Properties of glutamate uptake in salamander Müller cells

Glutamate is a major transmitter in the central nervous system. In order to understand its physiological and pathological actions it is necessary to investigate the properties of the uptake system which removes glutamate from the extracellular space. An important function of glutamate uptake is to maintain the extracellular glutamate concentration below neurotoxic levels. High affinity, sodium-dependent, electrogenic glutamate uptake was measured using whole-cell patch-clamp recording in acutely isolated retinal glial (Müller) cells of the salamander. The ionic dependence of the glutamate uptake current was investigated. Internal sodium and glutamate both inhibited the uptake current. It was activated by intracellular potassium and inhibited by extracellular potassium: therefore glutamate uptake involves counter transport of potassium ions. Replacement of intracellular or extracellular chloride had no effect on the uptake current. The uptake current was maximal near pH 7.3. Second messenger systems were manipulated in order to identify mechanisms regulating the uptake carrier. Variation of the intracellular calcium concentration or inclusion of cGMP, cAMP or GTP-γ-S in the patch pipette had little or no effect on the uptake current. Arachidonic acid induced a progressive and prolonged inhibition of the uptake current. A fluorescence assay was used to measure glutamate uptake, independently of the uptake current. Manoeuvres which changed the uptake current had similar effects on uptake as measured by the fluorescence method. This confirmed that glutamate uptake into Muller cells occurs predominantly by the high affinity, sodium-dependent system and that the uptake current reflects accurately the rate of glutamate uptake into the cell

1-Phenyl-2-trifluoro­methyl-4-quinolone

In the title mol­ecule, C16H10F3NO, the N-bound phenyl ring is oriented nearly orthogonal to the quinolinyl ring in order to avoid steric clashes with the trifluoro­methyl substituent [dihedral angle 89.7 (1)°]

Event-driven simulation scheme for spiking neural networks using lookup tables to characterize neuronal dynamics

Nearly all neuronal information processing and interneuronal communication in the brain involves action potentials, or spikes, which drive the short-term synaptic dynamics of neurons, but also their long-term dynamics, via synaptic plasticity. In many brain structures, action potential activity is considered to be sparse. This sparseness of activity has been exploited to reduce the computational cost of large-scale network simulations, through the development of event-driven simulation schemes. However, existing event-driven simulations schemes use extremely simplified neuronal models. Here, we implement and evaluate critically an event-driven algorithm (ED-LUT) that uses precalculated look-up tables to characterize synaptic and neuronal dynamics. This approach enables the use of more complex (and realistic) neuronal models or data in representing the neurons, while retaining the advantage of high-speed simulation. We demonstrate the method's application for neurons containing exponential synaptic conductances, thereby implementing shunting inhibition, a phenomenon that is critical to cellular computation. We also introduce an improved two-stage event-queue algorithm, which allows the simulations to scale efficiently to highly connected networks with arbitrary propagation delays. Finally, the scheme readily accommodates implementation of synaptic plasticity mechanisms that depend on spike timing, enabling future simulations to explore issues of long-term learning and adaptation in large-scale networks.This work has been supported by the EU projects SpikeFORCE (IST-2001-35271), SENSOPAC (IST-028056) and the Spanish National Grant (DPI-2004-07032

Photolysis of a Caged, Fast-Equilibrating Glutamate Receptor Antagonist, MNI-Caged γ-D-Glutamyl-Glycine, to Investigate Transmitter Dynamics and Receptor Properties at Glutamatergic Synapses

