Mind affects matter: Hindbrain GLP1 neurons link stress, physiology and behaviour.

NEW FINDINGS: What is the topic of this review? This Lecture covers the role of caudal brainstem GLP1 neurons in acute and chronic stress responses. What advances does it highlight? This Lecture focuses on the recent advances in our understanding of GLP1 neurons and their physiological role in many aspects of stress. Particular focus is given to the recent elucidation, in part, of the anatomical basis for recruitment of GLP1 neurons in response to acute stress. Finally, the potential, but at this time somewhat speculative, role of GLP1 neurons in chronic stress is discussed. ABSTRACT: The brain responds rapidly to stressful stimuli by increasing sympathetic outflow, activating the hypothalamic-pituitary-adrenal axis and eliciting avoidance behaviours to limit risks to safety. Stress responses are adaptive and essential but can become maladaptive when the stress is chronic, causing autonomic imbalance, hypothalamic-pituitary-adrenal axis hyper-reactivity and a state of hypervigilance. Ultimately, this contributes to the development of cardiovascular disease and affective disorders, including major depression and anxiety. Stress responses are often thought to be driven mainly by forebrain areas; however, the brainstem nucleus of the solitary tract (NTS) is ideally located to control both autonomic outflow and behaviour in response to stress. Here, I review the preclinical evidence that the NTS and its resident glucagon-like peptide-1 (GLP1)-expressing neurons are prominent mediators of stress responses. This Lecture introduces the reader to the idea of good and bad stress and outlines the types of stress that engage the NTS and GLP1 neurons. I describe in particular detail the recent studies by myself and others aimed at mapping sources of synaptic inputs to GLP1 neurons and consider the implications for our understanding of the role of GLP1 neurons in stress. This is followed by a discussion of the contribution of brain GLP1 and GLP1 neurons to behavioural and physiological stress responses. The evidence reviewed highlights a potentially prominent role for GLP1 neurons in the response of the brain to acute stress and reveals important unanswered questions regarding their role in chronic stress.

Synaptic Inputs to the Mouse Dorsal Vagal Complex and Its Resident Preproglucagon Neurons.

Stress responses are coordinated by widespread neural circuits. Homeostatic and psychogenic stressors activate preproglucagon (PPG) neurons in the caudal nucleus of the solitary tract (cNTS) that produce glucagon-like peptide-1; published work in rodents indicates that these neurons play a crucial role in stress responses. While the axonal targets of PPG neurons are well established, their afferent inputs are unknown. Here we use retrograde tracing with cholera toxin subunit b to show that the cNTS in male and female mice receives axonal inputs similar to those reported in rats. Monosynaptic and polysynaptic inputs specific to cNTS PPG neurons were revealed using Cre-conditional pseudorabies and rabies viruses. The most prominent sources of PPG monosynaptic input include the lateral (LH) and paraventricular (PVN) nuclei of the hypothalamus, parasubthalamic nucleus, lateral division of the central amygdala, and Barrington's nucleus (Bar). Additionally, PPG neurons receive monosynaptic vagal sensory input from the nodose ganglia and spinal sensory input from the dorsal horn. Sources of polysynaptic input to cNTS PPG neurons include the hippocampal formation, paraventricular thalamus, and prefrontal cortex. Finally, cNTS-projecting neurons within PVN, LH, and Bar express the activation marker cFOS in mice after restraint stress, identifying them as potential sources of neurogenic stress-induced recruitment of PPG neurons. In summary, cNTS PPG neurons in mice receive widespread monosynaptic and polysynaptic input from brain regions implicated in coordinating behavioral and physiological stress responses, as well as from vagal and spinal sensory neurons. Thus, PPG neurons are optimally positioned to integrate signals of homeostatic and psychogenic stress.SIGNIFICANCE STATEMENT Recent research has indicated a crucial role for glucagon-like peptide-1-producing preproglucagon (PPG) neurons in regulating both appetite and behavioral and autonomic responses to acute stress. Intriguingly, the central glucagon-like peptide-1 system defined in rodents is conserved in humans, highlighting the translational importance of understanding its anatomical organization. Findings reported here indicate that PPG neurons receive significant monosynaptic and polysynaptic input from brain regions implicated in autonomic and behavioral responses to stress, as well as direct input from vagal and spinal sensory neurons. Improved understanding of the neural pathways underlying the recruitment of PPG neurons may facilitate the development of novel therapies for the treatment of stress-related disorders.

