Unlocking opioid neuropeptide dynamics with genetically encoded biosensors.

Neuropeptides are ubiquitous in the nervous system. Research into neuropeptides has been limited by a lack of experimental tools that allow for the precise dissection of their complex and diverse dynamics in a circuit-specific manner. Opioid peptides modulate pain, reward and aversion and as such have high clinical relevance. To illuminate the spatiotemporal dynamics of endogenous opioid signaling in the brain, we developed a class of genetically encoded fluorescence sensors based on kappa, delta and mu opioid receptors: κLight, δLight and µLight, respectively. We characterized the pharmacological profiles of these sensors in mammalian cells and in dissociated neurons. We used κLight to identify electrical stimulation parameters that trigger endogenous opioid release and the spatiotemporal scale of dynorphin volume transmission in brain slices. Using in vivo fiber photometry in mice, we demonstrated the utility of these sensors in detecting optogenetically driven opioid release and observed differential opioid release dynamics in response to fearful and rewarding conditions.

Effects of chronic high fat diet on mediobasal hypothalamic satiety neuron function in POMC-Cre mice.

OBJECTIVE: The prevalence of obesity has increased over the past three decades. Proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (ARC) play a vital role in induction of satiety. Chronic consumption of high-fat diet is known to reduce hypothalamic neuronal sensitivity to hormones like leptin, thus contributing to the development and persistence of obesity. The functional and morphological effects of a high-calorie diet on POMC neurons and how these effects contribute to the development and maintenance of the obese phenotype are not fully understood. For this purpose, POMC-Cre transgenic mice model was exposed to high-fat diet (HFD) and at the end of a 3- and 6-month period, electrophysiological and morphological changes, and the role of POMC neurons in homeostatic nutrition and their response to leptin were thoroughly investigated. METHODS: Effects of HFD on POMC-satiety neurons in transgenic mice models exposed to chronic high-fat diet were investigated using electrophysiological (patch-clamp), chemogenetic and Cre recombinase advanced technological methods. Leptin, glucose and lipid profiles were determined and analyzed. RESULTS: In mice exposed to a high-fat diet for 6 months, no significant changes in POMC dendritic spine number or projection density from POMC neurons to the paraventricular hypothalamus (PVN), lateral hypothalamus (LH), and bed nucleus stria terminalis (BNST) were observed. It was revealed that leptin hormone did not change the electrophysiological activities of POMC neurons in mice fed with HFD for 6 months. In addition, chemogenetic stimulation of POMC neurons increased HFD consumption. In the 3-month HFD-fed group, POMC activation induced an orexigenic response in mice, whereas switching to a standard diet was found to abolish orexigenic behavior in POMC mice. CONCLUSIONS: Chronic high fat consumption disrupts the regulation of POMC neuron activation by leptin. Altered POMC neuron activation abolished the neuron's characteristic behavioral anorexigenic response. Change in nutritional content contributes to the reorganization of developing maladaptations.

Stress integration by an ascending adrenergic-melanocortin circuit.

Stress is thought to be an important contributing factor for eating disorders; however, neural substrates underlying the complex relationship between stress and appetite are not fully understood. Using in vivo recordings from awake behaving mice, we show that various acute stressors activate catecholaminergic nucleus tractus solitarius (NTSTH) projections in the paraventricular hypothalamus (PVH). Remarkably, the resulting adrenergic tone inhibits MC4R-expressing neurons (PVHMC4R), which are known for their role in feeding suppression. We found that PVHMC4R silencing encodes negative valence in sated mice and is required for avoidance induced by visceral malaise. Collectively, these findings establish PVHMC4R neurons as an effector of stress-activated brainstem adrenergic input in addition to the well-established hypothalamic-pituitary-adrenal axis. Convergent modulation of stress and feeding by PVHMC4R neurons implicates NTSTH → PVHMC4R input in stress-associated appetite disorders.

Opioidergic signaling contributes to food-mediated suppression of AgRP neurons.

