Actions and Interactions of Extracellular Potassium and Kainate on Expression of 13 γ-Aminobutyric Acid Type A Receptor Subunits in Cultured Mouse Cerebellar Granule Neurons

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Actions and Interactions of Extracellular Potassium and Kainate on Expression of 13 γ-Aminobutyric Acid Type A Receptor Subunits in Cultured Mouse Cerebellar Granule Neurons

2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês

10.1074/jbc.m300548200

ISSN

1083-351X

Autores

A. Christine Engblom, Flemming Fryd Johansen, Uffe Kristiansen,

Tópico(s)

Lipid Membrane Structure and Behavior

Resumo

Cerebellar granule neurons in culture are a popular model for studying neuronal signaling and development. Depolarizing concentrations of K+ are routinely used to enhance cell survival, and kainate is sometimes added to eliminate GABAergic neurons. We have investigated the effect of these measures on expression of mRNA for γ-aminobutyric acid type A (GABAA) receptor α1–6, औ1–3, γ1–3, and δ subunits in cultures of mouse cerebellar granule neurons grown for 7 or 12 days in vitro (DIV) using semiquantitative reverse transcription-PCR. We detected mRNA for the α1, α2, α5, α6, औ2, औ3, γ2, and δ subunits in all the cell cultures, but the expression levels of the α5-, α6-, and औ2-subunit mRNAs were significantly dependent on the composition of the culture medium. Both an increase of the extracellular K+ concentration from 5 to 25 mm and the addition of 50 ॖm kainate immediately depolarized the neurons but prolonged exposure (7–8 DIV)-induced compensatory hyperpolarization. 25 mmK+ caused a shift from α6 to α5 expression measured at 7 and 12 DIV, which was mimicked by kainate in 12 DIV cultures. The expression of औ2 was decreased by 25 mm K+ in 7 DIV cultures and by kainate in 12 DIV cultures. The effects on औ2 expression could not be ascribed to depolarization. Alterations of α6 mRNA expression were reflected in altered sensitivity to GABA and furosemide of the resulting receptors. Our study has shown that a depolarizing K+ concentration as well as kainate in the culture medium significantly disturbs maturation of GABAAreceptor subunit expression. Cerebellar granule neurons in culture are a popular model for studying neuronal signaling and development. Depolarizing concentrations of K+ are routinely used to enhance cell survival, and kainate is sometimes added to eliminate GABAergic neurons. We have investigated the effect of these measures on expression of mRNA for γ-aminobutyric acid type A (GABAA) receptor α1–6, औ1–3, γ1–3, and δ subunits in cultures of mouse cerebellar granule neurons grown for 7 or 12 days in vitro (DIV) using semiquantitative reverse transcription-PCR. We detected mRNA for the α1, α2, α5, α6, औ2, औ3, γ2, and δ subunits in all the cell cultures, but the expression levels of the α5-, α6-, and औ2-subunit mRNAs were significantly dependent on the composition of the culture medium. Both an increase of the extracellular K+ concentration from 5 to 25 mm and the addition of 50 ॖm kainate immediately depolarized the neurons but prolonged exposure (7–8 DIV)-induced compensatory hyperpolarization. 25 mmK+ caused a shift from α6 to α5 expression measured at 7 and 12 DIV, which was mimicked by kainate in 12 DIV cultures. The expression of औ2 was decreased by 25 mm K+ in 7 DIV cultures and by kainate in 12 DIV cultures. The effects on औ2 expression could not be ascribed to depolarization. Alterations of α6 mRNA expression were reflected in altered sensitivity to GABA and furosemide of the resulting receptors. Our study has shown that a depolarizing K+ concentration as well as kainate in the culture medium significantly disturbs maturation of GABAAreceptor subunit expression. γ-aminobutyric acid type A days in vitro 