SUMMARY 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.

SummaryGlucagon, 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.

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.DOI: http://dx.doi.org/10.7554/eLife.15271.001

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.

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.

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.

Single-channel conductance in Cys-loop channels is controlled by the nature of the amino acids in the narrowest parts of the ion conduction pathway, namely the second transmembrane domain (M2) and the intracellular helix. In cationic channels, such as Torpedo ACh nicotinic receptors, conductance is increased by negatively charged residues exposed to the extracellular vestibule. We now show that positively charged residues at the same loop 5 position boost also the conductance of anionic Cys-loop channels, such as glycine (␣1 and ␣1␤) and GABA A (␣1␤2␥2) receptors. Charge reversal mutations here produce a greater decrease on outward conductance, but their effect strongly depends on which subunit carries the mutation. In the glycine ␣1␤ receptor, replacing Lys with Glu in ␣1 reduces single-channel conductance by 41%, but has no effect in the ␤ subunit. By expressing concatameric receptors with constrained stoichiometry, we show that this asymmetry is not explained by the subunit copy number. A similar pattern is observed in the ␣1␤2␥2 GABA A receptor, where only mutations in ␣1 or ␤2 decreased conductance (to different extents). In both glycine and GABA receptors, the effect of mutations in different subunits does not sum linearly: mutations that had no detectable effect in isolation did enhance the effect of mutations carried by other subunits. As in the nicotinic receptor, charged residues in the extracellular vestibule of anionic Cys-loop channels influence elementary conductance. The size of this effect strongly depends on the direction of the ion flow and, unexpectedly, on the nature of the subunit that carries the residue.Channels in the nicotinic superfamily are formed by five subunits arranged quasi-symmetrically around the central pore. Structural information from the Torpedo acetylcholine nicotinic receptor and related prokaryotic channels, such as GLIC and ELIC (all cation-permeable), shows that the conduction path for ions starts with a wide vestibule formed by the extracellular domains (1-3). This tapers to a narrower transmembrane region, which is lined mainly by the second transmembrane domains (M2) of each of the five subunits. In eukaryotic channels, this is connected to the cytoplasm by openings in the subunits intracellular domains, which are formed by the M3-M4 loop. The rate of ion flow along this pathway (and therefore the single-channel conductance) is strongly affected by the nature of the residues that line it, especially those that line the narrowest parts. In particular, there are rings of charged amino acids at the extracellular and intracellular ends of the transmembrane part of the pore (positions Ϫ4Ј and 20Ј in the M2 domain (4) and around the fenestrations of the cytoplasmic domain (5). It was recently found that single-channel conductance can also be influenced by residues in the extracellular domain (ECD). The first clue came from the structure of a member of the family of ACh-binding proteins (AChBP), molluscan homopentameric soluble proteins, which are homologous to the ECD part...

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.