for fertility and are potently inhibited during negative energy balance; this inhibition occurs in the presence or absence of ovarian steroids. Preproglucagon-expressing neurons located in the brainstem send abundant fiber projections to the ARC where they release the anorexigenic neuropeptide, glucagon-like peptide-1 (GLP-1). The aim of these studies was to determine the interaction of the CNS GLP-1 system with ARC Kiss1 activity to potentially provide a link between systems that control energy balance with those that control reproductive neuroendocrine output.

The endogenous preproglucagon system is not essential for gut growth homeostasis in mice

Molecular Metabolism

2017 Apr 27

Wismann P, Barkholt P, Secher T, Vrang N, Hansen HB, Bekker Jeppesen P, Baggio LL, Koehler JA, Drucker DJ, Sandoval DA, Jelsing J.
PMID: - | DOI: 10.1016/j.molmet.2017.04.007

Abstract

Objective

The prevalence of obesity and related co-morbidities is reaching pandemic proportions. Today, the most effective obesity treatments are glucagon-like peptide 1 (GLP-1) analogs and bariatric surgery. Interestingly, both intervention paradigms have been associated with adaptive growth responses in the gut; however, intestinotrophic mechanisms associated with or secondary to medical or surgical obesity therapies are poorly understood. Therefore, the objective of this study was to assess the local basal endogenous and pharmacological intestinotrophic effects of glucagon-like peptides and bariatric surgery in mice.

Methods

We used in situ hybridization to provide a detailed and comparative anatomical map of the local distribution of GLP-1 receptor (Glp1r), GLP-2 receptor (Glp2r), and preproglucagon (Gcg) mRNA expression throughout the mouse gastrointestinal tract. Gut development in GLP-1R-, GLP-2R-, or GCG-deficient mice was compared to their corresponding wild-type controls, and intestinotrophic effects of GLP-1 and GLP-2 analogs were assessed in wild-type mice. Lastly, gut volume was determined in a mouse model of vertical sleeve gastrectomy (VSG).

Results

Comparison of Glp1r, Glp2r, and Gcg mRNA expression indicated a widespread, but distinct, distribution of these three transcripts throughout all compartments of the mouse gastrointestinal tract. While mice null for Glp1r or Gcg showed normal intestinal morphology, Glp2r^−/− mice exhibited a slight reduction in small intestinal mucosa volume. Pharmacological treatment with GLP-1 and GLP-2 analogs significantly increased gut volume. In contrast, VSG surgery had no effect on intestinal morphology.

Conclusion

The present study indicates that the endogenous preproglucagon system, exemplified by the entire GCG gene and the receptors for GLP-1 and GLP-2, does not play a major role in normal gut development in the mouse. Furthermore, elevation in local intestinal and circulating levels of GLP-1 and GLP-2 achieved after VSG has limited impact on intestinal morphometry. Hence, although exogenous treatment with GLP-1 and GLP-2 analogs enhances gut growth, the contributions of endogenously-secreted GLP-1 and GLP-2 to gut growth may be more modest and highly context-dependent.

GAD2-expression defines a class of excitatory lateral habenula neurons in mice that project to the raphe and pontine tegmentum

eNeuro

2020 Apr 22

Quina LA1, Walker A1, Morton G1, Han V1, Turner EE2,3
PMID: 32332079 | DOI: 10.1523/ENEURO.0527-19.2020