3-hydroxy-5-methyl-4-isoxazolepropionic acid N-methyl-d-aspartate artificial balanced salt solution concentration eliciting 507 of the maximum current analysis of variance equilibrium potential for K+ The γ-aminobutyric acid type A (GABAA)1 receptor is a transmitter-gated Cl− ion channel assembled from different subunits in a pentameric composition. In the mammalian central nervous system a large family of subunits exist that, based on homology, are grouped into types α1–6, औ1–3, γ1–3, δ, ε, and θ (1Barnard E.A. Skolnick P. Olsen R.W. Mohler H. Sieghart W. Biggio G. Braestrup C. Bateson A.N. Langer S.Z. Pharmacol. Rev. 1998; 50: 291-313PubMed Google Scholar, 2Bonnert T.P. McKernan R.M. Farrar S. Le Bourdellès B. Heavens R.P. Smith D.W. Hewson L. Rigby M.R. Sirinathsinghji D.J.S. Brown N. Wafford K.A. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9891-9896Crossref PubMed Scopus (279) Google Scholar, 3Korpi E.R. Gründer G. Lüddens H. Prog. Neurobiol. 2002; 67: 113-159Crossref PubMed Scopus (412) Google Scholar). The GABAA receptor subunits are expressed in a region- and age-specific manner (4Laurie D.J. Seeburg P.H. Wisden W. J. Neurosci. 1992; 12: 1063-1076Crossref PubMed Google Scholar, 5Laurie D.J. Wisden W. Seeburg P.H. J. Neurosci. 1992; 12: 4151-4172Crossref PubMed Google Scholar, 6Poulter M.O. Barker J.L. O'Carroll A.M. Lolait S.J. Mahan L.C. J. Neurosci. 1992; 12: 2888-2900Crossref PubMed Google Scholar, 7Pirker S. Schwarzer C. Wieselthaler A. Sieghart W. Sperk G. Neuroscience. 2000; 101: 815-850Crossref PubMed Scopus (1072) Google Scholar, 8Sinkkonen S.T. Hanna M.C. Kirkness E.F. Korpi E.R. J. Neurosci. 2000; 20: 3588-3595Crossref PubMed Google Scholar, 9Brooks-Kayal A.R. Shumate M.D. Jin H. Rikhter T.Y. Kelly M.E. Coulter D.A. J. Neurochem. 2001; 77: 1266-1278Crossref PubMed Scopus (107) Google Scholar). The different subunit combinations exhibit distinct properties that presumably underlie a precise physiological role for each subtype (Refs. 10Verdoorn T.A. Mol. Pharmacol. 1994; 45: 475-480PubMed Google Scholar, 11Korpi E.R. Kuner T. Seeburg P.H. Lüddens H. Mol. Pharmacol. 1995; 47: 283-289PubMed Google Scholar, 12Sieghart W. Pharmacol. Rev. 1995; 47: 181-234PubMed Google Scholar, 13Darlison M.G. Albrecht B.E. Semin. Neurosci. 1995; 7: 115-126Crossref Scopus (34) Google Scholar, 14McKernan R. Whiting P. Trends Neurosci. 1996; 19: 139-143Abstract Full Text PDF PubMed Scopus (1069) Google Scholar, 15Saxena N.C. Macdonald R.L. Mol. Pharmacol. 1996; 49: 567-579PubMed Google Scholar, 16Sieghart W. Fuchs K. Tretter V. Ebert V. Jechlinger M. Höger H. Adamiker D. Neurochem. Int. 1999; 34: 379-385Crossref PubMed Scopus (255) Google Scholar, 17Alsbo C.W. Kristiansen U. Moller F. Hansen S.L. Johansen F.F. Eur. J. Neurosci. 2001; 13: 1673-1682Crossref PubMed Scopus (18) Google Scholar, 18Hansen S.L. Ebert B. Fjalland B. Kristiansen U. Br. J. Pharmacol. 2001; 133: 539-549Crossref PubMed Scopus (20) Google Scholar; for review, see Ref. 19Rudolph U. Crestani F. Möhler H. Trends Pharmacol. Sci. 2001; 22: 188-194Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). Knowledge of the distribution of different GABAA receptor subtypes, both anatomically and developmentally, is therefore essential for understanding the physiological actions of GABA and the pharmacological actions of drugs that act on different GABAA receptor subtypes. Cerebellar granule neurons are used for studying neuronal signaling and development because they have a relatively simple morphology and receive most of their inhibitory input from one cell type (for review, see Ref. 20Wisden W. Korpi E.R. Bahn S. Neuropharmacology. 1996; 35: 1139-1160Crossref PubMed Scopus (110) Google Scholar). When cultured in serum-based medium, cerebellar granule neurons express a wide range of receptors and develop stimulus-coupled glutamate release (21Gallo V. Kingsbury A. Balazs R. Jorgensen O.S. J. Neurosci. 