Opioids are generally known to promote hedonic food consumption. Although much of the existing evidence is primarily based on studies of the mesolimbic pathway, endogenous opioids and their receptors are widely expressed in hypothalamic appetite circuits as well; however, their role in homeostatic feeding remains unclear. Using a fluorescent opioid sensor, deltaLight, here we report that mediobasal hypothalamic opioid levels increase by feeding, which directly and indirectly inhibits agouti-related protein (AgRP)-expressing neurons through the µ-opioid receptor (MOR). AgRP-specific MOR expression increases by energy surfeit and contributes to opioid-induced suppression of appetite. Conversely, its antagonists diminish suppression of AgRP neuron activity by food and satiety hormones. Mice with AgRP neuron-specific ablation of MOR expression have increased fat preference without increased motivation. These results suggest that post-ingestion release of endogenous opioids contributes to AgRP neuron inhibition to shape food choice through MOR signaling.

Postweaning Social Isolation Alters Puberty Onset by Suppressing Electrical Activity of Arcuate Kisspeptin Neurons.

INTRODUCTION: Postweaning social isolation (PWSI) in rodents is an advanced psychosocial stress model in early life. Some psychosocial stress, such as restrain and isolation, disrupts reproductive physiology in young and adult periods. Mechanisms of early-life stress effects on central regulation of reproduction need to be elucidated. We have investigated the effects of PWSI on function of arcuate kisspeptin (ARCKISS1) neurons by using electrophysiological techniques combining with monitoring of puberty onset and estrous cycle in male and female Kiss1-Cre mice. METHODS: Female mice were monitored for puberty onset with vaginal opening examination during social isolation. After isolation, the estrous cycle of female mice was monitored with vaginal cytology. Anxiety-like behavior of mice was determined by an elevated plus maze test. Effects of PWSI on electrophysiology of ARCKISS1 neurons were investigated by the patch clamp method after intracranial injection of AAV-GFP virus into arcuate nucleus of Kiss1-Cre mice after the isolation period. RESULTS: We found that both male and female isolated mice showed anxiety-like behavior. PWSI caused delay in vaginal opening and extension in estrous cycle length. Spontaneous-firing rates of ARCKISS1 neurons were significantly lower in the isolated male and female mice. The peak amplitude of inhibitory postsynaptic currents to ARCKISS1 neurons was higher in the isolated mice, while frequency of excitatory postsynaptic currents was higher in group-housed mice. CONCLUSION: These findings demonstrate that PWSI alters pre- and postpubertal reproductive physiology through metabolic and electrophysiological pathways.

AgRP neurons encode circadian feeding time.

Food intake follows a predictable daily pattern and synchronizes metabolic rhythms. Neurons expressing agouti-related protein (AgRP) read out physiological energetic state and elicit feeding, but the regulation of these neurons across daily timescales is poorly understood. Using a combination of neuron dynamics measurements and timed optogenetic activation in mice, we show that daily AgRP-neuron activity was not fully consistent with existing models of homeostatic regulation. Instead of operating as a 'deprivation counter', AgRP-neuron activity primarily followed the circadian rest-activity cycle through a process that required an intact suprachiasmatic nucleus and synchronization by light. Imposing novel feeding patterns through time-restricted food access or periodic AgRP-neuron stimulation was sufficient to resynchronize the daily AgRP-neuron activity rhythm and drive anticipatory-like behavior through a process that required DMHPDYN neurons. These results indicate that AgRP neurons integrate time-of-day information of past feeding experience with current metabolic needs to predict circadian feeding time.

Adrenergic modulation of melanocortin pathway by hunger signals.

Norepinephrine (NE) is a well-known appetite regulator, and the nor/adrenergic system is targeted by several anti-obesity drugs. To better understand the circuitry underlying adrenergic appetite control, here we investigated the paraventricular hypothalamic nucleus (PVN), a key brain region that integrates energy signals and receives dense nor/adrenergic input, using a mouse model. We found that PVN NE level increases with signals of energy deficit and decreases with food access. This pattern is recapitulated by the innervating catecholaminergic axon terminals originating from NTSTH-neurons. Optogenetic activation of rostral-NTSTH → PVN projection elicited strong motivation to eat comparable to overnight fasting whereas its inhibition attenuated both fasting-induced & hypoglycemic feeding. We found that NTSTH-axons functionally targeted PVNMC4R-neurons by predominantly inhibiting them, in part, through α1-AR mediated potentiation of GABA release from ARCAgRP presynaptic terminals. Furthermore, glucoprivation suppressed PVNMC4R activity, which was required for hypoglycemic feeding response. These results define an ascending nor/adrenergic circuit, NTSTH → PVNMC4R, that conveys peripheral hunger signals to melanocortin pathway.

