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Molecules and Membrane Activity: Single-Cell RT-PCR and Patch-Clamp Recording from Central Neurons

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Neuroanatomical Tract-Tracing 3

Abstract

This chapter summarizes methods for characterizing mRNA expression and electrophysiological properties of central neurons using patch-clamp recording and single-cell reverse-transcription/polymerase chain reaction (scRT-PCR). A simple scRT-PCR protocol can be used to identify neurons by the expression of phenotypic marker mRNAs. The combination of these methods allows for the correlation of functional properties with molecular expression. Somewhat more complex methods are available for quantitation of mRNA expression. Both traditional gel-based PCR identification and real-time fluorescent PCR identification methods can be employed. Advantages and requirements of various methods are discussed. Different types of tissue preparations are presented with emphasis on methods used in our laboratories for acutely dissociated or cultured basal forebrain and amygdala neurons. The basal forebrain contains a heterogeneous population of cholinergic and GABAergic neurons, while the amygdala displays neurons with a complex receptor subunit composition. Investigation of neurons with this type of molecular diversity benefits from techniques such as scRT-PCR for cell identification. We also illustrate how these PCR methods can be combined with more complex experimental protocols, such as calcium buffering measurements using fluorescent dyes in dissociated neurons from aged animals. The capacity to combine scRT-PCR with a variety of experimental protocols allows the identification of unique cell types and relationships between physiology and gene expression.

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References

  • Audinat, E., Lambolez, B., and Rossier, J., 1996, Functional and molecular analysis of glutamategated channels by patch-clamp and RT-PCR at the single-cell level, Neurochem. Int. 28:119–136.

    Article  PubMed  CAS  Google Scholar 

  • Bargas, J., Howe, A., Eberwine, J., Cao, Y., and Surmeier, D. J., 1994, Cellular and molecular characterization of Ca2+ currents in acutely isolated, adult rat neostriatal neurons, J. Neurosci. 14:6667–6686.

    PubMed  CAS  Google Scholar 

  • Bartus, R. T., Dean, R. L., III, Beer, B., and Lippa, A. S., 1982, The cholinergic hypothesis of geriatric memory dysfunction, Science 217:408–414.

    Article  PubMed  CAS  Google Scholar 

  • Bochet, P., Audinat, E., Lambolez, B., Crepel, F., Rossier, J., Iino, M., Tsuzuki, K., and Ozawa, S., 1994, Subunit composition at the single-cell level explains functional properties of a glutamate-gated channel, Neuron 12:383–388.

    Article  PubMed  CAS  Google Scholar 

  • Cao, Y., Wilcox, K. S., Martin, C. E., Rachinsky, T. L., Eberwine, J., and Dichter, M. A., 1996, Presence of mRNA for glutamic acid decarboxylase in both excitatory and inhibitory neurons, Proc. Natl. Acad. Sci. U.S.A. 93:9844–9849.

    Article  PubMed  CAS  Google Scholar 

  • Chen, K. C., Blalock, E. M., Thibault, O., Kaminker, P., and Landfield, P. W., 2000, Expression of alpha 1D subunit mRNA is correlated with L-type Ca2+ channel activity in single neurons of hippocampal “zipper” slices, Proc. Natl. Acad. Sci. U.S.A. 97:4357–4362.

    Article  PubMed  CAS  Google Scholar 

  • Chow, N., Cox, C., Callahan, L. M., Weimer, J. M., Guo, L., and Coleman, P. D., 1998, Expression profiles of multiple genes in single neurons of Alzheimer’s disease, Proc. Natl. Acad. Sci. U.S.A. 95:9620–9625.

    Article  PubMed  CAS  Google Scholar 

  • Coyle, J. T., Price, D. L., and DeLong, M. R., 1983, Alzheimer’s disease: a disorder of cortical cholinergic innervation, Science 219:1184–1190.

    Article  PubMed  CAS  Google Scholar 

  • Crino, P., and Eberwine, J., 1996, Molecular characterization of the dendritic growth cone: regulated mRNA transport and local protein synthesis, Neuron 17:1173–1187.

    Article  PubMed  CAS  Google Scholar 

  • Decker, M. W., 1987, The effects of aging on hippocampal and cortical projections of the forebrain cholinergic system, Brain Res. Rev. 12:423–438.

