Phospholipase C beta 1 in the dentate gyrus gates fear memory formation through regulation of neuronal excitability.

Memory processes rely on a molecular signaling system that balances the interplay between positive and negative modulators. Recent research has focused on identifying memory-regulating genes and their mechanisms. Phospholipase C beta 1 (PLCß1), highly expressed in the hippocampus, reportedly serves as a convergence point for signal transduction through G protein-coupled receptors. However, the detailed role of PLCß1 in memory function has not been elucidated. Here, we demonstrate that PLCß1 in the dentate gyrus functions as a memory suppressor. We reveal that mice lacking PLCß1 in the dentate gyrus exhibit a heightened fear response and impaired memory extinction, and this excessive fear response is repressed by upregulation of PLCß1 through its overexpression or activation using a newly developed optogenetic system. Last, our results demonstrate that PLCß1 overexpression partially inhibits exaggerated fear response caused by traumatic experience. Together, PLCß1 is crucial in regulating contextual fear memory formation and potentially enhancing the resilience to trauma-related conditions.

Erratum: Assessment Methods for Problematic Eating Behaviors in Children and Adolescents With Autism Spectrum Disorder.

[This corrects the article on p. 57 in vol. 35, PMID: 38204745.].

Assessment Methods for Problematic Eating Behaviors in Children and Adolescents With Autism Spectrum Disorder.

Autism spectrum disorder (ASD) can be associated with eating problems. However, currently, there is a lack of established guidelines for assessing and addressing eating behaviors in individuals with ASD. This gap in research exists due to the challenges associated with using traditional assessment methods, which may lead to discrepancies in responses and unintentional potential biases from caregivers. In this review, we provided a comprehensive overview of various eating behaviors commonly observed in individuals with ASD. These behaviors include 1) food neophobia, 2) selective eating, 3) binge eating, 4) food avoidance, 5) chewing and swallowing problems, 6) pica, 7) rumination, 8) rituals, and 9) problematic behaviors. Furthermore, we provide a perspective of utilizing digital tools: 1) augmentative and alternative communication; 2) ecological momentary assessment; and 3) video analysis, behavioral analysis, and facial expression analysis. This review explores existing assessment methods and suggests novel assessment aiding together.

Insulin Preferentially Regulates the Activity of Parasympathetic Preganglionic Neurons over Sympathetic Preganglionic Neurons.

BACKGRUOUND: Insulin is a peptide hormone that regulates post-prandial physiology, and it is well known that insulin controls homeostasis at least in part via the central nervous system. In particular, insulin alters the activity of neurons within the autonomic nervous system. However, currently available data are mostly from unidentified brainstem neurons of the dorsal motor nucleus of the vagus nerve (DMV). METHODS: In this study, we used several genetically engineered mouse models to label distinct populations of neurons within the brainstem and the spinal cord for whole-cell patch clamp recordings and to assess several in vivo metabolic functions. RESULTS: We first confirmed that insulin directly inhibited cholinergic (parasympathetic preganglionic) neurons in the DMV. We also found inhibitory effects of insulin on both the excitatory and inhibitory postsynaptic currents recorded in DMV cholinergic neurons. In addition, GABAergic neurons of the DMV and nucleus tractus solitarius were inhibited by insulin. However, insulin had no effects on the cholinergic sympathetic preganglionic neurons of the spinal cord. Finally, we obtained results suggesting that the insulininduced inhibition of parasympathetic preganglionic neurons may not play a critical role in the regulation of glucose homeostasis and gastrointestinal motility. CONCLUSION: Our results demonstrate that insulin inhibits parasympathetic neuronal circuitry in the brainstem, while not affecting sympathetic neuronal activity in the spinal cord.

Hypothalamic GABRA5-positive neurons control obesity via astrocytic GABA.

The lateral hypothalamic area (LHA) regulates food intake and energy balance. Although LHA neurons innervate adipose tissues, the identity of neurons that regulate fat is undefined. Here we show that GABRA5-positive neurons in LHA (GABRA5LHA) polysynaptically project to brown and white adipose tissues in the periphery. GABRA5LHA are a distinct subpopulation of GABAergic neurons and show decreased pacemaker firing in diet-induced obesity mouse models in males. Chemogenetic inhibition of GABRA5LHA suppresses fat thermogenesis and increases weight gain, whereas gene silencing of GABRA5 in LHA decreases weight gain. In the diet-induced obesity mouse model, GABRA5LHA are tonically inhibited by nearby reactive astrocytes releasing GABA, which is synthesized by monoamine oxidase B (Maob). Gene silencing of astrocytic Maob in LHA facilitates fat thermogenesis and reduces weight gain significantly without affecting food intake, which is recapitulated by administration of a Maob inhibitor, KDS2010. We propose that firing of GABRA5LHA suppresses fat accumulation and selective inhibition of astrocytic GABA is a molecular target for treating obesity.

