Abstract
One of the main challenges for a better understanding of signaling mechanisms in the normal and diseased central nervous system (CNS) is to unravel the molecular basics of function on the cellular and systemic levels. Several important cellular processes, such as proliferation, differentiation, and cell death, are directly or indirectly influenced or even controlled by the activity of ion channels and receptors. The patch-clamp technique has been proven a powerful method for studying functional peculiarities of such channels, which considerably improved our knowledge of mechanisms underlying neuronal excitation and inhibition.
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References
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds.) (1996) Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY.
Bordey, A. and Sontheimer, H. (1998) Properties of human glial cells associ ated with epileptic seizure foci. Epilepsy Res. 32, 286–303.
Brenner, C. A., Tam, A. W., Nelson, P. A., Engleman, E. G., Suzuki, N., Fry, K. E., and Larrick, J. W. (1989) Message amplification phenotyping (MAPPing): a technique to simultaneously measure multiple mRNAs from small numbers of cells. BioTechniques 7, 1096–1103.
Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.
Dilworth, D. D. and McCarrey, J. R. (1992) Single-step elimination of contaminating DNA prior to reverse transcriptase PCR. PCR Methods Appl. 1, 279–282.
Dragon, E. A. (1993) Handling reagents in the PCR laboratory. PCR Methods Appl. 3, S8–S9.
Fakler, B. and Ruppersberg, J. P. (1996) Functional and molecular diversity classifies the family of inward-rectifier K+channels. Cell. Physiol. Biochem. 6, 195–209.
Fink, L., Seeger, W., Ermert, L., Hänze, J., Stahl, U., Grimminger, F., et al. (1998) Real-time quantitative RT-PCR after laser-assisted cell picking. Nature Med. 4, 1329–1333.
Gurantz, D., Ribera, A. B., and Spitzer, N. C. (1996) Temporal regulation of Shaker- and Shab-like potassium channel gene expression in single embryonic spinal neurons during K+current development. J. Neurosci. 16, 3287–3295.
Halford, W. P. (1999) The essential prerequisites for quantitative RT-PCR. Nat. Biotechnol. 17, 835.
Hinterkeuser, S., Schröder, W., Hager, G., Seifert, G., Blümcke, I., Elger, C. E., et al. (2000) Astrocytes in the hippocampus of patients with temporal lobe epilepsy display changes in potassium conductances. Eur. J. Neurosci. 12, 2087–2096.
Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H. (1991) Detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88, 7276–7280.
Jauch, R., Gabriel, S., Kivi, A., and Heinemann, U. (1999) Spatial buffering of iontophoretically applied K+in rat hippocampal CA1. Pflugers Arch. 437, R121.
Jonas, P., Racca, C., Sakmann, B., Seeburg, P. H., and Monyer, H. (1994) Differences in Ca2+permeability of AMPA-type glutamate receptor channels in neocortical neurons caused by differential GluR-B subunit expression. Neuron 12, 1281–1289.
Kressin, K., Kuprijanova, E., Jabs, R., Seifert, G., and Steinhäuser, C. (1995) Developmental regulation of Na+and K+conductances in glial cells of mouse hippocampal brain slices. Glia 15, 173–187.
Lambolez, B., Audinat, E., Bochet, P., Crépel, F., and Rossier, J. (1992) AMPA receptor subunits expressed by single Purkinje cells. Neuron 9, 247–258.
Li, H. H., Gyllensten, U. B., Cui, X. F., Saiki, R. K., Erlich, H. A., and Arnheim, N. (1988) Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 335, 414–417.
Livak, K. J., Flood, S. J., Marmaro, J., Giusti, W., and Deetz, K. (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 4, 357–362.
Medhurst, A. D., Harrison, D. C., Read, S. J., Campbell, C. A., Robbins, M. J., and Pangalos, M. N. (2000) The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models. J. Neurosci. Methods 98, 9–20.
Monyer, H. and Jonas, P. (1995) Polymerase chain reaction analysis of ion channel expression in single neurons of brain slices, in Single-Channel Recording, 2nd ed. (Sakmann, B. and Neher, E., eds.), Plenum Press, New York, NY, pp. 357–373.
Newman, E. A. (1986) High potassium conductance in astrocyte endfeet. Science 233, 453–454.
Poopalasundaram, S., Knott, C., Shamotienko, O. G., Foran, P. G., Dolly, J. O., Ghiani, C. A., et al. (2000) Glial heterogeneity in expression of the inwardly rectifying K+channel, Kir4.1, in adult rat CNS. Glia 30, 362–372.
Reimann, F. and Ashcroft, F. M. (1999) Inwardly rectifying potassium channels. Curr. Opin. Cell. Biol. 11, 503–508.
Ruano, D., Lambolez, B., Rossier, J., Paternain, A. V., and Lerma, J. (1995) Kainate receptor subunits expressed in single cultured hippocampal neurons: molecular and functional variants by RNA editing. Neuron 14, 1009–1017.
Schröder, W., Hinterkeuser, S., Seifert, G., Schramm, J., Jabs, R., Wilkin, G. P., and Steinhäuser, C. (2000) Functional and molecular properties of human astrocytes in acute hippocampal slices obtained from patients with temporal lobe epilepsy. Epilepsia 41, S181–S184.
Seifert, G., Rehn, L., Weber, M., and Steinhäuser, C. (1997a) AMPA receptor subunits expressed by single astrocytes in the juvenile mouse hippocampus. Mol. Brain Res. 47, 286–294.
Seifert, G., Zhou, M., and Steinhäuser, C. (1997b) Analysis of AMPA receptor properties during postnatal development of mouse hippocampal astrocytes. J. Neurophysiol. 78, 2916–2923.
Seifert, G., Kuprijanova, E., Zhou, M., and Steinhäuser, C. (1999) Developmental changes in the expression of Shaker- and Shab-related K+channels in neurons of the rat trigeminal ganglion. Mol. Brain Res. 74, 55–68.
Seifert, G., Zhou, M., Dietrich, D., Schumacher, T. B., Dybek, A., Weiser, T., et al. (2000) Developmental regulation of AMPA-receptor properties in CA1 pyramidal neurons of rat hippocampus. Neuropharmacology 39, 931–942.
Steinhäuser, C., Berger, T., Frotscher, M., and Kettenmann, H. (1992) Heteroge-neity in the membrane current pattern of identified glial cells in the hippo-campal slice. Eur. J. Neurosci. 4, 472–484.
Steinhäuser, C., Kressin, K., Kuprijanova, E., Weber, M., and Seifert, G. (1994) Properties of voltage-activated sodium and potassium currents in mouse hippocampal glial cells in situ and after acute isolation from tissue slices. Pflugers Arch. 428, 610–620.
Stonehouse, A. H., Pringle, J. H., Norman, R. I., Stanfield, P. R., Conley, E. C., and Brammar, W. J. (1999) Characterisation of Kir2.0 proteins in the rat cerebellum and hippocampus by polyclonal antibodies. Histochem. Cell Biol. 112, 457–465.
von Eggeling, F. and Ballhausen, W. (1995) Freezing of isolated cells provides free mRNA for RT-PCR amplification. BioTechniques 18, 408–410.
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Seifert, G., Becker, A., Steinhäuser, C. (2002). Combining Patch-Clamp Techniques with RT-PCR. In: Walz, W., Boulton, A.A., Baker, G.B. (eds) Patch-Clamp Analysis. Neuromethods, vol 35. Humana Press. https://doi.org/10.1385/1-59259-276-7:301
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DOI: https://doi.org/10.1385/1-59259-276-7:301
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