236 research outputs found
Excitatory GABA: How a Correct Observation May Turn Out to be an Experimental Artifact
The concept of the excitatory action of GABA during early development is based on data obtained mainly in brain slice recordings. However, in vivo measurements as well as observations made in intact hippocampal preparations indicate that GABA is in fact inhibitory in rodents at early neonatal stages. The apparent excitatory action of GABA seems to stem from cellular injury due to the slicing procedure, which leads to accumulation of intracellular Cl− in injured neurons. This procedural artifact was shown to be attenuated through various manipulations such as addition of energy substrates more relevant to the in vivo situation. These observations question the very concept of excitatory GABA in immature neuronal networks
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. At a heterologous expression of different GlyR subunits it has been shown that blocking potency of NFA is more than one order higher for alpha2 GlyRs than for receptors formed by alpha1 subunit. To reveal the action of NFA on the synaptic activity we analyzed here the effects of NFA on the glycinergic inhibitory post-synaptic currents in the HMs from mouse brainstem slices. In the whole-cell patch clamp configuration, the amplitude and the frequency of glycinergic synaptic currents from two age groups have been analyzed: “neonate” (P2–P4) and “juvenile” (P7–P12). Addition of NFA in the presence of antagonists of glutamate and GABA receptors caused a decrease in the mean amplitude and frequency of synaptic events. The degree of the inhibition induced by NFA decreased with the postnatal development, being higher on the motoneurons from “neonate” brainstem slices in comparison with the “juvenile” age group. Analysis of the pair-pulse facilitation suggests the post-synaptic origin of NFA action. These observations provide evidence on the developmental changes in the inhibition by NFA of glycinergic synaptic transmission, which reflects increase in the alpha1 and decrease in the alpha2 GlyR subunits expression in synapses to hypoglossal motoneurons during the early stages of postnatal life
Optogenetics: Perspectives in biomedical research
© 2016, Nizhny Novgorod State Medical Academy. All rights reserved.Optogenetic tools, photochromic switches and genetically encoded biosensors revolutionized contemporary neuroscience research. These approaches provided unprecedented opportunities for monitoring and modulating the function of specific neurons and have literally shed light on the mechanisms of neuronal networks function in the brain. A number of light-sensitive biosensors for non-invasive monitoring of ions and enzymes have been developed. These molecular designs expand extremely rapidly and a number of new approaches for image analysis of various proteins in living cells have being proposed. In this review we discuss new tools for molecular imaging and remote activation of receptors, ionic channels and synaptic networks, as well as its potential for biomedical research
Frequency-Dependent Cannabinoid Receptor-Independent Modulation of Glycine Receptors by Endocannabinoid 2-AG
Endocannabinoids are known as retrograde messengers, being released from the postsynaptic neuron and acting on specific presynaptic G-protein-coupled cannabinoid (CB) receptors to decrease neurotransmitter release. Also, at physiologically relevant concentrations cannabinoids can directly modulate the function of voltage-gated and receptor-operated ion channels. Using patch-clamp recording we analyzed the consequences of the direct action of an endocannabinoid, 2-arachidonoylglycerol (2-AG), on the functional properties of glycine receptor channels (GlyRs) and ionic currents in glycinergic synapses. At physiologically relevant concentrations (0.1–1 μM), 2-AG directly affected the functions of recombinant homomeric α1H GlyR: it inhibited peak amplitude and dramatically enhanced desensitization. The action of 2-AG on GlyR-mediated currents developed rapidly, within ∼300 ms. Addition of 1 μM 2-AG strongly facilitated the depression of glycine-induced currents during repetitive (4–10 Hz) application of short (2 ms duration) pulses of glycine to outside-out patches. In brainstem slices from CB1 receptor knockout mice, 2-AG significantly decreased the extent of facilitation of synaptic currents in hypoglossal motoneurons during repetitive (10–20 Hz) stimulation. These observations suggest that endocannabinoids can modulate postsynaptic metaplasticity of glycinergic synaptic currents in a CB1 receptor-independent manner
Optogenetics and photopharmacology - effective tools for managing cell activity using light