    Article  CAS  Google Scholar 

  • Dixon, A. K., Richardson, P. J., Lee, K., Carter, N. P., and Freeman, T. C., 1998, Expression profiling of single cells using 3 prime end amplification (TPEA) PCR, Nucleic Acids Res. 26:4426–4431.

    Article  PubMed  CAS  Google Scholar 

  • Dixon, A. K., Richardson, P. J., Pinnock, R. D., and Lee, K., 2000, Gene-expression analysis at the single-cell level, Trends Pharmacol. Sci. 21:65–70.

    Article  PubMed  CAS  Google Scholar 

  • Eberwine, J., Kacharmina, J. E., Andrews, C., Miyashiro, K., McIntosh, T., Becker, K., Barrett, T., Hinkle, D., Dent, G., and Marciano, P., 2001, mRNA expression analysis of tissue sections and single cells, J. Neurosci. 21:8310–8314.

    PubMed  CAS  Google Scholar 

  • Eberwine, J., Yeh, H., Miyashiro, K., Cao, Y., Nair, S., Finnell, R., Zettel, M., and Coleman, P., 1992, Analysis of gene expression in single live neurons, Proc. Natl. Acad. Sci. U.S.A. 89:3010–3014.

    Article  PubMed  CAS  Google Scholar 

  • Edwards, M. C., and Gibbs, R. A., 1994, Multiplex PCR: advantages, development, and applications, PCR Methods Appl. 3:S65–S75.

    PubMed  CAS  Google Scholar 

  • Elowitz, M. B., Levine, A. J., Siggia, E. D., and Swain, P. S., 2002, Stochastic gene expression in a single cell, Science 297:1183–1186.

    Article  PubMed  CAS  Google Scholar 

  • Fanselow, M. S., and LeDoux, J. E., 1999, Why we think plasticity underlying Pavlovian fear conditioning occurs in the basolateral amygdala, Neuron 23:229–232.

    Article  PubMed  CAS  Google Scholar 

  • Fibiger, H. C., 1982, The organization and some projections of cholinergic neurons of the mammalian forebrain, Brain Res. 257:327–388.

    PubMed  CAS  Google Scholar 

  • Fischer, W., Gage, F. H., and Bjorklund, A., 1989, Degenerative changes in forebrain cholinergic nuclei correlate with cognitive impairments in aged rats. Eur. J. Neurosci. 1:34–45.

    Article  PubMed  Google Scholar 

  • Floyd, D. W., Jung, K. Y., and McCool, B. A., 2003, Chronic ethanol ingestion facilitates Nmethyl-d-aspartate receptor function and expression in rat lateral/basolateral amygdala neurons, J. Pharmacol. Exp. Ther. 307:1020–1029.

    Article  PubMed  CAS  Google Scholar 

  • Freeman, W. M., Walker, S. J., and Vrana, K. E., 1999, QuantitativeRT-PCR: pitfalls and potential, Biotechniques 26:112–125.

    PubMed  CAS  Google Scholar 

  • Gibson, U. E., Heid, C. A., and Williams, P. M., 1996, A novel method for real time quantitative RT-PCR, Genome Res. 6:995–1001.

    PubMed  CAS  Google Scholar 

  • Griffith, W. H., Jasek, M. C., Bain, S. H., and Murchison, D., 2000, Modification of ion channels and calcium homeostasis of basal forebrain neurons during aging, Behav. Brain Res. 115:219–233.

    Article  PubMed  CAS  Google Scholar 

  • Han, S-H., McCool, B. A., Murchison, D., Nahm, S. S., Parrish, A. R., and Griffith, W. H., 2002, Single-cell RT-PCR detects shifts in mRNA expression profiles of basal forebrain neurons during aging, Mol. Brain Res. 98:67–80.

    Article  PubMed  CAS  Google Scholar 

  • Han, S-H., Murchison, D., and Griffith, W. H., 2005, Low voltage-activated calcium and fast tetrodotoxin-resistant sodium currents define subtypes of cholinergic and noncholinergic neurons in rat basal forebrain, Mol. Brain Res. 134:226–238.

    Article  PubMed  CAS  Google Scholar 

  • Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M., 1996, Real time quantitative PCR, Genome Res. 6:986–994.

