Imaging Brain Slices

  • Ayumu Tashiro
  • Gloster Aaron
  • Dmitriy Aronov
  • Rosa Cossart
  • Daniella Dumitriu
  • Vivian Fenstermaker
  • Jesse Goldberg
  • Farid Hamzei-Sichani
  • Yuji Ikegaya
  • Sila Konur
  • Jason MacLean
  • Boaz Nemet
  • Volodymyr Nikolenko
  • Carlos Portera-Cailliau
  • Rafael Yuste

Abstract

Brain slices are convenient preparations to study synapses, neurons, and neural circuits because, while they are easily accessed by experimental manipulations such as drug applications, intracellular recordings, and optical imaging, they preserve many of the essential functional properties of these circuits. In this chapter, we describe techniques of live brain-slice imaging used in our laboratory. We cover in detail experimental protocols and know-how acquired over the years about preparing neocortical and hippocampal slices and slice cultures, loading neurons with dyes or using biolistic transfection techniques, two-photon and second harmonic imaging, morphological reconstructions, and image processing and analysis. These techniques are used to study the functional or morphological dynamics of synaptic structures, including dendritic spines and axon terminals, and to characterize circuit connectivity and dynamics.

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References

  1. Agmon, A., and Connors, B.W., 1991, Thalamocortical responses of mouse somatosensory (barrel) cortex in vitro, Neuroscience 41:365–379.CrossRefPubMedGoogle Scholar
  2. Alger, B.E., Dhanjal, S.S., Dingledine, R., Garthwaite, J., Henderson, G., King, G.L., Lipton, P., North, A., Schwartzkroin, P.A., Sears, T.A., Segal, M., Whittingham, T.S., and Williams, J., 1984, Brain slice methods, In: Brain Slices (R. Dingledine, ed.), Plenum Press, New York, pp. 381–437.Google Scholar
  3. Arnold, D., Feng, L., Kim, J., and Heintz, N., 1994, A strategy for the analysis of gene expression during neural development, Proc. Natl. Acad. Sci. USA 91:9970–9974.CrossRefPubMedGoogle Scholar
  4. Beierlein, M., Fall, C.P., Rinzel, J., and Yuste, R., 2002, Thalamocortical bursts trigger recurrent activity in neocortical networks: Layer 4 as a frequencydependent gate, J. Neurosci. 22:9885–9894.Google Scholar
  5. Brenner, S., 2002, Life sciences: Detective rummages, investigates, The Scientist, 16(6):15–16.Google Scholar
  6. Dunaevsky, A., Blazeski, R., Yuste, R., and Mason, C., 2001, Spine motility with synaptic contact, Nat. Neurosci. 4:685–686.Google Scholar
  7. Dunaevsky, A., Tashiro, A., Majewska, A., Mason, C.A., and Yuste, R., 1999 Developmental regulation of spine motility in mammalian CNS, Proc. Natl. Acad. Sci. USA 96:13438–13443.CrossRefPubMedGoogle Scholar
  8. Edwards, F.A., Konnerth, A., Sakmann, B., and Takahashi, T., 1989, A thin slice preparation for patch recordings from neurones of mamalian central nervous system, Pflugers Arch. 414:600–612.CrossRefPubMedGoogle Scholar
  9. Feng, G., Mellor, R.H., Bernstein, M., Keller-Peck, C., Nguyen, Q.T., Wallace, M., Nerbonne, J.M., Lichtman, J.W., and Sanes, J.R., 2000, Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP, Neuron 28:41–51.CrossRefPubMedGoogle Scholar
  10. Franklin, K.B.J., and Paxinos, G., 1997, The mouse brain in stereotaxic coordinates, Academic Press, San Diego, California.Google Scholar
  11. Gan, W.B., Grutzendler, J., Wong, W.T., Wong, R.O., and Lichtman, J.W., 2000, Multicolor “DiOlistic” labeling of the nervous system using lipophilic dye combinations, Neuron 27:219–225.CrossRefPubMedGoogle Scholar
  12. Grynkiewicz, G., Poenie, M., and Tsien, R.Y., 1985, A new generation of Ca2+ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260:3440–3450.PubMedGoogle Scholar
  13. Helmchen, F., Imoto, K., and Sakmann, B., 1996, Ca2+ buffering and action potential-evoked Ca2+ signalling in dendrites of pyramidal neurons, Biophys. J. 70:1069–1081.Google Scholar
  14. Honig, M.G., and Hume, R.I., 1989, DiI and DiO: Versatile fluorescent dyes for neuronal labelling and pathway tracing, Trends Neurosci. 334:333–340.CrossRefGoogle Scholar
  15. Kettunen, P., Demas, J., Lohmann, C., Kasthuri, N., Gong, Y., Wong, R.O., and Gan, W.B., 2002, Imaging calcium dynamics in the nervous system by means of ballistic delivery of indicators, J. Neurosci. Methods 119:37–43.CrossRefPubMedGoogle Scholar
  16. Kozloski, J., Hamzei-Sichani, F., and Yuste, R., 2001, Stereotyped position of local synaptic targets in neocortex, Science 293:868–872.CrossRefPubMedGoogle Scholar
  17. Lechleiter, J.D., Lin, D.T., and Sieneart, I., 2002, Multi-photon laser scanning microscopy using an acoustic optical deflector, Biophys. J. 83:2292–2299.Google Scholar
