Dataset Information

Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABA(A) receptors in cortical pyramidal cells.

ABSTRACT: Recently developed pharmacogenetic and optogenetic approaches, with their own advantages and disadvantages, have become indispensable tools in modern neuroscience. Here, we employed a previously described knock-in mouse line (GABA(A)R?2(77I)lox) in which the ?2 subunit of the GABA(A) receptor (GABA(A)R) was mutated to become zolpidem insensitive (?2(77I)) and used viral vectors to swap ?2(77I) with wild-type, zolpidem-sensitive ?2 subunits (?2(77F)). The verification of unaltered density and subcellular distribution of the virally introduced ?2 subunits requires their selective labelling. For this we generated six N- and six C-terminal-tagged ?2 subunits, with which cortical cultures of GABA(A)R?2(?/?) mice were transduced using lentiviruses. We found that the N-terminal AU1 tag resulted in excellent immunodetection and unimpaired synaptic localization. Unaltered kinetic properties of the AU1-tagged ?2 ((AU1)?2(77F)) channels were demonstrated with whole-cell patch-clamp recordings of spontaneous IPSCs from cultured cells. Next, we carried out stereotaxic injections of lenti- and adeno-associated viruses containing Cre-recombinase and the (AU1)?2(77F) subunit (Cre-2A-(AU1)?2(77F)) into the neocortex of GABA(A)R?2(77I)lox mice. Light microscopic immunofluorescence and electron microscopic freeze-fracture replica immunogold labelling demonstrated the efficient immunodetection of the AU1 tag and the normal enrichment of the (AU1)?2(77F) subunits in perisomatic GABAergic synapses. In line with this,miniature and action potential-evoked IPSCs whole-cell recorded from transduced cells had unaltered amplitudes, kinetics and restored zolpidem sensitivity. Our results obtained with a wide range of structural and functional verification methods reveal unaltered subcellular distributions and functional properties of ?2(77I) and (AU1)?2(77F) GABA(A)Rs in cortical pyramidal cells. This transgenic–viral pharmacogenetic approach has the advantage that it does not require any extrinsic protein that might endow some unforeseen alterations of the genetically modified cells. In addition, this virus-based approach opens up the possibility of modifying multiple cell types in distinct brain regions and performing alternative recombination-based intersectional genetic manipulations.

SUBMITTER: Sumegi M

PROVIDER: S-EPMC3413495 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABA(A) receptors in cortical pyramidal cells.

Sumegi Mate M Fukazawa Yugo Y Matsui Ko K Lorincz Andrea A Eyre Mark D MD Nusser Zoltan Z Shigemoto Ryuichi R

The Journal of physiology 20120220 7

Recently developed pharmacogenetic and optogenetic approaches, with their own advantages and disadvantages, have become indispensable tools in modern neuroscience. Here, we employed a previously described knock-in mouse line (GABA(A)Rγ2(77I)lox) in which the γ2 subunit of the GABA(A) receptor (GABA(A)R) was mutated to become zolpidem insensitive (γ2(77I)) and used viral vectors to swap γ2(77I) with wild-type, zolpidem-sensitive γ2 subunits (γ2(77F)). The verification of unaltered density and sub ...[more]

PMID: 22351636

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Project description:GABA (gamma-aminobutyric acid) is predominantly released by local interneurons in the cerebral cortex to particular subcellular domains of the target cells. This suggests that compartmentalized, synapse-specific action of GABA is required in cortical networks for phasic inhibition. However, GABA released at the synaptic cleft diffuses to receptors outside the postsynaptic density and thus tonically activates extrasynaptic GABA(A) and GABA(B) receptors, which include subtypes of both receptor families especially sensitive to low concentrations of GABA. The synaptic and extrasynaptic action of GABA corroborates the idea that neurons of the brain use synaptic (or wiring) transmission and non-synaptic (or volume) transmission for communication. However, re-uptake mechanisms restrict the spatial extent of extrasynaptic GABA-mediated effects, and it has been proposed that the concerted action of several presynaptic interneurons, the sustained firing of individual cells or an increase in release-site density is required to reach ambient GABA levels sufficient to activate extrasynaptic receptors. Here we show that individual neurogliaform cells release enough GABA for volume transmission within the axonal cloud and, thus, that neurogliaform cells do not require synapses to produce inhibitory responses in the overwhelming majority of nearby neurons. Neurogliaform cells suppress connections between other neurons acting on presynaptic terminals that do not receive synapses at all in the cerebral cortex. They also reach extrasynaptic, delta-subunit-containing GABA(A) (GABA(Adelta)) receptors responsible for tonic inhibition. We show that GABA(Adelta) receptors are localized to neurogliaform cells preferentially among cortical interneurons. Neurosteroids, which are modulators of GABA(Adelta) receptors, alter unitary GABA-mediated effects between neurogliaform cells. In contrast to the specifically placed synapses formed by other interneurons, the output of neurosteroid-sensitive neurogliaform cells represents the ultimate form of the lack of spatial specificity in GABA-mediated systems, leading to long-lasting network hyperpolarization combined with widespread suppression of communication in the local circuit.

Dataset Information

Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABA(A) receptors in cortical pyramidal cells.

Publications

Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABA(A) receptors in cortical pyramidal cells.

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