Fast uncaging of low affinity competitive receptor antagonists can in principle measure the timing and concentration dependence of transmitter action at receptors during synaptic transmission. Here, we describe the development, synthesis and characterization of MNI-caged γ-D-glutamyl-glycine (γ-DGG), which combines the fast photolysis and hydrolytic stability of nitroindoline cages with the well-characterized fast-equilibrating competitive glutamate receptor antagonist γ-DGG. At climbing fiber-Purkinje cell (CF-PC) synapses MNI-caged-γ-DGG was applied at concentrations up to 5 mM without affecting CF-PC transmission, permitting release of up to 1.5 mM γ-DGG in 1 ms in wide-field flashlamp photolysis. In steady-state conditions, photoreleased γ-DGG at 0.55–1.7 mM inhibited the CF first and second paired EPSCs by on average 30% and 60%, respectively, similar to reported values for bath applied γ-DGG. Photolysis of the L-isomer MNI-caged γ-L-glutamyl-glycine was ineffective. The time-course of receptor activation by synaptically released glutamate was investigated by timed photolysis of MNI-caged-γ-DGG at defined intervals following CF stimulation in the second EPSCs. Photorelease of γ-DGG prior to the stimulus and up to 3 ms after showed strong inhibition similar to steady-state inhibition; in contrast γ-DGG applied by a flash at 3–4 ms post-stimulus produced weaker and variable block, suggesting transmitter-receptor interaction occurs mainly in this time window. The data also show a small and lasting component of inhibition when γ-DGG was released at 4–7 ms post stimulus, near the peak of the CF-PC EPSC, or at 10–11 ms. This indicates that competition for binding and activation of AMPA receptors occurs also during the late phase of the EPSC, due to either delayed transmitter release or persistence of glutamate in the synaptic region. The results presented here first show that MNI-caged-γ-DGG has properties suitable for use as a synaptic probe at high concentration and that its photolysis can resolve timing and extent of transmitter activation of receptors in glutamatergic transmission

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

No Extraction of Spine Neck Resistance from Underdetermined Equations

International audienceFrom voltage-dye measurements of dendritic spine responses to uncaging of glutamate, Cartailler et al. (2018) report extracting a value of spine neck resistance by optimisation of an electrodiffusion model. This comment fo-cuses on flaws in the modelling and optimisation. It is based on a more exhaustive blog post (Barbour, 2018) and the authors' response (Cartailler and Holcman, 2018). Underdetermined equations The analysis starts with the voltage responses in the spine head (V h) and base dendrite (V d) as functions of time following uncaging of glutamate to activate a synaptic current. The voltage response could be described by Ohm's law (for clarity, the time dependence is removed by considering a single time point): V h − V d = V = IR n , (1) where the neck resistance R n is to be determined and the current I is unknown (at all time points). Solving this single equation for two unknowns would clearly require some additional information, but the authors have none. Instead of Ohm's law, the authors employ a more complex 'electrodiffu-sion' model, which describes the resistance in terms of ionic diffusivity and neck geometry. It also accounts for a nonlinearity that arises when strong currents flow and create ionic concentration gradients. The most useful form of the equation is on p10 of the authors' online response (Cartailler and Holcman, 2018), V = k B T e ln 1 + IL 2πF C p r 2 0 D p , (2)

Multiple climbing fibers signal to molecular layer interneurons exclusively via glutamate spillover

International audienceSpillover of glutamate under physiological conditions has only been established as an adjunct to conventional synaptic transmission. Here we describe a pure spillover connection between the climbing fiber and molecular layer interneurons in the rat cerebellar cortex. We show that, instead of acting via conventional synapses, multiple climbing fibers activate AMPA- and NMDA-type glutamate receptors on interneurons exclusively via spillover. Spillover from the climbing fiber represents a form of glutamatergic volume transmission that could be triggered in a regionalized manner by experimentally observed synchronous climbing fiber activity. Climbing fibers are known to direct parallel fiber synaptic plasticity in interneurons, so one function of this spillover is likely to involve controlling synaptic plasticity

Only negligible deviations from electroneutrality are expected in dendritic spines

International audienceThe central aim of the Opinion article by Holcman and Yuste (The new nanophysiology: regulation of ionic flow in neuronal subcompartments. Nat. Rev. Neurosci. 16, 685–692 (2015))1 is to suggest that revolutionary ionic and electrical behaviour will be identified and understood if we no longer apply the classical constraint of electroneutrality when modelling electrodiffusion in small neuronal structures such as dendritic spines. However, as I have argued online2, the authors misunderstand the origin of the electroneutrality approximation and present inconsistent calculations that are irrelevant in a biological context. The result is a misleading and deeply confusing Perspective