1987; 7: 2203-2213Crossref PubMed Google Scholar). In the developing cerebellum, granule neurons express GABAA receptor α2-, α3-, औ3-, γ1-, and γ2-subunit genes (5Laurie D.J. Wisden W. Seeburg P.H. J. Neurosci. 1992; 12: 4151-4172Crossref PubMed Google Scholar). These subunits are replaced in the adult cerebellum where α1, α6, औ2, औ3, γ2, and δ predominate (4Laurie D.J. Seeburg P.H. Wisden W. J. Neurosci. 1992; 12: 1063-1076Crossref PubMed Google Scholar,7Pirker S. Schwarzer C. Wieselthaler A. Sieghart W. Sperk G. Neuroscience. 2000; 101: 815-850Crossref PubMed Scopus (1072) Google Scholar, 22Montpied P. Yan G.M. Paul S.M. Morrow A.L. Dev. Neurosci. 1998; 20: 74-82Crossref PubMed Scopus (5) Google Scholar). The α6 subunit is expressed almost exclusively in cerebellar granule neurons, where it marks neuronal maturation. A similar development evidently occurs in cultures of rat cerebellar granule neurons (e.g. Refs. 23Thompson C.L. Stephenson F.A. J. Neurochem. 1994; 52: 2037-2044Google Scholar, 24Mathews G.C. Bolos-Sy A.M. Holland K.D. Isenberg K.E. Covey D.F. Ferrendelli J.A. Rothman S.M. Neuron. 1994; 13: 149-158Abstract Full Text PDF PubMed Scopus (77) Google Scholar, 25Caruncho H.J. Puia G. Mohler H. Costa E. Neuroscience. 1995; 67: 583-593Crossref PubMed Scopus (39) Google Scholar). It has been suggested that 45–597 of GABAA receptors in the cerebellum of the adult rodent contain α6 subunits (25Caruncho H.J. Puia G. Mohler H. Costa E. Neuroscience. 1995; 67: 583-593Crossref PubMed Scopus (39) Google Scholar, 26Quirk K. Gillar N.P. Ragan C.I. Whiting P.J. McKernan R.M. J. Biol. Chem. 1994; 269: 16020-16028Abstract Full Text PDF PubMed Google Scholar, 27Jechlinger M. Pelz R. Tretter V. Klausberger T. Sieghart W. J. Neurosci. 1998; 18: 2449-2457Crossref PubMed Google Scholar, 28Nusser Z. Ahmad Z. Tretter V. Fuchs K. Wisden W. Sieghart W. Somogyi P. Eur. J. Neurosci. 1999; 11: 1685-1697Crossref PubMed Scopus (95) Google Scholar). The most predominant combinations are proposed to be α1α6औ2/3γ2, α1औ2/3γ2, α6औ2/3γ2, α1α6औ2/3δ, and α6औ2/3δ (25Caruncho H.J. Puia G. Mohler H. Costa E. Neuroscience. 1995; 67: 583-593Crossref PubMed Scopus (39) Google Scholar, 27Jechlinger M. Pelz R. Tretter V. Klausberger T. Sieghart W. J. Neurosci. 1998; 18: 2449-2457Crossref PubMed Google Scholar, 29Pollard S. Thompson C.L. Stephenson F.A. J. Biol. Chem. 1995; 270: 21285-21290Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 30Sigel E. Baur R. J. Neurochem. 2000; 74: 2590-2596Crossref PubMed Scopus (38) Google Scholar); noteworthy is the finding that the δ subunit is found exclusively in combination with α6 subunits (26Quirk K. Gillar N.P. Ragan C.I. Whiting P.J. McKernan R.M. J. Biol. Chem. 1994; 269: 16020-16028Abstract Full Text PDF PubMed Google Scholar, 31Jones A. Korpi E.R. McKernan R.M. Pelz R. Nusser Z. Mäkelä R. Mellor J.R. J. Neurosci. 1997; 17: 1350-1362Crossref PubMed Google Scholar). It is well known that elevated extracellular K+concentrations or other calcium-elevating stimuli promote long term survival of rat cerebellar granule neurons in dissociated cultures (21Gallo V. Kingsbury A. Balazs R. Jorgensen O.S. J. Neurosci. 1987; 7: 2203-2213Crossref PubMed Google Scholar). The physiological extracellular K+ concentration is ∼ 5 mm, but cerebellar granule neuronal cultures are often maintained in 25 mm K+ to enhance survival (21Gallo V. Kingsbury A. Balazs R. Jorgensen O.S. J. Neurosci. 1987; 7: 2203-2213Crossref PubMed Google Scholar). This use of chronic depolarization is questionable, because it affects the subunit gene expression of neurotransmitter receptors and, hence, the receptor composition and function. More specifically, rat granule neurons do not correctly develop their AMPA or NMDA receptor subunit expression in 25 mm K+(32Bessho Y. Nawa H. Nakanishi S. Neuron. 