Serotonergic regulation of appetite and sodium appetite.

Serotonin is a neurotransmitter that is synthesized and released from the brainstem raphe nuclei to affect many brain functions. It is well known that the activity of raphe serotonergic neurons is changed in response to the changes in feeding status to regulate appetite via the serotonin receptors. Likewise, changes in volume status are known to alter the activity of raphe serotonergic neurons and drugs targeting serotonin receptors were shown to affect sodium appetite. Therefore, the central serotonin system appears to regulate ingestion of both food and salt, although neural mechanisms that induce appetite in response to hunger and sodium appetite in response to volume depletion are largely distinct from each other. In this review, we discuss our current knowledge regarding the regulation of ingestion - appetite and sodium appetite - by the central serotonin system.

GIRK2 potassium channels expressed by the AgRP neurons decrease adiposity and body weight in mice.

It is well known that the neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons increase appetite and decrease thermogenesis. Previous studies demonstrated that optogenetic and/or chemogenetic manipulations of NPY/AgRP neuronal activity alter food intake and/or energy expenditure (EE). However, little is known about intrinsic molecules regulating NPY/AgRP neuronal excitability to affect long-term metabolic function. Here, we found that the G protein-gated inwardly rectifying K+ (GIRK) channels are key to stabilize NPY/AgRP neurons and that NPY/AgRP neuron-selective deletion of the GIRK2 subunit results in a persistently increased excitability of the NPY/AgRP neurons. Interestingly, increased body weight and adiposity observed in the NPY/AgRP neuron-selective GIRK2 knockout mice were due to decreased sympathetic activity and EE, while food intake remained unchanged. The conditional knockout mice also showed compromised adaptation to coldness. In summary, our study identified GIRK2 as a key determinant of NPY/AgRP neuronal excitability and driver of EE in physiological and stress conditions.

Protocol to prepare mouse spinal cord for patch-clamp and histology experiments.

The spinal cord is a part of the central nervous system located within the spinal canal of the vertebrae. Here, we present a protocol to prepare mouse spinal cord sections for patch-clamp and histology experiments. We describe steps for isolating spinal cord from the spinal canal and obtaining acute slices for patch-clamp experiments. For histology experiments, we detail fixing spinal cord for cryosectioning and imaging. This protocol provides procedures to assess neuronal activity and protein expression of sympathetic preganglionic neurons. For complete details on the use and execution of this protocol, please refer to Ju et al.1.

Melanocortin-4 receptors activate sympathetic preganglionic neurons and elevate blood pressure via TRPV1.

Melanocortin-4 receptors (MC4Rs) expressed by the central nervous system are essential regulators of energy homeostasis, and Mc4r mutation is the most common cause of human monogenic obesity. Notably, patients with obesity carrying Mc4r mutations are protected against obesity-induced hypertension, and MC4R agonists elevate blood pressure (BP). Although increased sympathetic tone by MC4Rs is suggested to underlie this phenotype, the detailed mechanisms remain unclear. Here, we investigate how MC4Rs regulate the sympathetic preganglionic neurons and find that MC4Rs activate these neurons via the protein kinase A-dependent activation of the transient receptor potential vanilloid 1 (TRPV1) channel. Importantly, we demonstrate that the inhibition of TRPV1 prevents MC4R-induced elevation of BP but does not affect MC4R-induced anorexia. We further show that TRPV1 is responsible for MC4R-dependent activation of the sympathetic preganglionic neurons by high-fat diet. Together, our results provide insight into how MC4Rs regulate sympathetic function.

Leptin-inhibited neurons in the lateral parabrachial nucleus do not alter food intake or glucose balance.

The lateral parabrachial nucleus (LPBN) has been shown to be involved in the suppression of appetite at the pharmacological, optogenetic and chemogenetic levels. However, the signalling that mediates activation of these neurons in physiological conditions has been hindered by difficulties in segregating different cell populations in this region. Using reporter mice, we identify at the electrophysiological level the effects of an anorexic hormone, leptin, on leptin receptor (ObR)-expressing neurons in the LPBN (LPBNObR neurons). Application of leptin caused inhibition in a subpopulation of LPBNObR neurons. This effect was mediated by an increased potassium conductance and was also accompanied by a decrease in excitatory synaptic input onto these neurons. However, mimicking the inhibitory effects of leptin on LPBNObR neurons through chemogenetics led to no changes in feeding or glucose levels, which suggests that leptin action on LPBNObR neurons may not be sufficient to regulate these metabolic aspects.

