Certain neuron types fire spontaneously at high rates, an ability that is crucial for their function in brain circuits. The spontaneously active GABAergic neurons of the substantia nigra pars ...reticulata (SNr), a major output of the basal ganglia, provide tonic inhibition of downstream brain areas. A depolarizing 'leak' current supports this firing pattern, but its molecular basis remains poorly understood. To understand how SNr neurons maintain tonic activity, we used single-cell RNA sequencing to determine the transcriptome of individual mouse SNr neurons. We discovered that SNr neurons express the sodium leak channel, NALCN, and that SNr neurons lacking NALCN have impaired spontaneous firing. In addition, NALCN is involved in the modulation of excitability by changes in glycolysis and by activation of muscarinic acetylcholine receptors. Our findings suggest that disruption of NALCN could impair the basal ganglia circuit, which may underlie the severe motor deficits in humans carrying mutations in NALCN.
Activating mutations in the Kir6.2 (KCNJ11) subunit of the ATP-sensitive potassium channel cause neonatal diabetes (ND). Patients with severe mutations also suffer from neurological complications. ...Glibenclamide blocks the open KATP channels and is the treatment of choice for ND. However, although glibenclamide successfully restores normoglycaemia, it has a far more limited effect on the neurological problems. To assess the extent to which glibenclamide crosses the blood-brain barrier (BBB) in vivo, we quantified glibenclamide concentrations in plasma, cerebrospinal fluid (CSF), and brain tissue of rats, control mice, and mice expressing a human neonatal diabetes mutation (Kir6.2-V59M) selectively in neurones (nV59M mice). As only small sample volumes can be obtained from rodents, we developed a highly sensitive method of analysis, using liquid chromatography tandem mass spectrometry acquisition with pseudo-selected reaction monitoring, achieving a quantification limit of 10ng/ml (20nM) glibenclamide in a 30μl sample. Glibenclamide was not detectable in the CSF or brain of rats after implantation with subcutaneous glibenclamide pellets, despite high plasma concentrations. Further, one hour after a suprapharmacological glibenclamide dose was administered directly into the lateral ventricle of the brain, the plasma concentration was twice that of the CSF. This suggests the drug is rapidly exported from the CSF. Elacridar, an inhibitor of P-glycoprotein and breast cancer resistance protein (major multidrug resistance transporters at the BBB), did not affect glibenclamide levels in CSF and brain tissue. We also identified a reduced sensitivity to volatile anaesthetics in nV59M mice and showed this was not reversed by systemic delivery of glibenclamide. Our results therefore suggest that little glibenclamide reaches the central nervous system when given systemically, that glibenclamide is rapidly removed across the BBB when given intracranioventricularly, and that any glibenclamide that does enter (and is below our detection limit) is insufficient to influence neuronal function as assessed by anaesthesia sensitivity.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Brain metabolism can profoundly influence neuronal excitability. Mice with genetic deletion or alteration of Bad (BCL-2 agonist of cell death) exhibit altered brain-cell fuel metabolism, accompanied ...by resistance to acutely induced epileptic seizures; this seizure protection is mediated by ATP-sensitive potassium (KATP) channels. Here we investigated the effect of BAD manipulation on KATP channel activity and excitability in acute brain slices. We found that BAD’s influence on neuronal KATP channels was cell-autonomous and directly affected dentate granule neuron (DGN) excitability. To investigate the role of neuronal KATP channels in the anticonvulsant effects of BAD, we imaged calcium during picrotoxin-induced epileptiform activity in entorhinal-hippocampal slices. BAD knockout reduced epileptiform activity, and this effect was lost upon knockout or pharmacological inhibition of KATP channels. Targeted BAD knockout in DGNs alone was sufficient for the antiseizure effect in slices, consistent with a ‘dentate gate’ function that is reinforced by increased KATP channel activity.
Glucagon, secreted by pancreatic islet α cells, is the principal hyperglycemic hormone. In diabetes, glucagon secretion is not suppressed at high glucose, exacerbating the consequences of ...insufficient insulin secretion, and is inadequate at low glucose, potentially leading to fatal hypoglycemia. The causal mechanisms remain unknown. Here we show that α cell KATP-channel activity is very low under hypoglycemic conditions and that hyperglycemia, via elevated intracellular ATP/ADP, leads to complete inhibition. This produces membrane depolarization and voltage-dependent inactivation of the Na+ channels involved in action potential firing that, via reduced action potential height and Ca2+ entry, suppresses glucagon secretion. Maneuvers that increase KATP channel activity, such as metabolic inhibition, mimic the glucagon secretory defects associated with diabetes. Low concentrations of the KATP channel blocker tolbutamide partially restore glucose-regulated glucagon secretion in islets from type 2 diabetic organ donors. These data suggest that impaired metabolic control of the KATP channels underlies the defective glucose regulation of glucagon secretion in type 2 diabetes.
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•KATP channel closure stimulates insulin secretion but inhibits glucagon release•α cell depolarization reduces voltage-gated Ca2+ entry and glucagon release•An activating KATP channel mutation impairs glucagon release in mice•KATP channel closure corrects glucagon secretion defect in type 2 diabetic islets
Live cell imaging of cytosolic NADH/NAD + ratio in hepatocytes and liver slices Masia, Ricard; McCarty, William J; Lahmann, Carolina ...