Ghrelin Action in the PVH of Male Mice: Accessibility, Neuronal Targets, and CRH Neurons Activation.

The hormone ghrelin displays several well-characterized functions, including some with pharmaceutical interest. The receptor for ghrelin, the growth hormone secretagogue receptor (GHSR), is expressed in the hypothalamic paraventricular nucleus (PVH), a critical hub for the integration of metabolic, neuroendocrine, autonomic, and behavioral functions. Here, we performed a neuroanatomical and functional characterization of the neuronal types mediating ghrelin actions in the PVH of male mice. We found that fluorescent ghrelin mainly labels PVH neurons immunoreactive for nitric oxide synthase 1 (NOS1), which catalyze the production of nitric oxide [NO]). Centrally injected ghrelin increases c-Fos in NOS1 PVH neurons and NOS1 phosphorylation in the PVH. We also found that a high dose of systemically injected ghrelin increases the ghrelin level in the cerebrospinal fluid and in the periventricular PVH, and induces c-Fos in NOS1 PVH neurons. Such a high dose of systemically injected ghrelin activates a subset of NOS1 PVH neurons, which do not express oxytocin, via an arcuate nucleus-independent mechanism. Finally, we found that pharmacological inhibition of NO production fully abrogates ghrelin-induced increase of calcium concentration in corticotropin-releasing hormone neurons of the PVH whereas it partially impairs ghrelin-induced increase of plasma glucocorticoid levels. Thus, plasma ghrelin can directly target a subset of NO-producing neurons of the PVH that is involved in ghrelin-induced activation of the hypothalamic-pituitary-adrenal neuroendocrine axis.

Angiotensin AT1A receptor signal switching in Agouti-related peptide neurons mediates metabolic rate adaptation during obesity.

Resting metabolic rate (RMR) adaptation occurs during obesity and is hypothesized to contribute to failed weight management. Angiotensin II (Ang-II) type 1 (AT1A) receptors in Agouti-related peptide (AgRP) neurons contribute to the integrative control of RMR, and deletion of AT1A from AgRP neurons causes RMR adaptation. Extracellular patch-clamp recordings identify distinct cellular responses of individual AgRP neurons from lean mice to Ang-II: no response, inhibition via AT1A and Gαi, or stimulation via Ang-II type 2 (AT2) receptors and Gαq. Following diet-induced obesity, a subset of Ang-II/AT1A-inhibited AgRP neurons undergo a spontaneous G-protein "signal switch," whereby AT1A stop inhibiting the cell via Gαi and instead begin stimulating the cell via Gαq. DREADD-mediated activation of Gαi, but not Gαq, in AT1A-expressing AgRP cells stimulates RMR in lean and obese mice. Thus, loss of AT1A-Gαi coupling within the AT1A-expressing AgRP neuron subtype represents a molecular mechanism contributing to RMR adaptation.

Hunger potentiated.

Dieting often fails in the long run becuase of an ever-growing urge to eat. In this issue of Cell Metabolism, Grzelka et al. unveil a brain circuit that is potentiated during caloric restriction and incites rebound increases in food consumption and body weight.

Dorsal raphe serotonergic neurons suppress feeding through redundant forebrain circuits.