Delineating a serotonin 1B receptor circuit for appetite suppression in mice.

Triptans are a class of commonly prescribed antimigraine drugs. Here, we report a previously unrecognized role for them to suppress appetite in mice. In particular, frovatriptan treatment reduces food intake and body weight in diet-induced obese mice. Moreover, the anorectic effect depends on the serotonin (5-HT) 1B receptor (Htr1b). By ablating Htr1b in four different brain regions, we demonstrate that Htr1b engages in spatiotemporally segregated neural pathways to regulate postnatal growth and food intake. Moreover, Htr1b in AgRP neurons in the arcuate nucleus of the hypothalamus (ARH) contributes to the hypophagic effects of HTR1B agonists. To further study the anorexigenic Htr1b circuit, we generated Htr1b-Cre mice. We find that ARH Htr1b neurons bidirectionally regulate food intake in vivo. Furthermore, single-nucleus RNA sequencing analyses revealed that Htr1b marks a subset of AgRP neurons. Finally, we used an intersectional approach to specifically target these neurons (Htr1bAgRP neurons). We show that they regulate food intake, in part, through a Htr1bAgRP→PVH circuit.

Lomitapide, a cholesterol-lowering drug, is an anticancer agent that induces autophagic cell death via inhibiting mTOR.

Autophagy is a biological process that maintains cellular homeostasis and regulates the internal cellular environment. Hyperactivating autophagy to trigger cell death has been a suggested therapeutic strategy for cancer treatment. Mechanistic target of rapamycin (mTOR) is a crucial protein kinase that regulates autophagy; therefore, using a structure-based virtual screen analysis, we identified lomitapide, a cholesterol-lowering drug, as a potential mTOR complex 1 (mTORC1) inhibitor. Our results showed that lomitapide directly inhibits mTORC1 in vitro and induces autophagy-dependent cancer cell death by decreasing mTOR signaling, thereby inhibiting the downstream events associated with increased LC3 conversion in various cancer cells (e.g., HCT116 colorectal cancer cells) and tumor xenografts. Lomitapide also significantly suppresses the growth and viability along with elevated autophagy in patient-derived colorectal cancer organoids. Furthermore, a combination of lomitapide and immune checkpoint blocking antibodies synergistically inhibits tumor growth in murine MC38 or B16-F10 preclinical syngeneic tumor models. These results elucidate the direct, tumor-relevant immune-potentiating benefits of mTORC1 inhibition by lomitapide, which complement the current immune checkpoint blockade. This study highlights the potential repurposing of lomitapide as a new therapeutic option for cancer treatment.

Autonomic control of energy balance and glucose homeostasis.

Neurons in the central nervous system (CNS) communicate with peripheral organs largely via the autonomic nervous system (ANS). Through such communications, the sympathetic and parasympathetic efferent divisions of the ANS may affect thermogenesis and blood glucose levels. In contrast, peripheral organs send feedback to the CNS via hormones and autonomic afferent nerves. These humoral and neural feedbacks, as well as neural commands from higher brain centers directly or indirectly shape the metabolic function of autonomic neurons. Notably, recent developments in mouse genetics have enabled more detailed studies of ANS neurons and circuits, which have helped elucidate autonomic control of metabolism. Here, we will summarize the functional organization of the ANS and discuss recent updates on the roles of neural and humoral factors in the regulation of energy balance and glucose homeostasis by the ANS.

Protocol for sodium depletion and measurement of sodium appetite in mice.

Sodium appetite is a state that motivates animals to consume normally unappetizing concentrations of sodium. Here we describe a protocol to induce sodium appetite in mice by furosemide-induced diuresis and measure sodium intake using volumetric drinking tubes. This protocol induces sodium appetite rapidly and can be used to assess the effect of various treatments on sodium appetite. This protocol does not require electronic equipment and can be implemented easily. For complete details on the use and execution of this protocol, please refer to Park et al. (2020).

Gαi/o-coupled Htr2c in the paraventricular nucleus of the hypothalamus antagonizes the anorectic effect of serotonin agents.