American journal of physiology. Gastrointestinal and liver physiology/American journal of physiology: Gastrointestinal and liver physiology,
01/2018, Letnik:
314, Številka:
1
Journal Article
Recenzirano
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Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol ...dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD
ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD
ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD
ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD
ratio, and this increase is mitigated by the presence of NAD
-generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD
ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD
ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real-time measurements of the ratio in living, functioning liver cells, overcoming many limitations of previous methods for measuring this important redox parameter. The feasibility of using Peredox in liver slices is particularly attractive because slices allow preservation of hepatic microanatomy and metabolic zonation of hepatocytes.
Abstract Activating mutations in the Kir6.2 ( KCNJ11 ) subunit of the ATP-sensitive potassium channel cause neonatal diabetes. Many patients also suffer from neurological complications. By using mice ...carrying a human Kir6.2 mutation (Val59 to Met59 ; nV59M mice) targeted to neurones, we show that these mutations also result in altered anxiety behaviour. The light/dark box, successive alleys and elevated plus maze tasks revealed that nV59M mice have reduced anxiety related responses. Additionally, nV59M mice displayed enhanced basal locomotor activity and exploratory behaviour, as assessed by the low anxiety open-field test. These findings, in combination with previously reported hyperactivity of nV59M mice, appear to correlate with the increased impulsivity and inattentiveness reported in iDEND/DEND patients.
Loss-of-function mutations in the KATP channel genes KCNJ11 and ABCC8 cause neonatal hyperinsulinism in humans. Dominantly inherited mutations cause less severe disease, which may progress to glucose ...intolerance and diabetes in later life (e.g., SUR1-E1506K). We generated a mouse expressing SUR1-E1506K in place of SUR1. KATP channel inhibition by MgATP was enhanced in both homozygous (homE1506K) and heterozygous (hetE1506K) mutant mice, due to impaired channel activation by MgADP. As a consequence, mutant β-cells showed less on-cell KATP channel activity and fired action potentials in glucose-free solution. HomE1506K mice exhibited enhanced insulin secretion and lower fasting blood glucose within 8 weeks of birth, but reduced insulin secretion and impaired glucose tolerance at 6 months of age. These changes correlated with a lower insulin content; unlike wild-type or hetE1506K mice, insulin content did not increase with age in homE1506K mice. There was no difference in the number and size of islets or β-cells in the three types of mice, or evidence of β-cell proliferation. We conclude that the gradual development of glucose intolerance in patients with the SUR1-E1506K mutation might, as in the mouse model, result from impaired insulin secretion due a failure of insulin content to increase with age.
Proper brain function requires a substantial energy supply, up to 20% of whole-body energy in humans, and brain activation produces large dynamic variations in energy demand. While local increases in ...cerebral blood flow are well known, the cellular responses to energy demand are controversial. During brain excitation, glycolysis of glucose to lactate temporarily exceeds the rate of mitochondrial fuel oxidation; although the increased energy demand occurs mainly within neurons, some have suggested this glycolysis occurs mainly in astrocytes, which then shuttle lactate to neurons as their primary fuel. Using metabolic biosensors in acute hippocampal slices and brains of awake mice, we find that neuronal metabolic responses to stimulation do not depend on astrocytic stimulation by glutamate release, nor do they require neuronal uptake of lactate; instead they reflect increased direct glucose consumption by neurons. Neuronal glycolysis temporarily outstrips oxidative metabolism, and provides a rapid response to increased energy demand.
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•Neuronal stimulation in slices or in vivo transiently increases cytosolic NADH:NAD+•Lactate shuttling into neurons is not required for the cytosolic NADH transients•Stimulation increases neuronal glycolysis rather than lactate metabolism•After stimulation, neurons are likely net exporters of lactate
Using fluorescent biosensors of metabolism, Díaz-García, Mongeon, Lahmann et al. observe the metabolic response of neurons to stimulation in brain slices and in vivo. Neurons increase their cytosolic NADH redox by increasing glycolysis, consistent with the mismatch between glucose and oxygen utilization that enables functional brain imaging.
Glucose is an essential source of energy for the brain. Recently, the development of genetically encoded fluorescent biosensors has allowed real time visualization of glucose dynamics from individual ...neurons and astrocytes. A major difficulty for this approach, even for ratiometric sensors, is the lack of a practical method to convert such measurements into actual concentrations in ex vivo brain tissue or in vivo. Fluorescence lifetime imaging provides a strategy to overcome this. In a previous study, we reported the lifetime glucose sensor iGlucoSnFR‐TS (then called SweetieTS) for monitoring changes in neuronal glucose levels in response to stimulation. This genetically encoded sensor was generated by combining the Thermus thermophilus glucose‐binding protein with a circularly permuted variant of the monomeric fluorescent protein T‐Sapphire. Here, we provide more details on iGlucoSnFR‐TS design and characterization, as well as pH and temperature sensitivities. For accurate estimation of glucose concentrations, the sensor must be calibrated at the same temperature as the experiments. We find that when the extracellular glucose concentration is in the range 2–10 mM, the intracellular glucose concentration in hippocampal neurons from acute brain slices is ~20% of the nominal external glucose concentration (~0.4–2 mM). We also measured the cytosolic neuronal glucose concentration in vivo, finding a range of ~0.7–2.5 mM in cortical neurons from awake mice.