Ion channel-mediated uptake of cationic vital dyes into live cells: a potential source of error when assessing cell viability.

Ionic "vital dyes" are commonly used to assess cell viability based on the idea that their permeation is contingent on a loss of membrane integrity. However, the possibility that dye entry is conducted into live cells by endogenous membrane transporters must be recognized and controlled for. Several cation-selective plasma membrane-localized ion channels, including the adenosine 5'-triphosphate (ATP)-gated P2X receptors, have been reported to conduct entry of the DNA-binding fluorescence dye, YO-PRO-1, into live cells. Extracellular ATP often becomes elevated as a result of release from dying cells, and so it is possible that activation of P2X channels on neighboring live cells could lead to exaggerated estimation of cytotoxicity. Here, we screened a number of fluorescent vital dyes for ion channel-mediated uptake in HEK293 cells expressing recombinant P2X2, P2X7, or TRPV1 channels. Our data shows that activation of all three channels caused substantial uptake and nuclear accumulation of YO-PRO-1, 4',6-diamidino-2-phenylindole (DAPI), and Hoechst 33258 into transfected cells and did so well within the time period usually used for incubation of cells with vital dyes. In contrast, channel activation in the presence of propidium iodide and SYTOX Green caused no measurable uptake and accumulation during a 20-min exposure, suggesting that these dyes are not likely to exhibit measurable uptake through these particular ion channels during a conventional cell viability assay. Caution is encouraged when choosing and employing cationic dyes for the purpose of cell viability assessment, particularly when there is a likelihood of cells expressing ion channels permeable to large ions.

An atlas of GPCRs in dopamine neurons: Identification of the free fatty acid receptor 4 as a regulator of food and water intake.

Midbrain dopaminergic neurons (DANs) are subject to extensive metabotropic regulation, but the repertoire of G protein-coupled receptors (GPCRs) present in these neurons has not been mapped. Here, we isolate DANs from Dat-eGFP mice to generate a GPCR atlas by unbiased qPCR array expression analysis of 377 GPCRs. Combined with data mining of scRNA-seq databases, we identify multiple receptors in DAN subpopulations with 38 of these receptors representing the majority of transcripts. We identify 41 receptors expressed in midbrain DANs but not in non-DAN midbrain cells, including the free fatty acid receptor 4 (FFAR4). Functional expression of FFAR4 is validated by ex vivo Ca2+ imaging, and in vivo experiments support that FFAR4 negatively regulates food and water intake and bodyweight. In addition to providing a critical framework for understanding metabotropic DAN regulation, our data suggest fatty acid sensing by FFAR4 as a mechanism linking high-energy intake to the dopamine-reward pathway.

Hypothalamic hormone-sensitive lipase regulates appetite and energy homeostasis.

OBJECTIVE: The goal of this study was to investigate the importance of central hormone-sensitive lipase (HSL) expression in the regulation of food intake and body weight in mice to clarify whether intracellular lipolysis in the mammalian hypothalamus plays a role in regulating appetite. METHODS: Using pharmacological and genetic approaches, we investigated the role of HSL in the rodent brain in the regulation of feeding and energy homeostasis under basal conditions during acute stress and high-fat diet feeding. RESULTS: We found that HSL, a key enzyme in the catabolism of cellular lipid stores, is expressed in the appetite-regulating centers in the hypothalamus and is activated by acute stress through a mechanism similar to that observed in adipose tissue and skeletal muscle. Inhibition of HSL in rodent models by a synthetic ligand, global knockout, or brain-specific deletion of HSL prevents a decrease in food intake normally seen in response to acute stress and is associated with the increased expression of orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AgRP). Increased food intake can be reversed by adeno-associated virus-mediated reintroduction of HSL in neurons of the mediobasal hypothalamus. Importantly, metabolic stress induced by a high-fat diet also enhances the hyperphagic phenotype of HSL-deficient mice. Specific deletion of HSL in the ventromedial hypothalamic nucleus (VMH) or AgRP neurons reveals that HSL in the VMH plays a role in both acute stress-induced food intake and high-fat diet-induced obesity. CONCLUSIONS: Our results indicate that HSL activity in the mediobasal hypothalamus is involved in the acute reduction in food intake during the acute stress response and sensing of a high-fat diet.

RhoA in tyrosine hydroxylase neurones regulates food intake and body weight via altered sensitivity to peripheral hormones.

Dopamine-producing tyrosine hydroxylase (TH) neurones in the hypothalamic arcuate nucleus (ARC) have recently been shown to be involved in ghrelin signalling and body weight homeostasis. In the present study, we investigate the role of the intracellular regulator RhoA in hypothalamic TH neurones in response to peripheral hormones. Diet-induced obesity was found to be associated with increased phosphorylation of TH in ARC, indicating obesity-associated increased activity of ARC TH neurones. Mice in which RhoA was specifically knocked out in TH neurones (TH-RhoA-/- mice) were more sensitive to the orexigenic effect of peripherally administered ghrelin and displayed an abolished response to the anorexigenic hormone leptin. When TH-RhoA-/- mice were challenged with a high-fat high-sucrose (HFHS) diet, they became hyperphagic and gained more body weight and fat mass compared to wild-type control mice. Importantly, lack of RhoA prevented development of ghrelin resistance, which is normally observed in wild-type mice after long-term HFHS diet feeding. Patch-clamp electrophysiological analysis demonstrated increased ghrelin-induced excitability of TH neurones in lean TH-RhoA-/- mice compared to lean littermate control animals. Additionally, increased expression of the orexigenic hypothalamic neuropeptides agouti-related peptide and neuropeptide Y was observed in TH-RhoA-/- mice. Overall, our data indicate that TH neurones in ARC are important for the regulation of body weight homeostasis and that RhoA is both a central effector in these neurones and important for the development of obesity-induced ghrelin resistance. The obese phenotype of TH-RhoA-/- mice may be a result of increased sensitivity to ghrelin and decreased sensitivity to leptin, resulting in increased food intake.

Human glia can both induce and rescue aspects of disease phenotype in Huntington disease.

The causal contribution of glial pathology to Huntington disease (HD) has not been heavily explored. To define the contribution of glia to HD, we established human HD glial chimeras by neonatally engrafting immunodeficient mice with mutant huntingtin (mHTT)-expressing human glial progenitor cells (hGPCs), derived from either human embryonic stem cells or mHTT-transduced fetal hGPCs. Here we show that mHTT glia can impart disease phenotype to normal mice, since mice engrafted intrastriatally with mHTT hGPCs exhibit worse motor performance than controls, and striatal neurons in mHTT glial chimeras are hyperexcitable. Conversely, normal glia can ameliorate disease phenotype in transgenic HD mice, as striatal transplantation of normal glia rescues aspects of electrophysiological and behavioural phenotype, restores interstitial potassium homeostasis, slows disease progression and extends survival in R6/2 HD mice. These observations suggest a causal role for glia in HD, and further suggest a cell-based strategy for disease amelioration in this disorder.