The lateral habenula (LHb) sends complex projections to several areas of the mesopontine tegmentum, the raphe, and the hypothalamus. However, few markers have been available to distinguish subsets of LHb neurons that may serve these pathways. In order to address this complexity, we examined the mouse and rat LHb for neurons that express the GABA biosynthesis enzymes glutamate decarboxylase 1 and 2 (GAD1, GAD2), and the vesicular GABA transporter (VGAT). The mouse LHb contains a population of neurons that express GAD2, while the rat LHb contains discrete populations of neurons that express GAD1 and VGAT. However, we could not detect single neurons in either species that co-express a GABA synthetic enzyme and VGAT, suggesting that these LHb neurons do not use GABA for conventional synaptic transmission. Instead, all of the neuronal types expressing a GABAergic marker in both species showed co-expression of the glutamate transporter VGluT2. Anterograde tract-tracing of the projections of GAD2-expressing LHb neurons in Gad2Cre mice, combined with retrograde tracing from selected downstream nuclei, show that LHb-GAD2 neurons project selectively to the midline structures in the mesopontine tegmentum, including the median raphe and nucleus incertus, and only sparsely innervate the hypothalamus, rostromedial tegmental nucleus, and ventral tegmental area. Postsynaptic recording of LHb-GAD2 neuronal input to tegmental neurons confirms that glutamate, not GABA, is the fast neurotransmitter in this circuit. Thus GAD2 expression can serve as a marker for functional studies of excitatory neurons serving specific LHb output pathways in mice.SIGNFICANCE STATEMENT The lateral habenula provides a major link between subcortical forebrain areas and the dopamine (DA) and serotonin (5HT) systems of the midbrain and pons, and it has been implicated in reward mechanisms and the regulation of mood states. Few markers have been available for the specific cell types and complex output pathways of the lateral habenula. Here we examined the expression of genes mediating GABAergic and glutamatergic transmission in the mouse and rat LHb, where no neurons in either species expressed a full complement of GABAergic markers, and all expressed the glutamatergic marker VGluT2. Consistent with this, in mice the LHb GAD2 neurons are excitatory and appear to use only glutamate for fast synaptic transmission.

Expression of type one cannabinoid receptor in different subpopulation of kisspeptin neurons and kisspeptin afferents to GnRH neurons in female mice

Brain structure & function

2021 Jul 14

Wilheim, T;Nagy, K;Mohanraj, M;Ziarniak, K;Watanabe, M;Sliwowska, J;Kalló, I;
PMID: 34263407 | DOI: 10.1007/s00429-021-02339-z

The endocannabinoids have been shown to target the afferents of hypothalamic neurons via cannabinoid 1 receptor (CB1) and thereby to influence their excitability at various physiological and/or pathological processes. Kisspeptin (KP) neurons form afferents of multiple neuroendocrine cells and influence their activity via signaling through a variation of co-expressed classical neurotransmitters and neuropeptides. The differential potency of endocannabinoids to influence the release of classical transmitters or neuropeptides, and the ovarian cycle-dependent functioning of the endocannabinoid signaling in the gonadotropin-releasing hormone (GnRH) neurons initiated us to study whether (a) the different subpopulations of KP neurons express CB1 mRNAs, (b) the expression is influenced by estrogen, and (c) CB1-immunoreactivity is present in the KP afferents to GnRH neurons. The aim of the study was to investigate the site- and cell-specific expression of CB1 in female mice using multiple labeling in situ hybridization and immunofluorescent histochemical techniques. The results support that CB1 mRNAs are expressed by both the GABAergic and glutamatergic subpopulations of KP neurons, the receptor protein is detectable in two-thirds of the KP afferents to GnRH neurons, and the expression of CB1 mRNA shows an estrogen-dependency. The applied estrogen-treatment, known to induce proestrus, reduced the level of CB1 transcripts in the rostral periventricular area of the third ventricle and arcuate nucleus, and differently influenced its co-localization with vesicular GABA transporter or vesicular glutamate transporter-2 in KP neurons. This indicates a gonadal cycle-dependent role of endocannabinoid signaling in the neuronal circuits involving KP neurons.

Osmoadaptive GLP-1R signalling in hypothalamic neurones inhibits antidiuretic hormone synthesis and release

Molecular metabolism

2023 Feb 10

Greenwood, MP;Greenwood, M;Bárez-López, S;Hawkins, JW;Short, K;Tatovic, D;Murphy, D;
PMID: 36773648 | DOI: 10.1016/j.molmet.2023.101692

