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      Assessment of Methods for the Intracellular Blockade of GABA A Receptors

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      1 , 2 , 1 , 1 , *
      PLoS ONE
      Public Library of Science

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          Abstract

          Selective blockade of inhibitory synaptic transmission onto specific neurons is a useful tool for dissecting the excitatory and inhibitory synaptic components of ongoing network activity. To achieve this, intracellular recording with a patch solution capable of blocking GABA A receptors has advantages over other manipulations, such as pharmacological application of GABAergic antagonists or optogenetic inhibition of populations of interneurones, in that the majority of inhibitory transmission is unaffected and hence the remaining network activity preserved. Here, we assess three previously described methods to block inhibition: intracellular application of the molecules picrotoxin, 4,4’-dinitro-stilbene-2,2’-disulphonic acid (DNDS) and 4,4’-diisothiocyanostilbene-2,2’-disulphonic acid (DIDS). DNDS and picrotoxin were both found to be ineffective at blocking evoked, monosynaptic inhibitory postsynaptic currents (IPSCs) onto mouse CA1 pyramidal cells. An intracellular solution containing DIDS and caesium fluoride, but lacking nucleotides ATP and GTP, was effective at decreasing the amplitude of IPSCs. However, this effect was found to be independent of DIDS, and the absence of intracellular nucleotides, and was instead due to the presence of fluoride ions in this intracellular solution, which also blocked spontaneously occurring IPSCs during hippocampal sharp waves. Critically, intracellular fluoride ions also caused a decrease in both spontaneous and evoked excitatory synaptic currents and precluded the inclusion of nucleotides in the intracellular solution. Therefore, of the methods tested, only fluoride ions were effective for intracellular blockade of IPSCs but this approach has additional cellular effects reducing its selectivity and utility.

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          A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex.

          A key obstacle to understanding neural circuits in the cerebral cortex is that of unraveling the diversity of GABAergic interneurons. This diversity poses general questions for neural circuit analysis: how are these interneuron cell types generated and assembled into stereotyped local circuits and how do they differentially contribute to circuit operations that underlie cortical functions ranging from perception to cognition? Using genetic engineering in mice, we have generated and characterized approximately 20 Cre and inducible CreER knockin driver lines that reliably target major classes and lineages of GABAergic neurons. More select populations are captured by intersection of Cre and Flp drivers. Genetic targeting allows reliable identification, monitoring, and manipulation of cortical GABAergic neurons, thereby enabling a systematic and comprehensive analysis from cell fate specification, migration, and connectivity, to their functions in network dynamics and behavior. As such, this approach will accelerate the study of GABAergic circuits throughout the mammalian brain. Copyright © 2011 Elsevier Inc. All rights reserved.
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            Synaptic mechanisms of synchronized gamma oscillations in inhibitory interneuron networks.

            Gamma frequency oscillations are thought to provide a temporal structure for information processing in the brain. They contribute to cognitive functions, such as memory formation and sensory processing, and are disturbed in some psychiatric disorders. Fast-spiking, parvalbumin-expressing, soma-inhibiting interneurons have a key role in the generation of these oscillations. Experimental analysis in the hippocampus and the neocortex reveals that synapses among these interneurons are highly specialized. Computational analysis further suggests that synaptic specialization turns interneuron networks into robust gamma frequency oscillators.
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              The variable discharge of cortical neurons: implications for connectivity, computation, and information coding.

              Cortical neurons exhibit tremendous variability in the number and temporal distribution of spikes in their discharge patterns. Furthermore, this variability appears to be conserved over large regions of the cerebral cortex, suggesting that it is neither reduced nor expanded from stage to stage within a processing pathway. To investigate the principles underlying such statistical homogeneity, we have analyzed a model of synaptic integration incorporating a highly simplified integrate and fire mechanism with decay. We analyzed a "high-input regime" in which neurons receive hundreds of excitatory synaptic inputs during each interspike interval. To produce a graded response in this regime, the neuron must balance excitation with inhibition. We find that a simple integrate and fire mechanism with balanced excitation and inhibition produces a highly variable interspike interval, consistent with experimental data. Detailed information about the temporal pattern of synaptic inputs cannot be recovered from the pattern of output spikes, and we infer that cortical neurons are unlikely to transmit information in the temporal pattern of spike discharge. Rather, we suggest that quantities are represented as rate codes in ensembles of 50-100 neurons. These column-like ensembles tolerate large fractions of common synaptic input and yet covary only weakly in their spike discharge. We find that an ensemble of 100 neurons provides a reliable estimate of rate in just one interspike interval (10-50 msec). Finally, we derived an expression for the variance of the neural spike count that leads to a stable propagation of signal and noise in networks of neurons-that is, conditions that do not impose an accumulation or diminution of noise. The solution implies that single neurons perform simple algebra resembling averaging, and that more sophisticated computations arise by virtue of the anatomical convergence of novel combinations of inputs to the cortical column from external sources.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                8 August 2016
                2016
                : 11
                : 8
                : e0160900
                Affiliations
                [1 ]Centre for Synaptic Plasticity, School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom
                [2 ]School of Engineering Mathematics, University of Bristol, Bristol, United Kingdom
                Dalhousie University, CANADA
                Author notes

                Competing Interests: The authors have the following interests. This study was partly funded by Eli Lilly & Company. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

                • Conceptualization: LAA JRM.

                • Funding acquisition: JRM.

                • Investigation: LAA ESB.

                • Project administration: JRM.

                • Supervision: JRM.

                • Visualization: LAA ESB JRM.

                • Writing - original draft: LAA ESB JRM.

                • Writing - review & editing: LAA ESB JRM.

                Article
                PONE-D-16-23198
                10.1371/journal.pone.0160900
                4976935
                27501143
                d44a353b-d2f9-48cc-a3ce-f94c856c3316
                © 2016 Atherton et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 9 June 2016
                : 26 July 2016
                Page count
                Figures: 5, Tables: 1, Pages: 22
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100000266, Engineering and Physical Sciences Research Council;
                Award Recipient :
                Funded by: Eli Lilly and Company (US)
                Award Recipient :
                Funded by: Biotechnology and Biological Sciences Research Council (GB)
                Award Recipient :
                Funded by: Wellcome Trust (GB)
                Award Recipient :
                Funded by: Biotechnology and Biological Sciences Research Council (GB)
                Award Recipient :
                LAA supported by Engineering and Physical Sciences Research Council and Eli Lilly & Company, ESB supported by Biotechnology and Biological Sciences Research Council UK, and JRM supported by Wellcome Trust and Biotechnology and Biological Sciences Research Council UK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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