1994; 12: 87-95Abstract Full Text PDF PubMed Scopus (151) Google Scholar, 33Hack N.J. Sluiter A.A. Balázs R. Dev. Brain Res. 1995; 87: 55-61Crossref PubMed Scopus (39) Google Scholar, 34Vallano M.L. Lambolez B. Audinat E. Rossier J. J. Neurosci. 1996; 16: 631-639Crossref PubMed Google Scholar). In addition, the K+ concentration affects GABAA receptor subunit expression in rat (35Harris B.T. Costa E. Grayson D.R. Mol. Brain. Res. 1995; 28: 338-342Crossref PubMed Scopus (43) Google Scholar, 36Gault L.M. Siegel R.E. J. Neurosci. 1997; 17: 2391-2399Crossref PubMed Google Scholar) and mouse (37Mellor J.R. Merlo D. Jones A. Wisden W. Randall A.D. J. Neurosci. 1998; 18: 2822-2833Crossref PubMed Google Scholar) cerebellar granule neurons. Kainate is sometimes used to eliminate GABAergic neurons from cultures of cerebellar granule neurons (38Schousboe A. Meier E. Drejer J. Hertz L. Shahar A. de Vellis J. Vernadakis A. Haber B. A Dissection and Tissue Culture Manual of the Nervous System. Alan R. Liss, New York1989: 203-206Google Scholar). As a glutamate receptor agonist, it causes depolarization (39Dingledine R. Borges K. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar), but its effect on GABAA receptor subunit expression is not as well described as that of K+. The aim of this work was to investigate the effects of extracellular K+ and kainate on cell viability and on the expression of 13 different GABAA receptor subunit mRNAs in cultures of mouse cerebellar granule neurons. The role of membrane potential in mediating the effects of K+ and kainate was assessed by correlating the effects on membrane potential and on mRNA expression. Finally, the relative contribution of the α6 subunit to receptor function was estimated from the sensitivity of the receptors to GABA and to the α6 selective antagonist furosemide (11Korpi E.R. Kuner T. Seeburg P.H. Lüddens H. Mol. Pharmacol. 1995; 47: 283-289PubMed Google Scholar). Cerebellar granule neurons were prepared from 6–8-day-old NMRI mice (Taconic M&B) according to a procedure modified from Courtney et al. (40Courtney M.J. Åkerman K.E.O. Coffey E.T. J. Neurosci. 1997; 17: 4201-4211Crossref PubMed Google Scholar) and Schousboe et al. (38Schousboe A. Meier E. Drejer J. Hertz L. Shahar A. de Vellis J. Vernadakis A. Haber B. A Dissection and Tissue Culture Manual of the Nervous System. Alan R. Liss, New York1989: 203-206Google Scholar). Briefly, trypsin (0.25 mg/ml, Sigma)-dissociated and DNase (50 units/ml, Sigma)-treated cells were plated at ∼300,000 cells/cm2 in 35-mm Petri dishes coated with poly-d-lysine (Sigma). Cells were cultured in Dulbecco's minimum essential medium (Invitrogen) supplemented with 107 (v/v) fetal bovine serum (Invitrogen), 31 mm glucose, 0.2 mm glutamine (Sigma), 4 ॖg/liter insulin (Sigma), 7.3 ॖm p-aminobenzoic acid (Sigma), and 50 units/ml penicillin (LEO Pharma). As appropriate, the medium was further supplemented with 20 mm KCl (to a final K+ concentration of 25 mm) and/or 50 ॖm kainate (Sigma). Culture medium was replaced after ∼24 h with the inclusion of 10 ॖm cytosine arabinoside (Sigma) to reduce non-neuronal proliferation; after this treatment the medium was not changed. The cells were cultured in a humidified 57 CO2 atmosphere at 37 °C. The cultures were used in experiments within 12 days in vitro (DIV). The amount of viable cells in the cerebellar granule neuron cultures was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described (41Jensen J.B. Schousboe A. Pickering D.S. Neurochem. Int. 1998; 32: 505-513Crossref PubMed Scopus (50) Google Scholar). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is reduced to formazan by cells that have functional mitochondria; this process has been shown to correlate well with cell viability (42Mossman T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (45027) Google Scholar). The cultures were tested at 7 and 12 DIV. Reverse transcription-PCR was performed as previously described (43Johansen F.F. Lambolez B. Audinat E. Bochet P. Rossier J. Neurochem. Int. 1995; 26: 239-243Crossref PubMed Scopus (28) Google Scholar). The cultures were tested at 7 and 12 DIV. Primers (TableI) for the GABAA receptor subunits α1, α2, α3, α4, α5, α6, औ1, औ2, औ3, γ1, γ2, γ3, and δ were used. Briefly, RNA was isolated with RNeasy Mini Kit (Qiagen) after lysis of the cells, and the concentration and purification of RNA was measured on a spectrophotometer (Ultrospec4000, Pharmacia Biosciences). cDNA was synthesized using 0.25 ॖg of RNA, and 1 ॖl of the resulting mixture was used for PCR for each subunit. cDNA amplifications were performed in 42 cycles, and aliquots (15 of 100 ॖl in total) of the PCR products were taken from cycles 27, 30, 33, 36, 39, and 42. Relative intensity of the ethidium bromide-stained bands on gels were measured using computer-assisted image analysis and compared with the 400-bp band of the molecular weight marker (100 Base-Pair-Ladder, Amersham Biosciences) added in fixed amount for all gels. Because the efficiency of the PCR amplification is primer-dependent, the relative intensities could not be compared between different subunit mRNAs.Table IThe GABAA receptor subunits and the primer sequences for their mRNASubunitSequencePrimerα15′-AGCTATACCCCTAACTTAGCCAGG-3′Up5′-AGAAAGCGATTCTCAGTGCAGAGG-3′Lowα25′-ACAAGAAGCCAGAGAACAAGCCAG-3′Up5′-GAGGTCTACTGGTAAGCTCTACCA-3′Lowα35′-CAACATAGTGGGAACCACCTATGC-3′Up5′-GGGGTTTGGGATTTTGGATGCTTC-3′Lowα45′-AAATGCAGCTGAGACTATCTCTGC-3′Up5′-AGACAGTCTGTATTTCCATCACGG-3′Lowα55′-CAAGAAGGCCTTGGAAGCAGCTAA-3′Up5′-TCTTACTTTGGAGAGGTAGCCCCT-3′Lowα65′-AAGCCCCCGGTAGCAAAGTCAAAA-3′Up5′-TTCCTGGCTGCAAACTACTCGACA-3′Lowऔ15′-CCTGGAAATCAGGAATGAGACCAG-3′Up5′-GGAGTCTAAACCGAACCATGAGAC-3′Lowऔ25′-TGAGATGGCCACATCAGAAGCAGT-3′Up5′-TCATGGGAGGCTGGAGTTTAGTTC-3′Lowऔ35′-GAAATGAATGAGGTTGCAGGCAGC-3′Up5′-CAGGCAGGGTAATATTTCACTCAG-3′Lowγ15′-CAGAGACAGGAAGCTGAAAAGCAA-3′Up5′-CGAAGTGATTATATTGGACTAAGC-3′Lowγ25′-CTTCTTCGGATGTTTTCCTCTAAG-3′Up5′-TTCGTGAGATTCAGCGAATAAGAC-3′Lowγ35′-CACCACGGTGCTAACCATGACCAC-3′Up5′-TCCTCATAGCAGCAGAAGAAGCTC-3′Lowδ5′-TGAGGAACGCCATTGTCCTCTTCT-3′Up5′-ACCACCGCACGTGGTACATGTAAA-3′Low Open table in a new tab Before recordings, the culture medium was exchanged for an extracellular recording solution (artificial balanced salt solution (ABSS)), and the Petri dish with cells was transferred to an inverted phase-contrast Zeiss Axiovert 10 microscope stage. The cells were constantly perfused with ABSS (0.5 ml/min) at room temperature from a gravity-fed 7-barrel perfusion pipette (List) ∼100 ॖm from the recorded neuron. By switching application from one barrel to another, the extracellular solution surrounding the neuron was exchanged with a time constant of ∼50 ms. Individual cerebellar granule neurons were approached with micropipettes of 3–5-megaohm resistance manufactured from 1.5-mm-outer-diameter glass (World Precision Instruments). Standard patch clamp technique (44Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15066) Google Scholar) in current or voltage clamp mode was used to record from neurons in the whole-cell configuration using an EPC-9 amplifier (HEKA Elektronik). Whole-cell membrane currents and potentials were plotted on a low fidelity chart recorder during the experiment and stored on computer hard disk and video tape using a VR-100 digital data recorder (Instrutech). For recordings of the membrane potentials with extracellular K+ concentration = 5 mm (physiological K+), the cells were perfused with ABSS composed of 138.5 mm NaCl, 