    PubMed  CAS  Google Scholar 

  • Hinkle, D., Glanzer, J., Sarabi, A., Pajunen, T., Zielinski, J., Belt, B., Miyashiro, K., McIntosh, T., and Eberwine, J., 2004, Single neurons as experimental systems in molecular biology, Prog. Neurobiol. 72:129–142.

    Article  PubMed  CAS  Google Scholar 

  • Hsiao, S. H., DuBois, D. W., Miranda, R. C., and Frye, G. D., 2004, Critically timed ethanol exposure reduces GABAAR function on septal neurons developing in vivo but not in vitro, Brain Res. 1008:69–80.

    Article  PubMed  CAS  Google Scholar 

  • Johansen, F. F., Lambolez, B., Audinat, E., Bochet, P., and Rossier, J., 1995, Single cell RTPCR proceeds without the risk of genomic DNA amplification, Neurochem. Int. 26:239–243.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • Killcross, S., Robbins, T. W., and Everitt, B. J., 1997, Different types of fear-conditioned behaviour mediated by separate nuclei within amygdala, Nature 388:377–380.

    Article  PubMed  CAS  Google Scholar 

  • Lambolez, B., Audinat, E., Bochet, P., Crepel, F., and Rossier, J., 1992, AMPA receptor subunits expressed by single Purkinje cells, Neuron 9:247–258.

    Article  PubMed  CAS  Google Scholar 

  • Lindqvist, N., Vidal-Sanz, M., and Hallbook, F., 2002, Single cell RT-PCR analysis of tyrosine kinase receptor expression in adult rat retinal ganglion cells isolated by retinal sandwiching, Brain Res. Protoc. 10:75–83.

    Article  CAS  Google Scholar 

  • Liss, B., 2002, Improved quantitative real-time RT-PCR for expression profiling of individual cells, Nucleic Acids Res. 30:e89.

    Article  PubMed  Google Scholar 

  • Martina, M., Schultz, J. H., Ehmke, H., Monyer, H., and Jonas, P., 1998, Functional and molecular differences between voltage-gated K+ channels of fast-spiking interneurons and pyramidal neurons of rat hippocampus, J. Neurosci. 18:8111–8125.

    PubMed  CAS  Google Scholar 

  • McCool, B. A., and Farroni, J. S., 2001, Subunit composition of strychnine-sensitive glycine receptors expressed by adult rat basolateral amygdala neurons, Eur. J. Neurosci. 14:1082–1090.

    Article  PubMed  CAS  Google Scholar 

  • McCool, B. A., Frye, G. D., Pulido, M. D., and Botting, S. K., 2003, Effects of chronic ethanol consumption on rat GABA(A) and strychnine-sensitive glycine receptors expressed by lateral/basolateral amygdala neurons, Brain Res. 963:165–177.

    Article  PubMed  CAS  Google Scholar 

  • McDonald, A. J., 1985, Immunohistochemical identification of gamma-aminobutyric acidcontaining neurons in the rat basolateral amygdala, Neurosci. Lett. 53:203–207.

    Article  PubMed  CAS  Google Scholar 

  • McDonald, A. J., Beitz, A. J., Larson, A. A., Kuriyama, R., Sellitto, C., and Madl, J. E., 1989, Colocalization of glutamate and tubulin in putative excitatory neurons of the hippocampus and amygdala: an immunohistochemical study using monoclonal antibodies, Neuroscience 30:405–421.

    Article  PubMed  CAS  Google Scholar 

  • Medhurst, A. D., Harrison, D. C., Read, S. J., Campbell, C. A., Robbins, M. J., and Pangalos, M. N., 2000, The use ofTaqManRT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models, J. Neurosci. Methods 98:9–20.

    Article  PubMed  CAS  Google Scholar 

  • Mesulam, M. M., Mufson, E. J., Wainer, B. H., and Levey, A. I., 1983, Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Ch1–Ch6), Neuroscience 10:1185–1201.

    Article  PubMed  CAS  Google Scholar 

  • Miyashiro, K., Dichter, M., and Eberwine, J., 1994, Onthe nature and differential distribution of mRNAs in hippocampal neurites: implications for neuronal functioning, Proc. Natl. Acad. Sci. U.S.A. 91:10800–10804.

    Article  PubMed  CAS  Google Scholar 

  • Monyer, H., and Lambolez, B., 1995, Molecular biology and physiology at the single-cell level, Curr. Opin. Neurobiol. 5:382–387.