Preproglucagon neurons in the hindbrain have IL-6 receptor-α and show Ca2+ influx in response to IL-6.

Neuronal circuits in the hypothalamus and hindbrain are of importance for control of food intake, energy expenditure, and fat mass. We have recently shown that treatment with exendin-4 (Ex-4), an analog of the proglucagon-derived molecule glucagon-like peptide 1 (GLP-1), markedly increases mRNA expression of the cytokine interleukin-6 (IL-6) in the hypothalamus and hindbrain and that this increase partly mediates the suppression of food intake and body weight by Ex-4. Endogenous GLP-1 in the central nervous system (CNS) is produced by preproglucagon (PPG) neurons of the nucleus of the solitary tract (NTS) in the hindbrain. These neurons project to various parts of the brain, including the hypothalamus. Outside the brain, IL-6 stimulates GLP-1 secretion from the gut and pancreas. In this study, we aim to investigate whether IL-6 can affect GLP-1-producing PPG neurons in the nucleus of the solitary tract (NTS) in mouse hindbrain via the ligand binding part of the IL-6 receptor, IL-6 receptor-α (IL-6Rα). Using immunohistochemistry, we found that IL-6Rα was localized on PPG neurons of the NTS. Recordings of these neurons in GCaMP3/GLP-1 reporter mice showed that IL-6 enhances cytosolic Ca(2+) concentration in neurons capable of expressing PPG. We also show that the Ca(2+) increase originates from the extracellular space. Furthermore, we found that IL-6Rα was localized on cells in the caudal hindbrain expressing immunoreactive NeuN (a neuronal marker) or CNP:ase (an oligodendrocyte marker). In summary, IL-6Rα is present on PPG neurons in the NTS, and IL-6 can stimulate these cells by increasing influx of Ca(2+) to the cytosol from the extracellular space.

The ins and outs of the caudal nucleus of the solitary tract: An overview of cellular populations and anatomical connections.

The body and brain are in constant two-way communication. Driving this communication is a region in the lower brainstem: the dorsal vagal complex. Within the dorsal vagal complex, the caudal nucleus of the solitary tract (cNTS) is a major first stop for incoming information from the body to the brain carried by the vagus nerve. The anatomy of this region makes it ideally positioned to respond to signals of change in both emotional and bodily states. In turn, the cNTS controls the activity of regions throughout the brain that are involved in the control of both behaviour and physiology. This review is intended to help anyone with an interest in the cNTS. First, I provide an overview of the architecture of the cNTS and outline the wide range of neurotransmitters expressed in subsets of neurons in the cNTS. Next, in detail, I discuss the known inputs and outputs of the cNTS and briefly highlight what is known regarding the neurochemical makeup and function of those connections. Then, I discuss one group of cNTS neurons: glucagon-like peptide-1 (GLP-1)-expressing neurons. GLP-1 neurons serve as a good example of a group of cNTS neurons, which receive input from varied sources and have the ability to modulate both behaviour and physiology. Finally, I consider what we might learn about other cNTS neurons from our study of GLP-1 neurons and why it is important to remember that the manipulation of molecularly defined subsets of cNTS neurons is likely to affect physiology and behaviours beyond those monitored in individual experiments.

The role of nucleus of the solitary tract glucagon-like peptide-1 and prolactin-releasing peptide neurons in stress: anatomy, physiology and cellular interactions.

Neuroendocrine, behavioural and autonomic responses to stressful stimuli are orchestrated by complex neural circuits. The caudal nucleus of the solitary tract (cNTS) in the dorsomedial hindbrain is uniquely positioned to integrate signals of both interoceptive and psychogenic stress. Within the cNTS, glucagon-like peptide-1 (GLP-1) and prolactin-releasing peptide (PrRP) neurons play crucial roles in organising neural responses to a broad range of stressors. In this review we discuss the anatomical and functional overlap between PrRP and GLP-1 neurons. We outline their co-activation in response to stressful stimuli and their importance as mediators of behavioural and physiological stress responses. Finally, we review evidence that PrRP neurons are downstream of GLP-1 neurons and outline unexplored areas of the research field. Based on the current state-of-knowledge, PrRP and GLP-1 neurons may be compelling targets in the treatment of stress-related disorders. LINKED ARTICLES: This article is part of a themed issue on GLP1 receptor ligands (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.4/issuetoc.

