Light-Activated Agonist-Potentiator of GABA Receptors for Reversible Neuroinhibition in Wildtype Mice.

Gamma aminobutyric acid type A receptors (GABARs) play a key role in the mammalian central nervous system (CNS) as drivers of neuroinhibitory circuits, which are commonly targeted for therapeutic purposes with potentiator drugs. However, due to their widespread expression and strong inhibitory action, systemic pharmaceutical potentiation of GABARs inevitably causes adverse effects regardless of the drug selectivity. Therefore, therapeutic guidelines must often limit or exclude clinically available GABAR potentiators, despite their high efficacy, good biodistribution, and favorable molecular properties.

Analysis of the Effects of Pentose Phosphate Pathway Inhibition on the Generation of Reactive Oxygen Species and Epileptiform Activity in Hippocampal Slices.

The pentose phosphate pathway (PPP) is one of three major pathways involved in glucose metabolism, which is regulated by glucose-6-phosphate dehydrogenase (G6PD) controls NADPH formation. NADPH, in turn, regulates the balance of oxidative stress and reactive oxygen species (ROS) levels. G6PD dysfunction, affecting the PPP, is implicated in neurological disorders, including epilepsy.

Light-Controlled Modulation and Analysis of Neuronal Functions.

Light is an extraordinary tool allowing us to read out and control neuronal functions thanks to its unique properties: it has a great degree of bioorthogonality and is minimally invasive; it can be precisely delivered with high spatial and temporal precision; and it can be used simultaneously or consequently at multiple wavelengths and locations [...

Action of the Photochrome Glyght on GABAergic Synaptic Transmission in Mouse Brain Slices.

Glyght is a new photochromic compound described as an effective modulator of glycine receptors at heterologous expression, in brain slices and in zebrafish larvae. Glyght also caused weak inhibition of GABA-mediated currents in a cell line expressing α1/β2/γ2 GABA receptors. However, the effects of Glyght on GABAergic transmission in the brain have not been analysed, which does not allow a sufficiently comprehensive assessment of the effects of the compound on the nervous system.

Simultaneous Monitoring of pH and Chloride (Cl) in Brain Slices of Transgenic Mice.

Optosensorics is the direction of research possessing the possibility of non-invasive monitoring of the concentration of intracellular ions or activity of intracellular components using specific biosensors. In recent years, genetically encoded proteins have been used as effective optosensory means. These probes possess fluorophore groups capable of changing fluorescence when interacting with certain ions or molecules.

Subunit-Specific Photocontrol of Glycine Receptors by Azobenzene-Nitrazepam Photoswitcher.

Photopharmacology is a unique approach that through a combination of photochemistry methods and advanced life science techniques allows the study and control of specific biological processes, ranging from intracellular pathways to brain circuits. Recently, a first photochromic channel blocker of anion-selective GABA receptors, the azobenzene-nitrazepam-based photochromic compound (Azo-NZ1), has been described. In the present study, using patch-clamp technique in heterologous system and in mice brain slices, site-directed mutagenesis and molecular modeling we provide evidence of the interaction of Azo-NZ1 with glycine receptors (GlyRs) and determine the molecular basis of this interaction.

Photocontrol of Endogenous Glycine Receptors In Vivo.

Glycine receptors (GlyRs) are indispensable for maintaining excitatory/inhibitory balance in neuronal circuits that control reflexes and rhythmic motor behaviors. Here we have developed Glyght, a GlyR ligand controlled with light. It is selective over other Cys-loop receptors, is active in vivo, and displays an allosteric mechanism of action.

Optical Control of GABA Receptors with a Fulgimide-Based Potentiator.

Optogenetic and photopharmacological tools to manipulate neuronal inhibition have limited efficacy and reversibility. We report the design, synthesis, and biological evaluation of Fulgazepam, a fulgimide derivative of benzodiazepine that behaves as a pure potentiator of ionotropic γ-aminobutyric acid receptors (GABA Rs) and displays full and reversible photoswitching in vitro and in vivo. The compound enables high-resolution studies of GABAergic neurotransmission, and phototherapies based on localized, acute, and reversible neuroinhibition.

CLARITY analysis of the Cl/pH sensor expression in the brain of transgenic mice.

Genetically encoded biosensors are widely used in cell biology for the non-invasive imaging of concentrations of ions or the activity of enzymes, to evaluate the distribution of small molecules, proteins and organelles, and to image protein interactions in living cells. These fluorescent molecules can be used either by transient expression in cultured cells or in entire organisms or through stable expression by producing transgenic animals characterized by genetically encoded and heritable biosensors. Using the mouse Thy1 mini-promoter, we generated a line of transgenic mice expressing a genetically encoded sensor for the simultaneous measurements of intracellular Cl and pH.

Azologization of serotonin 5-HT receptor antagonists.

The serotonin 5-hydroxytryptamine 3 receptor (5-HTR) plays a unique role within the seven classes of the serotonin receptor family, as it represents the only ionotropic receptor, while the other six members are G protein-coupled receptors (GPCRs). The 5-HT receptor is related to chemo-/radiotherapy provoked emesis and dysfunction leads to neurodevelopmental disorders and psychopathologies. Since the development of the first serotonin receptor antagonist in the early 1990s, the range of highly selective and potent drugs expanded based on various chemical structures.