OBJECTIVE: Serotonin (5HT) is a well-known anorexigenic molecule, and 5HT neurons of dorsal raphe nucleus (DRN) have been implicated in suppression of feeding; however, the downstream circuitry is poorly understood. Here we explored major projections of DRN5HT neurons for their capacity to modulate feeding. METHODS: We used optogenetics to selectively activate DRN5HT axonal projections in hypothalamic and extrahypothalamic areas and monitored food intake. We next used fiber photometry to image the activity dynamics of DRN5HT axons and 5HT levels in projection areas in response feeding and metabolic hormones. Finally, we used electrophysiology to determine how DRN5HT axons affect downstream neuron activity. RESULTS: We found that selective activation of DRN5HT axons in (DRN5HT → LH) and (DRN5HT → BNST) suppresses feeding whereas activating medial hypothalamic projections has no effect. Using in vivo imaging, we found that food access and satiety hormones activate DRN5HT projections to LH where they also rapidly increase extracellular 5HT levels. Optogenetic mapping revealed that DRN5HT → LHvGAT and DRN5HT → LHvGlut2 connections are primarily inhibitory and excitatory respectively. Further, in addition to its direct action on LH neurons, we found that 5HT suppresses GABA release from presynaptic terminals arriving from AgRP neurons. CONCLUSIONS: These findings define functionally redundant forebrain circuits through which DRN5HT neurons suppress feeding and reveal that these projections can be modulated by metabolic hormones.

RGS6 mediates exercise-induced recovery of hippocampal neurogenesis, learning, and memory in an Alzheimer's mouse model.

Hippocampal neuronal loss causes cognitive dysfunction in Alzheimer's disease (AD). Adult hippocampal neurogenesis (AHN) is reduced in AD patients. Exercise stimulates AHN in rodents and improves memory and slows cognitive decline in AD patients. However, the molecular pathways for exercise-induced AHN and improved cognition in AD are poorly understood. Here, we show that voluntary running in APP SWE mice restores their hippocampal cognitive impairments to that of control mice. This cognitive rescue was abolished by RGS6 deletion in dentate gyrus (DG) neuronal progenitors (NPs), which also abolished running-mediated increases in AHN. AHN was reduced in sedentary APP SWE mice versus control mice, with basal AHN reduced by RGS6 deletion in DG NPs. RGS6 expression is significantly lower in the DG of AD patients. Thus, RGS6 mediates exercise-induced rescue of impaired cognition and AHN in AD mice, identifying RGS6 in DG NPs as a potential target to combat hippocampal neuron loss in AD. Teaser: RGS6 expression in hippocampal NPCs promotes voluntary running-induced neurogenesis and restored cognition in APP SWE mice. Field Codes: RGS6, Alzheimer's disease, adult hippocampal neurogenesis, neural precursor cells, dentate gyrus, exercise, learning/memory.

FGF21 suppresses alcohol consumption through an amygdalo-striatal circuit.

Excessive alcohol consumption is a major health and social issue in our society. Pharmacologic administration of the endocrine hormone fibroblast growth factor 21 (FGF21) suppresses alcohol consumption through actions in the brain in rodents, and genome-wide association studies have identified single nucleotide polymorphisms in genes involved with FGF21 signaling as being associated with increased alcohol consumption in humans. However, the neural circuit(s) through which FGF21 signals to suppress alcohol consumption are unknown, as are its effects on alcohol consumption in higher organisms. Here, we demonstrate that administration of an FGF21 analog to alcohol-preferring non-human primates reduces alcohol intake by 50%. Further, we reveal that FGF21 suppresses alcohol consumption through a projection-specific subpopulation of KLB-expressing neurons in the basolateral amygdala. Our results illustrate how FGF21 suppresses alcohol consumption through a specific population of neurons in the brain and demonstrate its therapeutic potential in non-human primate models of excessive alcohol consumption.

Presynaptic store-operated Ca2+ entry drives excitatory spontaneous neurotransmission and augments endoplasmic reticulum stress.