5 mm KCl, 1.25 mmNa2HPO4, 2 mm MgSO4, 2 mm CaCl2, 10 mm glucose, and 10 mm HEPES, pH 7.35. For recordings with extracellular K+ concentration = 25 mm (depolarizing K+) the concentration of NaCl was reduced to 118.5 mm, and the concentration of KCl was increased to 25 mm. Kainate was dissolved in ABSS to a final concentration of 50 ॖm, where indicated. The intrapipette solution contained 10 mm NaCl, 130 mm potassium gluconate, 1 mm MgCl2, 1 mmCaCl2, 10 mm EGTA, 2 mm MgATP, and 10 mm HEPES, pH 7.3. Initially the cells were perfused with “physiological” ABSS containing 5 mm K+without kainate. The perfusion was then switched to ABSS with either 25 mm K+ or 50 ॖm kainate or both. After a new stable membrane potential was reached, the perfusion was switched back to physiological ABSS. Each of the depolarizing solutions was tested at least twice on each cell. Membrane potentials were corrected for liquid junction potentials. In experiments addressing the GABA concentration-response relationships or furosemide sensitivities, the ABSS contained 140 mmNaCl, 3.5 mm KCl, 1.25 mmNa2HPO4, 2 mm MgSO4, 2 mm CaCl2, 10 mm glucose, and 10 mm HEPES, pH 7.35. The intrapipette solution contained 140 mm KCl, 1mm MgCl2, 1 mmCaCl2, 10 mm EGTA, 2mm MgATP, and 10 mm HEPES, pH 7.3. The neurons were voltage-clamped at −60 mV. The high intracellular Cl− concentration shifted the Cl− reversal potential to ∼0 mV and substantially increased the currents recorded at −60 mV. Series resistance was 657 compensated. GABA (Sigma) was dissolved in distilled water at a concentration at least 100× greater than that required for perfusion and diluted with ABSS. Furosemide (Sigma) was dissolved in Me2SO and diluted with ABSS; the content of Me2SO in the final solution was at most 0.17 and had no effect of its own on membrane current. Different concentrations of GABA were applied for 5 s at 1-min intervals. Furosemide was preapplied for 10 s immediately before the application of a premixed solution of GABA and furosemide. Between drug applications the cell was perfused with ABSS. Membrane currents and potentials were analyzed using Pulse (HEKA Elektronik) and Igor Pro (Wavemetrics) software. Current responses were quantified by measuring the peak current during application of GABA or GABA plus furosemide. GABA concentration-response relationships were fitted to the equation,I=Imax×[GABA]nEC50n+[GABA]nEquation 1 where I is the peak membrane current induced by the GABA concentration, [GABA], Imax is the maximum peak current that GABA can induce, EC50 is the GABA concentration eliciting 507 of Imax, andn is the Hill coefficient. Data were described using mean and S.E. or 957 confidence intervals. Mean values were compared using either Student'st test or analysis of variance (ANOVA); when relevant, the Tukey test was used as a means of post-hoc multiple comparisons. Two-way ANOVA was used to separate the effects of two variables and determine their interaction. Probabilities (p) < 0.05 were considered statistically significant. The term “occlusion” refers to situations where the effect due to simultaneous variation of two variables was smaller than the sum of the effects due to variation of each variable separately. Fig.1 shows the influence of the culture medium as well as the extracellular recording solution (ABSS) on the membrane potentials of the neurons. The same K+ and kainate concentrations were used for the culture media and extracellular recording solutions: 5 or 25 mm K+ and 0 or 50 ॖm kainate. A two-way ANOVA was used to estimate the effects of 1) culture medium and 2) extracellular recording solution on the membrane potential. At 2–3 DIV the membrane potential was independent of the culture medium but significantly dependent on the extracellular recording solution (p = 0.002). This dependence was further analyzed with a two-way ANOVA of the effects of 1) K+ concentration and 2) kainate concentration in the extracellular recording solution. Both K+ and kainate had highly significant depolarizing effects (p < 0.001 for both) and a significant interaction (p = 0.026), suggesting that the depolarizing effect of simultaneously increasing both K+ or kainate concentrations to 25 mm and 50 ॖm, respectively, was not as great as the sum of the depolarizations caused by increasing K+ or kainate separately (occlusion). Indeed, the combined effect of K+ and kainate did not differ significantly from the individual effects (total occlusion). After 7–8 DIV both culture medium and extracellular recording solution showed significant effects (p < 0.001 for both) without interaction, i.e. their effects were additive (two-way ANOVA). The effect of culture medium was further analyzed with two-way ANOVA. Increased K+ (p = 0.004) or kainate (p = 0.034) in the culture medium significantly hyperpolarized the membrane potentials without interaction. The effect of recording solution was also further analyzed. After 7–8 DIV (as at 2–3 DIV), both increased K+ (p < 0.001) and kainate (p < 0.001) concentrations were significantly depolarizing. The combined effect of increased K+ and kainate concentration was smaller than the sum of the individual effects (p < 0.001) but larger than any of the individual effects (p < 0.001, Tukey test), which were not significantly different. Thus, at this point the effects of K+ and kainate concentrations in the extracellular recording solution were only partially occluding. In conclusion, the immediate effects of increasing either K+ (from 5 to 25 mm) or the kainate concentrations (from 0 to 50 ॖm) in the extracellular recording solution were depolarizations of similar magnitude that were occluding and independent of the culture medium. Prolonged exposure to high K+ or kainate concentrations in the culture medium gave rise to hyperpolarization relative to cells grown in a physiological K+ concentration without kainate. This suggests that the neurons develop a compensatory hyperpolarizing mechanism when exposed to depolarizing conditions for an extended period. The membrane potentials of neurons in the different culture media may influence mRNA expression. These membrane potentials were estimated from measurements in extracellular recording solution with the same K+ and kainate concentration as in the culture medium (Fig.1, columns marked with a number sign). At 2–3 DIV elevated K+ concentration significantly depolarized the membrane potential (two-way ANOVA, p = 0.005); on the other hand, the kainate effect was not significant. After 7–8 DIV both K+ (p < 0.001) and kainate (p < 0.001) were significantly depolarizing, but their combined effect was smaller than the sum of the effects of increasing K+ or kainate separately (two-way ANOVA, p< 0.001); moreover, the combined effect was not significantly larger than the individual effects (total occlusion, Tukey test). No spontaneous synaptic potentials or currents were observed in any of the recorded neurons at 2–3 or 7–8 DIV. In addition, no action potentials were observed, even after depolarizations were induced with K+ or kainate. The survival of mouse cerebellar granule neurons in culture was strongly dependent on the concentration of K+in the culture medium as indicated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Fig. 2). 