A photoswitchable GABA receptor channel blocker.

Background And Purpose: Anion-selective Cys-loop receptors (GABA and glycine receptors) provide the main inhibitory drive in the CNS. Both types of receptor operate via chloride-selective ion channels, though with different kinetics, pharmacological profiles, and localization. Disequilibrium in their function leads to a variety of disorders, which are often treated with allosteric modulators.

Activation of nicotinamide adenine dinucleotide phosphate oxidase is the primary trigger of epileptic seizures in rodent models.

Objective: Despite decades of epilepsy research, 30% of focal epilepsies remain resistant to antiseizure drugs, with effective drug development impeded by lack of understanding on how seizures are initiated. Here, we report the mechanism of seizure onset relevant to most seizures that are characteristic of focal epilepsies.

Methods: Electric and metabolic network parameters were measured using several seizure models in mouse hippocampal slices and acutely induced seizures in rats in vivo to determine metabolic events occurring at seizure onset.

Developmental Changes in the Inhibition of Glycinergic Synaptic Currents by Niflumic Acid in Hypoglossal Motoneurons.

Mammalian brainstem hypoglossal motoneurones (HMs) receive powerful synaptic glycinergic inputs and are involved in a variety of motor functions, including respiration, chewing, sucking, swallowing, and phonation. During the early postnatal development, subunit composition of chloride-permeable glycine receptors (GlyRs) changes leading to a decrease of "fetal" alpha2 and elevation of "adult" alpha1 GlyR subunits. It has been recently demonstrated that niflumic acid (NFA), a member of the fenamate class of non-steroidal anti-inflammatory drugs, is an efficient subunits-specific blocker of GlyRs.

Light-induced regulation of ligand-gated channel activity.

Unlabelled: The control of ligand-gated receptors with light using photochromic compounds has evolved from the first handcrafted examples to accurate, engineered receptors, whose development is supported by rational design, high-resolution protein structures, comparative pharmacology and molecular biology manipulations. Photoswitchable regulators have been designed and characterized for a large number of ligand-gated receptors in the mammalian nervous system, including nicotinic acetylcholine, glutamate and GABA receptors. They provide a well-equipped toolbox to investigate synaptic and neuronal circuits in all-optical experiments.

Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid.

Niflumic acid (NFA) is a member of the fenamate class of nonsteroidal anti-inflammatory drugs. This compound and its derivatives are used worldwide clinically for the relief of chronic and acute pain. NFA is also a commonly used blocker of voltage-gated chloride channels.

Defective synaptic connectivity and axonal neuropathology in a human iPSC-based model of familial Parkinson's disease.

α-Synuclein (αSyn) is the major gene linked to sporadic Parkinson's disease (PD), whereas the G209A (p.A53T) αSyn mutation causes a familial form of PD characterized by early onset and a generally severe phenotype, including nonmotor manifestations. Here we generated de novo induced pluripotent stem cells (iPSCs) from patients harboring the p.

Selective potentiation of alpha 1 glycine receptors by ginkgolic acid.

Glycine receptors (GlyRs) belong to the superfamily of pentameric cys-loop receptor-operated channels and are involved in numerous physiological functions, including movement, vision, and pain. In search for compounds performing subunit-specific modulation of GlyRs we studied action of ginkgolic acid, an abundant Ginkgo biloba product. Using patch-clamp recordings, we analyzed the effects of ginkgolic acid in concentrations from 30 nM to 25 μM on α1-α3 and α1/β, α2/β configurations of GlyR and on GABAARs expressed in cultured CHO-K1 cells and mouse neuroblastoma (N2a) cells.

Efficient and cost-effective generation of mature neurons from human induced pluripotent stem cells.

For years, our ability to study pathological changes in neurological diseases has been hampered by the lack of relevant models until the recent groundbreaking work from Yamanaka's group showing that it is feasible to generate induced pluripotent stem cells (iPSCs) from human somatic cells and to redirect the fate of these iPSCs into differentiated cells. In particular, much interest has focused on the ability to differentiate human iPSCs into neuronal progenitors and functional neurons for relevance to a large number of pathologies including mental retardation and behavioral or degenerative syndromes. Current differentiation protocols are time-consuming and generate limited amounts of cells, hindering use on a large scale.

Crystal structures of free and antagonist-bound states of human α9 nicotinic receptor extracellular domain.

We determined the X-ray crystal structures of the extracellular domain (ECD) of the monomeric state of human neuronal α9 nicotinic acetylcholine receptor (nAChR) and of its complexes with the antagonists methyllycaconitine and α-bungarotoxin at resolutions of 1.8 Å, 1.7 Å and 2.

Reactive oxygen species initiate a metabolic collapse in hippocampal slices: potential trigger of cortical spreading depression.

Excessive accumulation of reactive oxygen species (ROS) underlies oxidative damage. We find that in hippocampal slices, decreased activity of glucose-based antioxidant system induces a massive, abrupt, and detrimental change in cellular functions. We call this phenomenon metabolic collapse (MC).

Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride.

Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli].