Skip to main content
SpringerLink
Log in

Morphological and Neurochemical Characterization of Electrophysiologically Identified Cells

  • Protocol
Receptor and Ion Channel Detection in the Brain

Part of the book series: Neuromethods ((NM,volume 110))

Abstract

It is now well known that there are many neuron subtypes in brain. For instance, a few subtypes of pyramidal cell in each layer and 10 or more non-pyramidal cell subtypes are found in neocortex. Their activity and functional role in the microcircuit are different among each cell subtype. Therefore, neuron subtype identification is very important to understand the functional role of the recorded neurons whose physiological firing properties are studied. Neuronal subtypes are morphologically and neurochemically distinct, so histological and immunohistochemical staining of the recorded cells promotes the cell identification. In this chapter, histological tissue preparation methods, for chemical marker identification, dendritic and axonal arborization tracing analysis, and observation by electron microscopy including block face scanning microscopy, of biocytin-injected recorded cells are described.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Jones EG (1984) Laminar distribution of cortical efferent cells. In: Cerebral Cortex, vol 1. Cellular Components of the Cerebral Cortex, Plenum, New York, pp 521–553

    Google Scholar 

  2. Hirai Y, Morishima M, Karube F, Kawaguchi Y (2012) Specialized cortical subnetworks differentially connect frontal cortex to parahippocampal areas. J Neurosci 32:1898–1913

    Article  CAS  PubMed  Google Scholar 

  3. Kawaguchi Y, Kubota Y (1997) GABAergic cell subtypes and their synaptic connections in rat frontal cortex. Cereb Cortex 7:476–486

    Article  CAS  PubMed  Google Scholar 

  4. Kubota Y (2014) Untangling GABAergic wiring in the cortical microcircuit. Curr Opin Neurobiol 26:7–14

    Article  CAS  PubMed  Google Scholar 

  5. Kubota Y, Shigematsu N, Karube F, Sekigawa A, Kato S, Yamaguchi N, Hirai Y, Morishima M, Kawaguchi Y (2011) Selective coexpression of multiple chemical markers defines discrete populations of neocortical GABAergic neurons. Cereb Cortex 21:1803–1817

    Article  PubMed  Google Scholar 

  6. Morishima M, Kawaguchi Y (2006) Recurrent connection patterns of corticostriatal pyramidal cells in frontal cortex. J Neurosci 26:4394–4405

    Article  CAS  PubMed  Google Scholar 

  7. Otsuka T, Kawaguchi Y (2011) Cell diversity and connection specificity between callosal projection neurons in the frontal cortex. J Neurosci 31:3862–3870

    Article  CAS  PubMed  Google Scholar 

  8. Uematsu M, Hirai Y, Karube F, Ebihara S, Kato M, Abe K, Obata K, Yoshida S, Hirabayashi M, Yanagawa Y et al (2008) Quantitative chemical composition of cortical GABAergic neurons revealed in transgenic venus-expressing rats. Cereb Cortex 18:315–330

    Article  PubMed  Google Scholar 

  9. Ueta Y, Otsuka T, Morishima M, Ushimaru M, Kawaguchi Y (2014) Multiple layer 5 pyramidal cell subtypes relay cortical feedback from secondary to primary motor areas in rats. Cereb Cortex 24:2362–2376

    Article  PubMed  Google Scholar 

  10. Morita K, Morishima M, Sakai K, Kawaguchi Y (2012) Reinforcement learning: computing the temporal difference of values via distinct corticostriatal pathways. Trends Neurosci 35:457–467

    Article  CAS  PubMed  Google Scholar 

  11. Groh A, Meyer HS, Schmidt EF, Heintz N, Sakmann B, Krieger P (2010) Cell-type specific properties of pyramidal neurons in neocortex underlying a layout that is modifiable depending on the cortical area. Cereb Cortex 20:826–836

    Article  PubMed  Google Scholar 

  12. Otsuka T, Kawaguchi Y (2008) Firing-pattern-dependent specificity of cortical excitatory feed-forward subnetworks. J Neurosci 28:11186–11195