25 mmK+ significantly (p < 0.001) increased the viability of mouse cerebellar granule neurons in culture regardless of the presence or absence of kainate both at 7 and 12 DIV. At 7 DIV kainate significantly (p < 0.01) increased cell viability in cultures grown in 5 mm but not in 25 mm K+, indicating that the effects of K+ and kainate on viability were not additive. The effect of kainate on cell viability was not detectable after 12 DIV. Of the six GABAA receptor α subunit mRNAs tested for, the α1, α2, α5, and α6 mRNAs were expressed in detectable amounts, whereas α3 mRNA and α4 mRNA could not be detected. The level of α5 mRNA was significantly affected by K+and kainate (Fig. 3). At 7 DIV α5 expression was enhanced with 25 mm K+ in the culture medium relative to 5 mm K+(p < 0.001). At 12 DIV both high K+(p < 0.01) and kainate (p < 0.01) enhanced the expression of α5 to similar levels, but the combination of K+ and kainate did not further increase α5. The α6 mRNA was reciprocally affected (Fig. 3). At 7 DIV expression was significantly decreased in 25 mmK+ compared with 5 mm K+(p < 0.001). At 12 DIV both high K+(p < 0.05) and kainate (p < 0.05) inhibited α6 expression to the same extent and with additive effects. The expression of α1 and α2 mRNA was not significantly affected by the concentration of K+ or kainate in the culture media (results not shown). Of the three GABAA receptor औ subunit mRNAs studied, the औ2 and औ3 were expressed in significant quantities, whereas expression of औ1 was not detected. Elevation of the extracellular K+concentration to 25 mm significantly (p < 0.001) decreased the expression of औ2 mRNA in 7 DIV but not in 12 DIV cultures (Fig. 3). Kainate had no effect on neurons cultured for 7 days, but after 12 DIV it significantly (p < 0.05) decreased the expression of औ2 independently of the K+concentration. Expression of औ3 mRNA was not significantly affected by increased K+ or the addition of kainate (results not shown). Of the three GABAA receptor γ subunits, only γ2 was detected. The δ subunit mRNA was also found in significant amounts. Neither 25 mm K+ nor kainate had a significant effect on expression of γ2 mRNA or δ mRNA (results not shown). To investigate whether differences in mRNA expression between cultures were reflected in the functional properties of the derived membrane-bound receptors, we determined GABA concentration-response relationships resulting from the different culture conditions (Fig. 4). The EC50 values and Hill coefficients are listed in TableII. Neurons cultured for 10–12 DIV in 5 mm K+ without kainate had a significantly lower EC50 value than neurons cultured in 25 mmK+ without kainate (p = 0.005). The Hill coefficients were not significantly different from each other.Table IICharacteristics of GABA-induced currents in cerebellar granule neurons cultured in different mediaCulture mediumEC50 valueHill coefficientsॖm25 mmK+/kainate55 (47–64)0.97 (0.86–1.07)25 mm K+65 (51–84)0.85 (0.72–0.99)5 mmK+/kainate55 (42–72)0.89 (0.72–1.07)5 mmK+36 (27–48)2-aThe EC50 of neurons grown in 5 mmK+ without kainate was significantly smaller compared to neurons grown in 25 mm K+ without kainate (p = 0.005).0.73 (0.60–0.86)The values were estimated by non-linear regression analyses of the concentration-response relationships for GABA-induced peak currents shown in Fig. 4. Numbers in parentheses are 957 confidence intervals for n = 19-29 neurons tested/culture medium.2-a The EC50 of neurons grown in 5 mmK+ without kainate was significantly smaller compared to neurons grown in 25 mm K+ without kainate (p = 0.005). Open table in a new tab The values were estimated by non-linear regression analyses of the concentration-response relationships for GABA-induced peak currents shown in Fig. 4. Numbers in parentheses are 957 confidence intervals for n = 19-29 neurons tested/culture medium. Furosemide has been shown to be a specific antagonist at GABA

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Actions and Interactions of Extracellular Potassium and Kainate on Expression of 13 γ-Aminobutyric Acid Type A Receptor Subunits in Cultured Mouse Cerebellar Granule Neurons