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AMPA receptor regulation at the mRNA and protein level in rat primary cortical cultures.

ABSTRACT: Ionotropic glutamate ?-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are the major mediators of fast synaptic neurotransmission. In this work, we used primary cortical cultures from rats as a model system to study AMPA receptor regulation during in vitro cell maturation and after synaptic activity modifications. The levels of AMPA receptor mRNA and protein, along with the alternative splicing and RNA editing of the AMPA receptor subunit (GluR1-4) mRNAs, were analyzed in immature (DIV5) and mature (DIV26) rat neuronal cultures. We observed an increase in the expression of all four AMPA receptor subunits during in vitro neuronal maturation. This finding might be due to the formation of new synapses between neurons during the development of a complex neuronal network. We also analyzed the effects of stimulation (KCl and glutamate) and inhibition (APV/TTX) on rat mature neuronal cultures (DIV26): stimulation with KCl led to an overall down-regulation of GluR1 and GluR3 AMPA receptor subunits and an up-regulation of the GluR2 subunit. Similarly, glutamate treatment induced a significant down-regulation of GluR1 together with an up-regulation of GluR2. In contrast, the chronic blockade of neuronal activity that resulted from APV/TTX treatment up-regulated GluR1 and GluR3 with a parallel down-regulation of GluR2 and GluR4. RNA editing at the R/G site increased during neuronal cell maturation for all AMPA receptors (from 8-39% at DIV5 to 28-67% at DIV26). Unexpectedly, all the treatments tested induced a marked reduction (ranging from -9% to -52%) of R/G editing levels in mature neurons, primarily for the mRNA flip variant. In summary, we showed that cultured rat cortical neurons are able to vary the stoichiometric ratios of the AMPA receptor subunits and to control post-transcriptional processes to adapt fast synaptic transmission under different environmental conditions.

SUBMITTER: Orlandi C

PROVIDER: S-EPMC3178644 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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AMPA receptor regulation at the mRNA and protein level in rat primary cortical cultures.

Orlandi Cesare C La Via Luca L Bonini Daniela D Mora Cristina C Russo Isabella I Barbon Alessandro A Barlati Sergio S

PloS one 20110922 9

Ionotropic glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are the major mediators of fast synaptic neurotransmission. In this work, we used primary cortical cultures from rats as a model system to study AMPA receptor regulation during in vitro cell maturation and after synaptic activity modifications. The levels of AMPA receptor mRNA and protein, along with the alternative splicing and RNA editing of the AMPA receptor subunit (GluR1-4) mRNAs, were analyzed in im ...[more]

PMID: 21966506

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Project description:BackgroundCortical cultures grown long-term on multi-electrode arrays (MEAs) are frequently and extensively used as models of cortical networks in studies of neuronal firing activity, neuropharmacology, toxicology and mechanisms underlying synaptic plasticity. However, in contrast to the predominantly asynchronous neuronal firing activity exhibited by intact cortex, electrophysiological activity of mature cortical cultures is dominated by spontaneous epileptiform-like global burst events which hinders their effective use in network-level studies, particularly for neurally-controlled animat ('artificial animal') applications. Thus, the identification of culture features that can be exploited to produce neuronal activity more representative of that seen in vivo could increase the utility and relevance of studies that employ these preparations. Acetylcholine has a recognised neuromodulatory role affecting excitability, rhythmicity, plasticity and information flow in vivo although its endogenous production by cortical cultures and subsequent functional influence upon neuronal excitability remains unknown.ResultsConsequently, using MEA electrophysiological recording supported by immunohistochemical and RT-qPCR methods, we demonstrate for the first time, the presence of intrinsic cholinergic neurons and significant, endogenous cholinergic tone in cortical cultures with a characterisation of the muscarinic and nicotinic components that underlie modulation of spontaneous neuronal activity. We found that tonic muscarinic ACh receptor (mAChR) activation affects global excitability and burst event regularity in a culture age-dependent manner whilst, in contrast, tonic nicotinic ACh receptor (nAChR) activation can modulate burst duration and the proportion of spikes occurring within bursts in a spatio-temporal fashion.ConclusionsWe suggest that the presence of significant endogenous cholinergic tone in cortical cultures and the comparability of its modulatory effects to those seen in intact brain tissues support emerging, exploitable commonalities between in vivo and in vitro preparations. We conclude that experimental manipulation of endogenous cholinergic tone could offer a novel opportunity to improve the use of cortical cultures for studies of network-level mechanisms in a manner that remains largely consistent with its functional role.

Project description:Cortical rat neuronal culture, lysis and proteolytic digestion Primary rat cortical neurons were generated from E18 Sprague-Dawley rat embryos with minor modifications (Swanger, Mattheyses et al. 2015, Henderson, Greathouse et al. 2019). Neurons were cultured at a density of 4x105 cells per well in 12-well culture plates (Fisher Scientific, catalog no. 353043). Neurons were cultured in Neurobasal medium (Fisher Scientific, catalog no. 21103-049) supplemented with B27 (Fisher Scientific, catalog no.17504-044). Culture maintenance included a half media change every 2-3 days. At DIV 14, neurons were either treated with 150 ng/mL recombinant NRN1 protein (Abcam, ab69755) or vehicle treated with diH2O for 6 hours. NRN1 concentration was chosen based on published data that identified a plateau in exogenous NRN1 induced effects on transient potassium currents at 150 ng/mL (Yao et al., 2012). After 6 hours neurons were washed 2x with 1 mL 1X phosphate-buffered saline (PBS). To harvest cells, 1 mL 1X PBS + protease inhibitor (Fisher Scientific, catalog no. 78426) was added and cells were centrifuged for 2300rpm for 5 minutes at 4°C. Cell pellets were lysed in 200uL 8M urea buffer and HALT protease and phosphatase inhibitor cocktail (1x final concentration). Lysates were sonicated with a probe sonicator 3 times for 10 s with 10 s intervals at 30% amplitude and cleared of cellular debris by centrifugation in a tabletop centrifuge at 18,000 rcf for 3 minutes at 4° C. Protein concentration was determined by BCA assay and one-dimensional SDS-PAGE gels were run followed by Coomassie blue staining as quality control for protein integrity and equal loading before proceeding to protein digestion. Protein homogenates (50 µg) were diluted with 50 mM NH4HCO3 to a final concentration of less than 2 M urea and then treated with 1 mM dithiothreitol (DTT) at 25°C for 30 minutes, followed by 5 mM iodoacetimide (IAA) at 25°C for 30 minutes in the dark. Protein was digested with 1:100 (w/w) lysyl endopeptidase (Wako) at 25°C for 2 hours and further digested overnight with 1:50 (w/w) trypsin (Pierce) at 25°C. Resulting peptides were desalted with a Sep-Pak C18 column (Waters) and dried under vacuum.

Dataset Information

AMPA receptor regulation at the mRNA and protein level in rat primary cortical cultures.

Publications

AMPA receptor regulation at the mRNA and protein level in rat primary cortical cultures.

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