The excessive release of the antidiuretic hormone vasopressin is implicated in many diseases including cardiovascular disease, diabetes, obesity, and metabolic syndrome. Once thought to be elevated as a consequence of diseases, data now supports a more causative role. We have previously identified CREB3L1 as a transcription factor that co-ordinates vasopressin synthesis and release in the hypothalamus. The objective here was to identify mechanisms orchestrated by CREB3L1 that co-ordinate vasopressin release.We mined Creb3l1 knockdown SON RNA-seq data to identify downstream target genes. We proceeded to investigate the expression of these genes and associated pathways in the supraoptic nucleus of the hypothalamus in response to physiological and pharmacological stimulation. We used viruses to selectively knockdown gene expression in the supraoptic nucleus and assessed physiological and metabolic parameters. We adopted a phosphoproteomics strategy to investigate mechanisms that facilitate hormone release by the pituitary gland.We discovered glucagon like peptide 1 receptor (Glp1r) as a downstream target gene and found increased expression in stimulated vasopressin neurones. Selective knockdown of supraoptic nucleus Glp1rs resulted in decreased food intake and body weight. Treatment with GLP-1R agonist liraglutide decreased vasopressin synthesis and release. Quantitative phosphoproteomics of the pituitary neurointermediate lobe revealed that liraglutide initiates hyperphosphorylation of presynapse active zone proteins that control vasopressin exocytosis.In summary, we show that GLP-1R signalling inhibits the vasopressin system. Our data advises that hydration status may influence the pharmacodynamics of GLP-1R agonists so should be considered in current therapeutic strategies.

Involvement of the ghrelin system in the maintenance and reinstatement of cocaine-motivated behaviors: a role of adrenergic action at peripheral β1 receptors

Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology

2021 Dec 18

You, ZB;Galaj, E;Alén, F;Wang, B;Bi, GH;Moore, AR;Buck, T;Crissman, M;Pari, S;Xi, ZX;Leggio, L;Wise, RA;Gardner, EL;
PMID: 34923576 | DOI: 10.1038/s41386-021-01249-2

Cocaine addiction is a significant medical and public concern. Despite decades of research effort, development of pharmacotherapy for cocaine use disorder remains largely unsuccessful. This may be partially due to insufficient understanding of the complex biological mechanisms involved in the pathophysiology of this disorder. In the present study, we show that: (1) elevation of ghrelin by cocaine plays a critical role in maintenance of cocaine self-administration and cocaine-seeking motivated by cocaine-conditioned stimuli; (2) acquisition of cocaine-taking behavior is associated with the acquisition of stimulatory effects of cocaine by cocaine-conditioned stimuli on ghrelin secretion, and with an upregulation of ghrelin receptor mRNA levels in the ventral tegmental area (VTA); (3) blockade of ghrelin signaling by pretreatment with JMV2959, a selective ghrelin receptor antagonist, dose-dependently inhibits reinstatement of cocaine-seeking triggered by either cocaine or yohimbine in behaviorally extinguished animals with a history of cocaine self-administration; (4) JMV2959 pretreatment also inhibits brain stimulation reward (BSR) and cocaine-potentiated BSR maintained by optogenetic stimulation of VTA dopamine neurons in DAT-Cre mice; (5) blockade of peripheral adrenergic β1 receptors by atenolol potently attenuates the elevation in circulating ghrelin induced by cocaine and inhibits cocaine self-administration and cocaine reinstatement triggered by cocaine. These findings demonstrate that the endogenous ghrelin system plays an important role in cocaine-related addictive behaviors and suggest that manipulating and targeting this system may be viable for mitigating cocaine use disorder.

GLP-1 neurons form a local synaptic circuit within the rodent nucleus of the solitary tract.

J Comp Neurol.

2018 Jul 17

Patrick Card J, Johnson AL, Llewellyn-Smith IJ, Zheng H, Anand R, Brierley DI, Trapp S, Rinaman L.
PMID: 30019398 | DOI: 10.1002/cne.24482