    Article  PubMed  CAS  Google Scholar 

  • Monyer, H., and Markram, H., 2004, Interneuron diversity series: molecular and genetic tools to study GABAergic interneuron diversity and function, Trends Neurosci. 27:90–97.

    Article  PubMed  CAS  Google Scholar 

  • Mufson, E. J., Counts, S. E., and Ginsberg, S. D., 2002, Gene expression profiles of cholinergic nucleus basalis neurons in Alzheimer’s disease, Neurochem. Res. 27:1035–1048.

    Article  PubMed  CAS  Google Scholar 

  • Murchison, D., and Griffith, W. H., 1996, High-voltage activated calcium currents in basal forebrain neurons during aging, J. Neurophysiol. 76:158–174.

    PubMed  CAS  Google Scholar 

  • Murchison, D., and Griffith, W. H., 1998, Increased calcium buffering in basal forebrain neurons during aging, J. Neurophysiol. 80:350–364.

    PubMed  CAS  Google Scholar 

  • Olton, D. S., Wenk, G. L., and Markowska, A. M., 1991, Basal forebrain, memory and attention, In: Richardson, R. T. (ed.), Activation to Acquisition: Functional Aspects of the Basal Forebrain Cholinergic System, Boston: Birkhauser, pp. 247–262.

    Google Scholar 

  • Panula, P., Revuelta, A.V., Cheney, D. L., Wu, J. Y., and Costa, E., 1984, An immunohistochemical study on the location of GABAergic neurons in rat septum, J. Comp. Neurol. 222:69–80.

    Article  PubMed  CAS  Google Scholar 

  • Pape, J. R., Skynner, M. J., Sim, J. A., and Herbison, A. E., 2001, Profiling gamma-aminobutyric acid (GABA(A)) receptor subunit mRNA expression in postnatal gonadotropin-releasing hormone (GnRH) neurons of the male mouse with single cell RT-PCR, Neuroendocrinology 74:300–308.

    Article  PubMed  CAS  Google Scholar 

  • Phillips, J. K., and Lipski, J., 2000, Single-cell RT-PCR as a tool to study gene expression in central and peripheral autonomic neurones, Auton. Neurosci. 86:1–12.

    Article  PubMed  CAS  Google Scholar 

  • Plant, T. D., Schirra, C., Katz, E., Uchitel, O. D., and Konnerth, A., 1998, Single-cell RT-PCR and functional characterization of Ca2+ channels in motoneurons of the rat facial nucleus, J. Neurosci. 18:9573–9584.

    PubMed  CAS  Google Scholar 

  • Roy, S. W., Fedorov, A., and Gilbert, W., 2003, Large-scale comparison of intron positions in mammalian genes shows intron loss but no gain, Proc. Natl. Acad. Sci. U.S.A. 100:7158–7162.

    Article  PubMed  CAS  Google Scholar 

  • Ruano, D., Perrais, D., Rossier, J., and Ropert, N., 1997, Expression of GABA(A) receptor subunit mRNAs by layer V pyramidal cells of the rat primary visual cortex. Eur. J. Neurosci. 9:857–862.

    Article  PubMed  CAS  Google Scholar 

  • Rye, D. B., Wainer, B. H., Mesulam, M. M., Mufson, E. J., and Saper, C. B., 1984, Cortical projections arising from the basal forebrain: a study of cholinergic and noncholinergic components employing combined retrograde tracing and immunohistochemical localization of choline acetyltransferase, Neuroscience 13:627–643.

    Article  PubMed  CAS  Google Scholar 

  • Santi, M. R., Vicini, S., Eldadah, B., and Neale, J. H., 1994, Analysis by polymerase chain reaction of alpha 1 and alpha 6 GABAA receptor subunit mRNAs in individual cerebellar neurons after whole-cell recordings, J. Neurochem. 63:2357–2360.

    Article  PubMed  CAS  Google Scholar 

  • Sarter, M., and Bruno, J. P., 2000, Cortical cholinergic inputs mediating arousal, attentional processing and dreaming: differential afferent regulation of the basal forebrain by telencephalic and brainstem afferents, Neuroscience 95:933–952.

    Article  PubMed  CAS  Google Scholar 

  • Sarter, M., and Bruno, J. P., 2002, The neglected constituent of the basal forebrain corticopetal projection system: GABAergic projections, Eur. J. Neurosci. 15:1867–1873.