Central and peripheral GLP-1 systems independently suppress eating.

The anorexigenic peptide glucagon-like peptide-1 (GLP-1) is secreted from gut enteroendocrine cells and brain preproglucagon (PPG) neurons, which, respectively, define the peripheral and central GLP-1 systems. PPG neurons in the nucleus tractus solitarii (NTS) are widely assumed to link the peripheral and central GLP-1 systems in a unified gut-brain satiation circuit. However, direct evidence for this hypothesis is lacking, and the necessary circuitry remains to be demonstrated. Here we show that PPGNTS neurons encode satiation in mice, consistent with vagal signalling of gastrointestinal distension. However, PPGNTS neurons predominantly receive vagal input from oxytocin-receptor-expressing vagal neurons, rather than those expressing GLP-1 receptors. PPGNTS neurons are not necessary for eating suppression by GLP-1 receptor agonists, and concurrent PPGNTS neuron activation suppresses eating more potently than semaglutide alone. We conclude that central and peripheral GLP-1 systems suppress eating via independent gut-brain circuits, providing a rationale for pharmacological activation of PPGNTS neurons in combination with GLP-1 receptor agonists as an obesity treatment strategy.

PPG neurons in the nucleus of the solitary tract modulate heart rate but do not mediate GLP-1 receptor agonist-induced tachycardia in mice.

OBJECTIVE: Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are used as anti-diabetic drugs and are approved for obesity treatment. However, GLP-1RAs also affect heart rate (HR) and arterial blood pressure (ABP) in rodents and humans. Although the activation of GLP-1 receptors (GLP-1R) is known to increase HR, the circuits recruited are unclear, and in particular, it is unknown whether GLP-1RAs activate preproglucagon (PPG) neurons, the brain source of GLP-1, to elicit these effects. METHODS: We investigated the effect of GLP-1RAs on heart rate in anaesthetized adult mice. In a separate study, we manipulated the activity of nucleus tractus solitarius (NTS) PPG neurons (PPGNTS) in awake, freely behaving transgenic Glu-Cre mice implanted with biotelemetry probes and injected with AAV-DIO-hM3Dq:mCherry or AAV-mCherry-FLEX-DTA. RESULTS: Systemic administration of the GLP-1RA Ex-4 increased resting HR in anaesthetized or conscious mice, but had no effect on ABP in conscious mice. This effect was abolished by ß-adrenoceptor blockade with atenolol, but unaffected by the muscarinic antagonist atropine. Furthermore, Ex-4-induced tachycardia persisted when PPGNTS neurons were ablated, and Ex-4 did not induce expression of the neuronal activity marker cFos in PPGNTS neurons. PPGNTS ablation or acute chemogenetic inhibition of these neurons via hM4Di receptors had no effect on resting HR. In contrast, chemogenetic activation of PPGNTS neurons increased resting HR. Furthermore, the application of GLP-1 within the subarachnoid space of the middle thoracic spinal cord, a major projection target of PPG neurons, increased HR. CONCLUSIONS: These results demonstrate that both systemic application of Ex-4 or GLP-1 and chemogenetic activation of PPGNTS neurons increases HR. Ex-4 increases the activity of cardiac sympathetic preganglionic neurons of the spinal cord without recruitment of PPGNTS neurons, and thus likely recapitulates the physiological effects of PPG neuron activation. These neurons therefore do not play a significant role in controlling resting HR and ABP but are capable of inducing tachycardia and so are likely involved in cardiovascular responses to acute stress.

Endogenous GLP-1 in lateral septum promotes satiety and suppresses motivation for food in mice.