Glutamatergic neurons that express pre-proglucagon (PPG) and are immunopositive (+) for glucagon-like peptide-1 (i.e., GLP-1+ neurons) are located within the caudal nucleus of the solitary tract (cNTS) and medullary reticular formation in rats and mice. GLP-1 neurons give rise to an extensive central network in which GLP-1 receptor (R) signaling suppresses food intake, attenuates rewarding, increases avoidance, and stimulates stress responses, partly via . GLP-1R signaling within the cNTS. In mice, noradrenergic (A2) cNTS neurons express GLP-1R, whereas PPG neurons do not. In the present study, confocal microscopy in rats confirmed that prolactin-releasing peptide (PrRP)+ A2 neurons are closely apposed by GLP-1+ axonal varicosities. Surprisingly, GLP-1+ appositions were also observed on dendrites of PPG/GLP-1+ neurons in both species, and electron microscopy in rats revealed that GLP-1+ boutons form asymmetric synaptic contacts with GLP-1+ dendrites. However, RNAscope confirmed that rat GLP-1 neurons do not express GLP-1R mRNA. Similarly, Ca2+ imaging of somatic and dendritic responses in mouse ex vivo slices confirmed that PPG neurons do not respond directly to GLP-1, and a mouse cross-breeding strategy revealed that fewer than 1% of PPG neurons co-express GLP-1R. Collectively, these data suggest that GLP-1R signaling pathways modulate the activity of PrRP+ A2 neurons, and also reveal a local "feed-forward" synaptic network among GLP-1 neurons that apparently does not utilize GLP-1R signaling. This local GLP-1 network may instead use glutamatergic signaling to facilitate dynamic and potentially selective recruitment of GLP-1 neural populations that shape behavioral and physiological responses to internal and external challenges.

Neurons innervating both the central amygdala and the ventral tegmental area encode different emotional valences

Frontiers in neuroscience

2023 May 05

Liu, A;Cheng, Y;Huang, J;
PMID: 37214399 | DOI: 10.3389/fnins.2023.1178693

Mammals are frequently exposed to various environmental stimuli, and to determine whether to approach or avoid these stimuli, the brain must assign emotional valence to them. Therefore, it is crucial to investigate the neural circuitry mechanisms involved in the mammalian brain's processing of emotional valence. Although the central amygdala (CeA) and the ventral tegmental area (VTA) individually encode different or even opposing emotional valences, it is unclear whether there are common upstream input neurons that innervate and control both these regions, and it is interesting to know what emotional valences of these common upstream neurons. In this study, we identify three major brain regions containing neurons that project to both the CeA and the VTA, including the posterior bed nucleus of the stria terminalis (pBNST), the pedunculopontine tegmental nucleus (PPTg), and the anterior part of the basomedial amygdala (BMA). We discover that these neural populations encode distinct emotional valences. Activating neurons in the pBNST produces positive valence, enabling mice to overcome their innate avoidance behavior. Conversely, activating neurons in the PPTg produces negative valence and induces anxiety-like behaviors in mice. Neuronal activity in the BMA, on the other hand, does not influence valence processing. Thus, our study has discovered three neural populations that project to both the CeA and the VTA and has revealed the distinct emotional valences these populations encode. These results provide new insights into the neurological mechanisms involved in emotional regulation.

Edinger-Westphal peptidergic neurons enable maternal preparatory nesting

Neuron

2022 Feb 01

Topilko, T;Diaz, SL;Pacheco, CM;Verny, F;Rousseau, CV;Kirst, C;Deleuze, C;Gaspar, P;Renier, N;
PMID: 35123655 | DOI: 10.1016/j.neuron.2022.01.012

Optimizing reproductive fitness in mammalians requires behavioral adaptations during pregnancy. Maternal preparatory nesting is an essential behavior for the survival of the upcoming litter. Brain-wide immediate early gene mapping in mice evoked by nesting sequences revealed that phases of nest construction strongly activate peptidergic neurons of the Edinger-Westphal nucleus in pregnant mice. Genetic ablation, bidirectional neuromodulation, and in vitro and in vivo activity recordings demonstrated that these neurons are essential to modulate arousal before sleep to promote nesting specifically. We show that these neurons enable the behavioral effects of progesterone on preparatory nesting by modulating a broad network of downstream targets. Our study deciphers the role of midbrain CART+ neurons in behavioral adaptations during pregnancy vital for reproductive fitness.