Contemporary research has been enriched by the new directions in which the light plays a key role as a tool for modulation of cellular activity and invasive monitoring of intracellular ions and other components. The main advantages of these approaches are the possibilities to precisely control the intensity, spectral characteristics and durations of light signals in space and time. This review summarizes the key areas, optogenetics and photopharmacology, - directions that allow to control cellular activity with light. Optogenetics is the use of light-sensitive transmembrane proteins capable of exciting or inhibiting cellular activity under illumination by different wavelengths. In 2003 a light-sensitive protein canalo-rodopsine was isolated and cloned which is capable of inducing ion currents and changing cellular rest potential with its excitation under the blue light when embedded into the neurons or other cell types. Inhibition of cellular activity is caused by expression of other lightsensitive proteins - chloride or hydrogen pumps, or anion-selective ion channels. These principles turns out to be efficacious for the study of the functions of solitary cells and neural nets as well as for the control of living organisms behavior but their use in medicine is complicated because of necessary genetic manipulations. Photopharmacology is based on creating and using of chemical compounds changing conformations and/or activity under the light. Photochromic compounds with the use of photosensitive switches are capable of selective activation or inhibition of the activity of functionally important proteins - receptors, ion channels, enzymes, etc. The principles and the potential use of optogenetics and photopharmacology in the analysis of the neuronal functions and the perspectives for new approaches to treat some diseases of the nervous system are discussed
About a new method to measure fractional Ca2+ currents through ligand-gated ion channels
[No abstract available
Optical Control of GABAA 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 A 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
Molecular probes and switches for functional analysis of receptors, ion channels and synaptic networks
Photochromic switches and genetically encoded biosensors have become powerful tools for monitoring and modulating the activity of neurons and neuronal networks. Our understanding of the mechanisms underlying the development and functioning of the nervous system has greatly advanced in recent years thanks to advancements in these effective molecular and genetic tools. The idea of this Special Research Issue of Frontiers in Molecular Neuroscience is to provide an overview of the approaches in this area of research, and to present new applications in molecular imaging of ions and remote activation of receptors, ionic channels and synaptic networks. The issue contains experimental and methodological papers as well as review articles dealing with molecular tools for investigation and modulation of neuronal function. It can be divided into two main sections: (i) genetically encoded probes for non-invasive monitoring of ions and ATP; and (ii) optogenetic and optopharmacologic tools for control of neuronal activity with light
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 GABAA-mediated currents in a cell line expressing α1/β2/γ2 GABAA 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. Therefore, in this study using whole-cell patch-clamp recording, we analysed the Glyght (100 µM) action on evoked GABAergic inhibitory postsynaptic currents (eIPSCs) in mice hippocampal slices. Two populations of cells were found: the first responded by reducing the GABAergic eIPSCs’ amplitude, whereas the second showed no sensitivity to the compound. Glyght did not affect the ionic currents’ amplitude induced by GABA application, suggesting the absence of action on postsynaptic GABA receptors. Additionally, Glyght had no impact on the paired-pulse modulation of GABAergic eIPSCs, indicating that Glyght does not modulate the neurotransmitter release mechanisms. In the presence of strychnine, an antagonist of glycine receptors, the Glyght effect on GABAergic synaptic transmission was absent. Our results suggest that Glyght can modulate GABAergic synaptic transmission via action on extrasynaptic glycine receptors
Subunit-specific photocontrol of glycine receptors by azobenzene-nitrazepam photoswitcher
© 2021 Maleeva et al. 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 GABAA 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. Glycinergic synaptic neurotransmission determines an important inhibitory drive in the vertebrate nervous system and plays a crucial role in the control of neuronal circuits in the spinal cord and brain stem. GlyRs are involved in locomotion, pain sensation, breathing, and auditory function, as well as in the development of such disorders as hyperekplexia, epilepsy, and autism. Here, we demonstrate that Azo-NZ1 blocks in a UV-dependent manner the activity of a2 GlyRs (GlyR2), while being barely active on a1 GlyRs (GlyR1). The site of Azo-NZ1 action is in the chloride-selective pore of GlyR at the 2’ position of transmembrane helix 2 and amino acids forming this site determine the difference in Azo-NZ1 blocking activity between GlyR2 and GlyR1. This subunit-specific modulation is also shown on motoneurons of brainstem slices from neonatal mice that switch during development from expressing “fetal” GlyR2 to “adult” GlyR1 receptors
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