Store-operated calcium entry (SOCE) is activated by depletion of Ca2+ from the endoplasmic reticulum (ER) and mediated by stromal interaction molecule (STIM) proteins. Here, we show that in rat and mouse hippocampal neurons, acute ER Ca2+ depletion increases presynaptic Ca2+ levels and glutamate release through a pathway dependent on STIM2 and the synaptic Ca2+ sensor synaptotagmin-7 (syt7). In contrast, synaptotagmin-1 (syt1) can suppress SOCE-mediated spontaneous release, and STIM2 is required for the increase in spontaneous release seen during syt1 loss of function. We also demonstrate that chronic ER stress activates the same pathway leading to syt7-dependent potentiation of spontaneous glutamate release. During ER stress, inhibition of SOCE or syt7-driven fusion partially restored basal neurotransmission and decreased expression of pro-apoptotic markers, indicating that these processes participate in the amplification of ER-stress-related damage. Taken together, we propose that presynaptic SOCE links ER stress and augmented spontaneous neurotransmission, which may, in turn, facilitate neurodegeneration.

Endoscopic Ultrasound-Guided Fine Needle Aspiration Using a 22-G Needle for Hepatic Lesions: Single-Center Experience.

BACKGROUND/AIMS: Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has been accepted as a reliable tool in diagnosing and staging intra-abdominal tumors. In this study, we aimed to investigate the performance of EUS-FNA in the evaluation of liver masses and its impact on patient management and procedure-related complications retrospectively. METHODS: Data of patients who underwent EUS-FNA biopsies due to liver masses between November 2017 and July 2018 were retrieved retrospectively. Biopsies were performed using 22-G needles. The demographics, EUS-FNA results, sensitivity and specificity of the procedure, negative predictive value, positive predictive value, and specimen sufficiency rates were assessed. RESULTS: A total of 25 patients (10 females) were included in the study. The mean age was 62.73±15.2 years. The mean size of the masses was 34.50±16.04 mm. The technical success rate was 88%. During the EUS-FNA procedure, each patient had only one pass with 94.45% of aspirate sufficiency rate and 86.3% of biopsy sufficiency rate. The diagnostic accuracy rate was 86.3%. There were no complications. CONCLUSION: For the evaluation of liver masses, EUS-FNA using a 22-G needle with even one pass had high aspiration and biopsy success rates accompanied with high diagnostic accuracy rates.

FGF21 Signals to Glutamatergic Neurons in the Ventromedial Hypothalamus to Suppress Carbohydrate Intake.

Fibroblast growth factor 21 (FGF21) is an endocrine hormone produced by the liver that regulates nutrient and metabolic homeostasis. FGF21 production is increased in response to macronutrient imbalance and signals to the brain to suppress sugar intake and sweet-taste preference. However, the central targets mediating these effects have been unclear. Here, we identify FGF21 target cells in the hypothalamus and reveal that FGF21 signaling to glutamatergic neurons is both necessary and sufficient to mediate FGF21-induced sugar suppression and sweet-taste preference. Moreover, we show that FGF21 acts directly in the ventromedial hypothalamus (VMH) to specifically regulate sucrose intake, but not non-nutritive sweet-taste preference, body weight, or energy expenditure. Finally, our data demonstrate that FGF21 affects neuronal activity by increasing activation and excitability of neurons in the VMH. Thus, FGF21 signaling to glutamatergic neurons in the VMH is an important component of the neurocircuitry that functions to regulate sucrose intake.

NTS Catecholamine Neurons Mediate Hypoglycemic Hunger via Medial Hypothalamic Feeding Pathways.

Glucose is the essential energy source for the brain, whose deficit, triggered by energy deprivation or therapeutic agents, can be fatal. Increased appetite is the key behavioral defense against hypoglycemia; however, the central pathways involved are not well understood. Here, we describe a glucoprivic feeding pathway by tyrosine hydroxylase (TH)-expressing neurons from nucleus of solitary tract (NTS), which project densely to the hypothalamus and elicit feeding through bidirectional adrenergic modulation of agouti-related peptide (AgRP)- and proopiomelanocortin (POMC)-expressing neurons. Acute chemogenetic inhibition of arcuate nucleus (ARC)-projecting NTSTH neurons or their target, AgRP neurons, impaired glucoprivic feeding induced by 2-Deoxy-D-glucose (2DG) injection. Neuroanatomical tracing results suggested that ARC-projecting orexigenic NTSTH neurons are largely distinct from neighboring catecholamine neurons projecting to parabrachial nucleus (PBN) that promotes satiety. Collectively, we describe a circuit organization in which an ascending pathway from brainstem stimulates appetite through key hunger neurons in the hypothalamus in response to hypoglycemia.