The anorexigenic effect of serotonergic compounds has largely been attributed to activation of serotonin 2C receptors (Htr2cs). Using mouse genetic models in which Htr2c can be selectively deleted or restored (in Htr2c-null mice), we investigate the role of Htr2c in forebrain Sim1 neurons. Unexpectedly, we find that Htr2c acts in these neurons to promote food intake and counteract the anorectic effect of serotonergic appetite suppressants. Furthermore, Htr2c marks a subset of Sim1 neurons in the paraventricular nucleus of the hypothalamus (PVH). Chemogenetic activation of these neurons in adult mice suppresses hunger, whereas their silencing promotes feeding. In support of an orexigenic role of PVH Htr2c, whole-cell patch-clamp experiments demonstrate that activation of Htr2c inhibits PVH neurons. Intriguingly, this inhibition is due to Gαi/o-dependent activation of ATP-sensitive K+ conductance, a mechanism of action not identified previously in the mammalian nervous system.

Generation of caudal-type serotonin neurons and hindbrain-fate organoids from hPSCs.

Serotonin (5-HT) neurons, the major components of the raphe nuclei, arise from ventral hindbrain progenitors. Based on anatomical location and axonal projection, 5-HT neurons are coarsely divided into rostral and caudal groups. Here, we propose a novel strategy to generate hindbrain 5-HT neurons from human pluripotent stem cells (hPSCs), which involves the formation of ventral-type neural progenitor cells and stimulation of the hindbrain 5-HT neural development. A caudalizing agent, retinoid acid, was used to direct the cells into the hindbrain cell fate. Approximately 30%-40% of hPSCs successfully developed into 5-HT-expressing neurons using our protocol, with the majority acquiring a caudal rhombomere identity (r5-8). We further modified our monolayer differentiation system to generate 5-HT neuron-enriched hindbrain-like organoids. We also suggest downstream applications of our 5-HT monolayer and organoid cultures to study neuronal response to gut microbiota. Our methodology could become a powerful tool for future studies related to 5-HT neurotransmission.

Non-Cell Autonomous Epileptogenesis in Focal Cortical Dysplasia.

OBJECTIVE: Low-level somatic mosaicism in the brain has been shown to be a major genetic cause of intractable focal epilepsy. However, how a relatively few mutation-carrying neurons are able to induce epileptogenesis at the local network level remains poorly understood. METHODS: To probe the origin of epileptogenesis, we measured the excitability of neurons with MTOR mutation and nearby nonmutated neurons recorded by whole-cell patch-clamp and array-based electrodes comparing the topographic distribution of mutation. Computational simulation is used to understand neural network-level changes based on electrophysiological properties. To examine the underlying mechanism, we measured inhibitory and excitatory synaptic inputs in mutated neurons and nearby neurons by electrophysiological and histological methods using the mouse model and postoperative human brain tissue for cortical dysplasia. To explain non-cell-autonomous hyperexcitability, an inhibitor of adenosine kinase was injected into mice to enhance adenosine signaling and to mitigate hyperactivity of nearby nonmutated neurons. RESULTS: We generated mice with a low-level somatic mutation in MTOR presenting spontaneous seizures. The seizure-triggering hyperexcitability originated from nonmutated neurons near mutation-carrying neurons, which proved to be less excitable than nonmutated neurons. Interestingly, the net balance between excitatory and inhibitory synaptic inputs onto mutated neurons remained unchanged. Additionally, we found that inhibition of adenosine kinase, which affects adenosine metabolism and neuronal excitability, reduced the hyperexcitability of nonmutated neurons. INTERPRETATION: This study shows that neurons carrying somatic mutations in MTOR lead to focal epileptogenesis via non-cell-autonomous hyperexcitability of nearby nonmutated neurons. ANN NEUROL 2021;90:285-299.

The atypical antipsychotic risperidone targets hypothalamic melanocortin 4 receptors to cause weight gain.

Atypical antipsychotics such as risperidone cause drug-induced metabolic syndrome. However, the underlying mechanisms remain largely unknown. Here, we report a new mouse model that reliably reproduces risperidone-induced weight gain, adiposity, and glucose intolerance. We found that risperidone treatment acutely altered energy balance in C57BL/6 mice and that hyperphagia accounted for most of the weight gain. Transcriptomic analyses in the hypothalamus of risperidone-fed mice revealed that risperidone treatment reduced the expression of Mc4r. Furthermore, Mc4r in Sim1 neurons was necessary for risperidone-induced hyperphagia and weight gain. Moreover, we found that the same pathway underlies the obesogenic effect of olanzapine-another commonly prescribed antipsychotic drug. Remarkably, whole-cell patch-clamp recording demonstrated that risperidone acutely inhibited the activity of hypothalamic Mc4r neurons via the opening of a postsynaptic potassium conductance. Finally, we showed that treatment with setmelanotide, an MC4R-specific agonist, mitigated hyperphagia and obesity in both risperidone- and olanzapine-fed mice.