  18. Lewis, A., Khatchatouriants, A., Treinin, M., Chen, Z., Peleg, G., Friedman, N., Bouevitch, O., Rothman, Z., Loew, L., and Sheves, M., 1999, Second harmonic generation of biological interfaces: Probing membrane proteins and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans, Chem. Phys. 245:133–144.CrossRefGoogle Scholar
  19. Lio, P., 2003, Wavelets in bioinformatics and computational biology: state of art and perspectives, Bioinformatics 19:2–9.CrossRefPubMedGoogle Scholar
  20. Lo, D.C., McAllister, A.K., and Katz, L.C., 1994, Neuronal transfection in brain slices using particle-mediated gene transfer, Neuron 13:1263–1268.CrossRefPubMedGoogle Scholar
  21. Lohmann, C., Myhr, K.L., and Wong, R.O., 2002, Transmitter-evoked local calcium release stabilizes developing dendrites, Nature 418:177–181.CrossRefPubMedGoogle Scholar
  22. Majewska, A., Tashiro, A., and Yuste, R., 2000a, Regulation of spine calcium compartmentalization by rapid spine motility, J. Neurosci. 20:8262–8268.Google Scholar
  23. Majewska, A., Yiu, G., and Yuste, R., 2000b, A custom-made two-photon microscope and deconvolution system, Eur. J. Physiol. 441:398–409.CrossRefGoogle Scholar
  24. Nikolenko, V., Nemet, B., and Yuste, R., 2003, A custom two-photon and second harmonic microscope, Methods. 30(1):3–15.CrossRefPubMedGoogle Scholar
  25. Peterlin, Z.A., Kozloski, J., Mao, B., Tsiola, A., and Yuste, R., 2000, Optical probing of neuronal circuits with calcium indicators, Proc. Natl. Acad. Sci. USA 97:3619–3624.CrossRefPubMedGoogle Scholar
  26. Sheenen, W.J.J.M., Makings, L.R., Gross, L.R., Pozzan, T., and Tsien, R.Y., 1996, Photodegration of indo-1 and its effect on apparent Ca concentrations, Chem. Biol. 3:765–774.Google Scholar
  27. Smetters, D.K., Majewska, A., and Yuste, R., 1999, Detecting action potentials in neuronal populations with calcium imaging, Methods 18:215–221.CrossRefPubMedGoogle Scholar
  28. Tashiro, A., and Yuste, R., 2004, Regulation of dendritic spine motility and stability by Rac1 and Rho kinase: evidence for two forms of spine motility, Mol. Cell Neurosci. 26(3):429–440.CrossRefGoogle Scholar
  29. Tashiro, A., Dunaersky, A., Blazeski, R., Mason, C.A., and Yuste, R., 2003, Bidirectional regulation of hippocampal mossy fiber filopodial motility in kainate receptors: a two-step model of synaptogenesis, Neuron, 38(5):773–784.CrossRefPubMedGoogle Scholar
  30. Tashiro, A., Minden, A., and Yuste, R., 2000, Regulation of dendritic spine morphology by the Rho family of small GTPases: Antagonistic roles of Rac and Rho, Cerebral Cortex 10:927–938.CrossRefPubMedGoogle Scholar
  31. Tsai, P.S., Nishimura, N., Yoder, E.J., White, A., Doluick, E., and Kleinfeld, D., 2002, Principles, design and construction of a two-photon scanning microscope for in vitro and in vivo studies, in Methods for in vivo Optical Imaging (R. Frostig, ed.), CRC Press, pp. 113–171.Google Scholar
  32. Tsien, R.Y., 1989, Fluorescent probes of cell signaling, Ann. Rev. Neurosci. 12:227–253.CrossRefPubMedGoogle Scholar
  33. Tsiola, A., and Yuste, R., 2003, Classification of neurons in the mouse primary visual cortex, J. Comp. Neurol. 461(4):415–428.CrossRefPubMedGoogle Scholar
  34. Yuste, R., 2000a, Loading populations neurons in slices with AM calcium indicators, In: Imaging Neurons: A Laboratory Manual (R. Yuste, F. Lanni, A. Konnerth, eds.), Cold Spring Harbor Press, Cold Spring Harbor, New York, pp. 34.31–34.39.Google Scholar
  35. Yuste, R., 2000b, Imaging Neurons: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York.Google Scholar
  36. Yuste, R., and Denk, W., 1995, Dendritic spines as basic units of synaptic integration, Nature 375:682–684.CrossRefPubMedGoogle Scholar
  37. Yuste, R., and Katz, L.C., 1989, Transmitter-induced changes in intracellular free calcium in brain slice of developing neocortex, Soc. Neurosci. Abstr. 4.5:2.Google Scholar
  38. Yuste, R., and Katz, L.C., 1991, Control of postsynaptic Ca2+ influx in developing neocortex by excitatory and inhibitory neurotransmitters, Neuron 6:333–344.CrossRefPubMedGoogle Scholar
  39. Zilles, K., and Wree, A., 1985, Cortex: A real and laminar structure, In: The Rat Nervous System (G. Paxinos, ed.), Academic Press, Sydney, pp. 375–415.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2006

Authors and Affiliations

  • Ayumu Tashiro
    • 1
  • Gloster Aaron
    • 1
  • Dmitriy Aronov
    • 1
  • Rosa Cossart
    • 1
  • Daniella Dumitriu
    • 1
  • Vivian Fenstermaker
    • 1
  • Jesse Goldberg
    • 1
  • Farid Hamzei-Sichani
    • 1
  • Yuji Ikegaya
    • 1
  • Sila Konur
    • 1
  • Jason MacLean
    • 1
  • Boaz Nemet
    • 1
  • Volodymyr Nikolenko
    • 1
  • Carlos Portera-Cailliau
    • 1
  • Rafael Yuste
    • 1
  1. 1.HHMI, Columbia UniversityNew YorkNew York

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