Glucagon-like peptide 1 receptors (GLP-1R) are expressed in the lateral septum (LS) of rats and mice, and we have published that endogenous LS GLP-1 affects feeding and motivation for food in rats. Here we asked if these effects are also observed in mice. In separate dose-response studies using male C57Bl6J mice, intra-LS GLP-1 or the GLP-1R antagonist Exendin 9 (Ex9) was delivered shortly before dark onset, at doses subthreshold for effect when injected intracerebroventricularly (icv). Intra-LS GLP-1 significantly suppressed chow intake early in the dark phase and tended to reduce overnight intake. However, blockade of LS GLP-1R with Ex9 had no effect on ad libitum dark onset chow intake. We then asked if LS GLP-1R blockade blunts nutrient preload-induced intake suppression. Mice were trained to consume Ensure immediately before dark onset, which suppressed subsequent chow intake, and intra-LS Ex9 attenuated that preload-induced intake suppression. We also found that restraint stress robustly activates hindbrain GLP-1-producing neurons, and that LS GLP-1R blockade attenuates 30-min restraint stress-induced hypophagia in mice. Furthermore, we have reported that in the rat, GLP-1R in the dorsal subregion of the LS (dLS) affect motivation for food. We examined this in food-restricted mice responding for sucrose pellets on a progressive ratio (PR) schedule. Intra-dLS GLP-1R stimulation significantly suppressed, and Ex9 significantly increased, operant responding, and the Ex9 effect remained after mice returned to ad libitum conditions. Similarly, we found that stimulation of dLS GLP-1 suppressed licking for sucrose and conversely, Ex9 increased licking under ad libitum feeding conditions. Together, our data suggest that endogenous activation of LS GLP-1R plays a role in feeding in mice under some but not all conditions, and that these receptors strongly influence motivation for food.

Preproglucagon Neurons in the Nucleus of the Solitary Tract Are the Main Source of Brain GLP-1, Mediate Stress-Induced Hypophagia, and Limit Unusually Large Intakes of Food.

Centrally administered glucagon-like peptide 1 (GLP-1) supresses food intake. Here we demonstrate that GLP-1-producing (PPG) neurons in the nucleus tractus solitarii (NTS) are the predominant source of endogenous GLP-1 within the brain. Selective ablation of NTS PPG neurons by viral expression of diphtheria toxin subunit A substantially reduced active GLP-1 concentrations in brain and spinal cord. Contrary to expectations, this loss of central GLP-1 had no significant effect on the ad libitum feeding of mice, affecting neither daily chow intake nor body weight or glucose tolerance. Only after bigger challenges to homeostasis were PPG neurons necessary for food intake control. PPG-ablated mice increased food intake after a prolonged fast and after a liquid diet preload. Consistent with our ablation data, acute inhibition of hM4Di-expressing PPG neurons did not affect ad libitum feeding; however, it increased refeeding intake after fast and blocked stress-induced hypophagia. Additionally, chemogenetic PPG neuron activation through hM3Dq caused a strong acute anorectic effect. We conclude that PPG neurons are not involved in primary intake regulation but form part of a secondary satiation/satiety circuit, which is activated by both psychogenic stress and large meals. Given their hypophagic capacity, PPG neurons might be an attractive drug target in obesity treatment.

Serotonergic modulation of the activity of GLP-1 producing neurons in the nucleus of the solitary tract in mouse.

OBJECTIVE: Glucagon-like peptide-1 (GLP-1) and 5-HT are potent regulators of food intake within the brain. GLP-1 is expressed by preproglucagon (PPG) neurons in the nucleus tractus solitarius (NTS). We have previously shown that PPG neurons innervate 5-HT neurons in the ventral brainstem. Here, we investigate whether PPG neurons receive serotonergic input and respond to 5-HT. METHODS: We employed immunohistochemistry to reveal serotonergic innervation of PPG neurons. We investigated the responsiveness of PPG neurons to 5-HT using in vitro Ca2+ imaging in brainstem slices from transgenic mice expressing the Ca2+ indicator, GCaMP3, in PPG neurons, and cell-attached patch-clamp recordings. RESULTS: Close appositions from 5-HT-immunoreactive axons occurred on many PPG neurons. Application of 20 µM 5-HT produced robust Ca2+ responses in NTS PPG dendrites but little change in somata. Dendritic Ca2+ spikes were concentration-dependent (2, 20, and 200 µM) and unaffected by blockade of glutamatergic transmission, suggesting 5-HT receptors on PPG neurons. Neither activation nor blockade of 5-HT3 receptors affected [Ca2+]i. In contrast, inhibition of 5-HT2 receptors attenuated increases in intracellular Ca2+ and 5-HT2C receptor activation produced Ca2+ spikes. Patch-clamp recordings revealed that 44% of cells decreased their firing rate under 5-HT, an effect blocked by 5-HT1A receptor antagonism. CONCLUSIONS: PPG neurons respond directly to 5-HT with a 5-HT2C receptor-dependent increase in dendritic [Ca2+]i. Electrical responses to 5-HT revealed additional inhibitory effects due to somatic 5-HT1A receptors. Reciprocal innervation between 5-HT and PPG neurons suggests that the coordinated activity of these brainstem neurons may play a role in the regulation of food intake.