Experience of the Endoscopists Matters in Endoscopic Retrograde Cholangiopancreatography in Billroth II Gastrectomy Patients.

BACKGROUND/AIMS: Altered anatomy is a challenge in endoscopic retrograde cholangiopancreatography (ERCP) for patients with Billroth II anastomosis. In this study, we investigated the overall success and role of endoscopist experience. METHODS: Data of patients who underwent ERCP between 2014 and 2018 after a previous Billroth II operation were retrieved retrospectively from 2 tertiary ERCP centers. The procedures were performed by 2 endoscopists with different levels of experience. Clinical success was defined as extraction of the stone, placement of a stent through a malignant stricture, and clinical and laboratory improvements in patients. RESULTS: Seventy-five patients were included. The technical success rate was 83% for the experienced endoscopist and 75% for the inexperienced endoscopist (p=0.46). The mean (±standard deviation) procedure time was 23.8±5.7 min for the experienced endoscopist and 40.68±6.07 min for the inexperienced endoscopist (p<0.001). In total, 3 perforations (4%) were found. The rate of afferent loop perforation was 6.25% (1/16) for the inexperienced endoscopist and 0% (0/59) for the experienced endoscopist (p=0.053). CONCLUSION: ERCP in patients who had undergone Billroth II gastrectomy was time consuming for the inexperienced endoscopist who should beware of the unique adverse events related to ERCP in patients with altered anatomy.

MCH Neuron Activity Is Sufficient for Reward and Reinforces Feeding.

BACKGROUND: Melanin-concentrating hormone (MCH)-expressing neurons have been implicated in regulation of energy homeostasis and reward, yet the role of their electrical activity in short-term appetite and reward modulation has not been fully understood. OBJECTIVES: We investigated short-term behavioral and physiological effects of MCH neuron activity manipulations. METHODS: We used optogenetic and chemogenetic approaches in Pmch-cre transgenic mice to acutely stimulate/inhibit MCH neuronal activity while probing feeding, locomotor activity, anxiety-like behaviors, glucose homeostasis, and reward. RESULTS: MCH neuron activity is neither required nor sufficient for short-term appetite unless stimulation is temporally paired with consumption. MCH neuronal activation does not affect short-term locomotor activity, but inhibition improves glucose tolerance and is mildly anxiolytic. Finally, using two different operant tasks, we showed that activation of MCH neurons alone is sufficient to induce reward. CONCLUSIONS: Our results confirm diverse behavioral/physiological functions of MCH neurons and suggest a direct role in reward function.

Totally robotic complete mesocolic excision for right-sided colon cancer.

Complexity and operative risks of complete mesocolic excision (CME) seem to be important drawbacks to generalize this procedure in the surgical treatment of right colon cancer. Robotic systems have been developed to improve quality and outcomes of minimal invasive surgery. The aim of this study was to evaluate the feasibility of robotic right-sided CME and present our initial experience. A retrospective review of 37 patients undergoing totally robotic right-sided CME between February 2015 and November 2017 was performed. All the operations were carried out using the key principles of both CME with intracorporeal anastomosis and no-touch technique. Data on perioperative clinical findings and short-term outcomes were analyzed. There were 20 men and 17 women with a mean age of 64.4 ± 13.5 years and a body mass index of 26.8 ± 5.7 kg/m2. The mean operative time and estimated blood loss were 289.8 ± 85.3 min and 77.4 ± 70.5 ml, respectively. Conversion to laparoscopy occurred in one patient (2.7%). All the surgical margins were clear and the mesocolic plane surgery was achieved in 27 (72.9%) of the cases. The mean number of harvested lymph nodes was 41.8 ± 11.9 (median, 40; range 22-65). The mean length of hospital stay was 6.6 ± 3.7 days. The intraoperative and postoperative complication rates were 5.4 and 21.6%, respectively. We believe that use of robot for right-sided CME is feasible and appears to provide remarkably a high number of harvested lymph nodes with good specimen quality.