Proglucagon signalling in the rat Dorsomedial Hypothalamus- Physiology and high-fat diet-mediated alterations

Molecular and cellular neurosciences

2023 Jun 08

Sanetra, AM;Palus-Chramiec, K;Chrobok, L;Jeczmien-Lazur, JS;Klich, JD;Lewandowski, MH;
PMID: 37295578 | DOI: 10.1016/j.mcn.2023.103873

A relatively new pharmacological target in obesity treatment has been the preproglucagon (PPG) signalling, predominantly with glucagon-like peptide (GLP) 1 receptor agonists. As far as the PPG role within the digestive system is well recognised, its actions in the brain remain understudied. Here, we investigated PPG signalling in the Dorsomedial Hypothalamus (DMH), a structure involved in feeding regulation and metabolism, using in situ hybridisation, electrophysiology, and immunohistochemistry. Our experiments were performed on animals fed both control, and high-fat diet (HFD), uncovering HFD-mediated alterations. First, sensitivity to exendin-4 (Exn4, a GLP1R agonist) was shown to increase under HFD, with a higher number of responsive neurons. The amplitude of the response to both Exn4 and oxyntomodulin (Oxm) was also altered, diminishing its relationship with the cells' spontaneous firing rate. Not only neuronal sensitivity, but also GLP1 presence, and therefore possibly release, was influenced by HFD. Immunofluorescent labelling of the GLP1 showed changes in its density depending on the metabolic state (fasted/fed), but this effect was eliminated by HFD feeding. Interestingly, these dietary differences were absent after a period of restricted feeding, allowing for an anticipation of the alternating metabolic states, which suggests possible prevention of such outcome.

Lateral septum as a melanocortin downstream site in obesity development

Cell reports

2023 May 11

Xu, Y;Jiang, Z;Li, H;Cai, J;Jiang, Y;Otiz-Guzman, J;Xu, Y;Arenkiel, BR;Tong, Q;
PMID: 37171957 | DOI: 10.1016/j.celrep.2023.112502

The melanocortin pathway is well established to be critical for body-weight regulation in both rodents and humans. Despite extensive studies focusing on this pathway, the downstream brain sites that mediate its action are not clear. Here, we found that, among the known paraventricular hypothalamic (PVH) neuron groups, those expressing melanocortin receptors 4 (PVHMc4R) preferably project to the ventral part of the lateral septum (LSv), a brain region known to be involved in emotional behaviors. Photostimulation of PVHMc4R neuron terminals in the LSv reduces feeding and causes aversion, whereas deletion of Mc4Rs or disruption of glutamate release from LSv-projecting PVH neurons causes obesity. In addition, disruption of AMPA receptor function in PVH-projected LSv neurons causes obesity. Importantly, chronic inhibition of PVH- or PVHMc4R-projected LSv neurons causes obesity associated with reduced energy expenditure. Thus, the LSv functions as an important node in mediating melanocortin action on body-weight regulation.

Postnatal age-differential ASD-like transcriptomic, synaptic, and behavioral deficits in Myt1l-mutant mice

Cell reports

2022 Sep 20

Kim, S;Oh, H;Choi, SH;Yoo, YE;Noh, YW;Cho, Y;Im, GH;Lee, C;Oh, Y;Yang, E;Kim, G;Chung, WS;Kim, H;Kang, H;Bae, Y;Kim, SG;Kim, E;
PMID: 36130507 | DOI: 10.1016/j.celrep.2022.111398

Myelin transcription factor 1 like (Myt1l), a zinc-finger transcription factor, promotes neuronal differentiation and is implicated in autism spectrum disorder (ASD) and intellectual disability. However, it remains unclear whether Myt1l promotes neuronal differentiation in vivo and its deficiency in mice leads to disease-related phenotypes. Here, we report that Myt1l-heterozygous mutant (Myt1l-HT) mice display postnatal age-differential ASD-related phenotypes: newborn Myt1l-HT mice, with strong Myt1l expression, show ASD-like transcriptomic changes involving decreased synaptic gene expression and prefrontal excitatory synaptic transmission and altered righting reflex. Juvenile Myt1l-HT mice, with markedly decreased Myt1l expression, display reverse ASD-like transcriptomes, increased prefrontal excitatory transmission, and largely normal behaviors. Adult Myt1l-HT mice show ASD-like transcriptomes involving astrocytic and microglial gene upregulation, increased prefrontal inhibitory transmission, and behavioral deficits. Therefore, Myt1l haploinsufficiency leads to ASD-related phenotypes in newborn mice, which are temporarily normalized in juveniles but re-appear in adults, pointing to continuing phenotypic changes long after a marked decrease of Myt1l expression in juveniles.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
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tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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