The physiological role of the brain GLP-1 system in stress.

Glucagon-like peptide-1 (GLP-1) within the brain is a potent regulator of food intake and most studies have investigated the anorexic effects of central GLP-1. A range of brain regions have now been found to be involved in GLP-1 mediated anorexia, including some which are not traditionally associated with appetite regulation. However, a change in food intake can be indicative of not only reduced energy demand, but also changes in the organism's motivation to eat following stressful stimuli. In fact, acute stress is well-known to reduce food intake. Recently, more research has focused on the role of GLP-1 in stress and the central GLP-1 system has been found to be activated in response to stressful stimuli. The source of GLP-1 within the brain, the preproglucagon (PPG) neurons, are ideally situated in the brainstem to receive and relay signals of stress and our recent data on the projection pattern of the PPG neurons to the spinal cord suggest a potential strong link with the sympathetic nervous system. We review here the role of central GLP-1 in the regulation of stress responses and discuss the potential involvement of the endogenous source of GLP-1 within the brain, the PPG neurons.

Distribution and characterisation of Glucagon-like peptide-1 receptor expressing cells in the mouse brain.

OBJECTIVE: Although Glucagon-like peptide 1 is a key regulator of energy metabolism and food intake, the precise location of GLP-1 receptors and the physiological relevance of certain populations is debatable. This study investigated the novel GLP-1R-Cre mouse as a functional tool to address this question. METHODS: Mice expressing Cre-recombinase under the Glp1r promoter were crossed with either a ROSA26 eYFP or tdRFP reporter strain to identify GLP-1R expressing cells. Patch-clamp recordings were performed on tdRFP-positive neurons in acute coronal brain slices from adult mice and selective targeting of GLP-1R cells in vivo was achieved using viral gene delivery. RESULTS: Large numbers of eYFP or tdRFP immunoreactive cells were found in the circumventricular organs, amygdala, hypothalamic nuclei and the ventrolateral medulla. Smaller numbers were observed in the nucleus of the solitary tract and the thalamic paraventricular nucleus. However, tdRFP positive neurons were also found in areas without preproglucagon-neuronal projections like hippocampus and cortex. GLP-1R cells were not immunoreactive for GFAP or parvalbumin although some were catecholaminergic. GLP-1R expression was confirmed in whole-cell recordings from BNST, hippocampus and PVN, where 100 nM GLP-1 elicited a reversible inward current or depolarisation. Additionally, a unilateral stereotaxic injection of a cre-dependent AAV into the PVN demonstrated that tdRFP-positive cells express cre-recombinase facilitating virally-mediated eYFP expression. CONCLUSIONS: This study is a comprehensive description and phenotypic analysis of GLP-1R expression in the mouse CNS. We demonstrate the power of combining the GLP-1R-CRE mouse with a virus to generate a selective molecular handle enabling future in vivo investigation as to their physiological importance.

PI3K/mTORC2 regulates TGF-ß/Activin signalling by modulating Smad2/3 activity via linker phosphorylation.

Crosstalk between the phosphatidylinositol 3-kinase (PI3K) and the transforming growth factor-ß signalling pathways play an important role in regulating many cellular functions. However, the molecular mechanisms underpinning this crosstalk remain unclear. Here, we report that PI3K signalling antagonizes the Activin-induced definitive endoderm (DE) differentiation of human embryonic stem cells by attenuating the duration of Smad2/3 activation via the mechanistic target of rapamycin complex 2 (mTORC2). Activation of mTORC2 regulates the phosphorylation of the Smad2/3-T220/T179 linker residue independent of Akt, CDK and Erk activity. This phosphorylation primes receptor-activated Smad2/3 for recruitment of the E3 ubiquitin ligase Nedd4L, which in turn leads to their degradation. Inhibition of PI3K/mTORC2 reduces this phosphorylation and increases the duration of Smad2/3 activity, promoting a more robust mesendoderm and endoderm differentiation. These findings present a new and direct crosstalk mechanism between these two pathways in which mTORC2 functions as a novel and critical mediator.