    Article  PubMed  Google Scholar 

  • See, R. E., Fuchs, R. A., Ledford, C. C., and McLaughlin, J., 2003, Drug addiction, relapse, and the amygdala, Ann. NY. Acad. Sci. 985:294–307.

    Article  PubMed  CAS  Google Scholar 

  • Song, W. J., Tkatch, T., Baranauskas, G., Ichinohe, N., Kitai, S. T., and Surmeier, D. J., 1998, Somatodendritic depolarization-activated potassium currents in rat neostriatal cholinergic interneurons are predominantly of the A type and attributable to coexpression of Kv4.2 and Kv4.1 subunits, J. Neurosci. 18:3124–3137.

    PubMed  CAS  Google Scholar 

  • Stahlberg, A., Hakansson, J., Xian, X., Semb, H., and Kubista, M., 2004, Properties of the reverse transcription reaction in mRNA quantification, Clin. Chem. 50:509–515.

    Article  PubMed  CAS  Google Scholar 

  • Subramaniam, S., 1998, The biology workbench—a seamless database and analysis environment for the biologist, Proteins 32:1–2.

    Article  PubMed  CAS  Google Scholar 

  • Sucher, N. J., and Deitcher, D. L., 1995, PCR and patch-clamp analysis of single neurons, Neuron 14:1095–1100.

    Article  PubMed  CAS  Google Scholar 

  • Sucher, N. J., Deitcher, D. L., Baro, D. J., Warrick, R. M., and Guenther, E., 2000, Genes and channels: patch/voltage-clamp analysis and single-cell RT-PCR, Cell Tissue Res. 302:295–307.

    Article  PubMed  CAS  Google Scholar 

  • Surmeier, D. J., Song, W. J., and Yan, Z., 1996, Coordinated expression of dopamine receptors in neostriatal medium spiny neurons, J. Neurosci. 16:6579–6591.

    PubMed  CAS  Google Scholar 

  • Tkatch, T., Baranauskas, G., and Surmeier, D. J., 1998, Basal forebrain neurons adjacent to the globus pallidus co-express GABAergic and cholinergic marker mRNAs, Neuroreport 9:1935–1939.

    PubMed  CAS  Google Scholar 

  • Whitcombe, D., Brownie, J., Gillard, H. L., McKechnie, D., Theaker, J., Newton, C. R., and Little, S., 1998, A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping, Clin. Chem. 44:918–923.

    PubMed  CAS  Google Scholar 

  • Yan, Z., and Surmeier, D. J., 1996, Muscarinic (m2/m4) receptors reduce N-and P-type Ca2+ currents in rat neostriatal cholinergic interneurons through a fast, membrane-delimited, G-protein pathway, J. Neurosci. 16:2592–2604.

    PubMed  CAS  Google Scholar 

  • Zaborszky, L., Carlsen, J., Brashear, H. R., and Heimer, L., 1986, Cholinergic and GABAergic afferents to the olfactory bulb in the rat with special emphasis on the projection neurons in the nucleus of the horizontal limb of the diagonal band, J. Comp. Neurol. 243:488–509.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Medical Pharmacology and Toxicology, College of Medicine, Texas A&M University System Health Science Center, College Station, TX, 77843-1114

    William H. Griffith & David Murchison

  2. Department of Pharmacology, University of California, Irvine, CA, 92697-4625

    Sun-Ho Han

  3. Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC, 27157-1083

    Brian A. McCool

Authors
  1. William H. Griffith
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  2. Sun-Ho Han
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  3. Brian A. McCool
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  4. David Murchison
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Editors and Affiliations

  1. Center for Molecular and Behavioral Neuroscience, Rutgers University, Newark, NJ, USA

    Laszlo Zaborszky

  2. Department of Anatomy, Vrije University Medical Center, Amsterdam, The Netherlands

    Floris G. Wouterlood

  3. Neurosciences Division, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain

    José Luis Lanciego

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Griffith, W.H., Han, SH., McCool, B.A., Murchison, D. (2006). Molecules and Membrane Activity: Single-Cell RT-PCR and Patch-Clamp Recording from Central Neurons. In: Zaborszky, L., Wouterlood, F.G., Lanciego, J.L. (eds) Neuroanatomical Tract-Tracing 3. Springer, Boston, MA . https://doi.org/10.1007/0-387-28942-9_5

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