    Article  CAS  PubMed  Google Scholar 

  13. Morishima M, Morita K, Kubota Y, Kawaguchi Y (2011) Highly differentiated projection-specific cortical subnetworks. J Neurosci 31:10380–10391

    Article  CAS  PubMed  Google Scholar 

  14. Ueta Y, Hirai Y, Otsuka T, Kawaguchi Y (2013) Direction- and distance-dependent interareal connectivity of pyramidal cell subpopulations in the rat frontal cortex. Front Neural Circuits 7:164

    Article  PubMed Central  PubMed  Google Scholar 

  15. Klausberger T, Somogyi P (2008) Neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations. Science 321:53–57

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Jiang X, Wang G, Lee AJ, Stornetta RL, Zhu JJ (2013) The organization of two new cortical interneuronal circuits. Nat Neurosci 16:210–218

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Lee S, Kruglikov I, Huang ZJ, Fishell G, Rudy B (2013) A disinhibitory circuit mediates motor integration in the somatosensory cortex. Nat Neurosci 16:1662–1670

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Puig MV, Ushimaru M, Kawaguchi Y (2008) Two distinct activity patterns of fast-spiking interneurons during neocortical UP states. Proc Natl Acad Sci U S A 105:8428–8433

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Yoshiyuki Kubota, Satoru Kondo, Masaki Nomura, Sayuri Hatada, Noboru Yamaguchi, Alsayed A. Mohamed, Fuyuki Karube, Joachim Lubke, Yasuo Kawaguchi (2015) Functional effects of distinct innervation styles of pyramidal cells by fast spiking cortical interneurons eLife (2015) eLife.07919

    Google Scholar 

  20. Jensen FE, Harris KM (1989) Preservation of neuronal ultrastructure in hippocampal slices using rapid microwave-enhanced fixation. J Neurosci Methods 29:217–230

    Article  CAS  PubMed  Google Scholar 

  21. Login GR, Dvorak AM (1988) Microwave fixation provides excellent preservation of tissue, cells and antigens for light and electron microscopy. Histochem J 20:373–387

    Article  CAS  PubMed  Google Scholar 

  22. Kubota Y, Kawaguchi Y (2000) Dependence of GABAergic synaptic areas on the interneuron type and target size. J Neurosci 20:375–386

    CAS  PubMed  Google Scholar 

  23. Somogyi P, Takagi H (1982) A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry. Neuroscience 7:1779–1783

    Article  CAS  PubMed  Google Scholar 

  24. Kawaguchi Y (2009) Anatomical and histological analysis of neurons recorded with electrophysiological method using whole cell electrode. In: Shin patch clamp jikkenn gijutsuho, 2009/10/26 edn, pp 118–131. Yoshioka Shoten in Japanese

    Google Scholar 

  25. Alcantara S, Ruiz M, D’Arcangelo G, Ezan F, de Lecea L, Curran T, Sotelo C, Soriano E (1998) Regional and cellular patterns of reelin mRNA expression in the forebrain of the developing and adult mouse. J Neurosci 18:7779–7799

    CAS  PubMed  Google Scholar 

  26. Deerinck T, Bushong EA, Lev-Ram V, Shu X, Tsien RY, Ellisman MH (2010) Enhancing serial block-face scanning electron microscopy to enable high resolution 3-D nanohistology of cells and tissues. Microsc Microanal 16:1138–1139

    Article  CAS  Google Scholar 

  27. Kawaguchi Y, Kubota Y (1993) Correlation of physiological subgroupings of nonpyramidal cells with parvalbumin- and calbindinD28k-immunoreactive neurons in layer V of rat frontal cortex. J Neurophysiol 70:387–396

    CAS  PubMed  Google Scholar 

  28. Kawaguchi Y, Kubota Y (1998) Neurochemical features and synaptic connections of large physiologically-identified GABAergic cells in the rat frontal cortex. Neuroscience 85:677–701

    Article  CAS  PubMed  Google Scholar 

  29. Kawaguchi Y (1993) Groupings of nonpyramidal and pyramidal cells with specific physiological and morphological characteristics in rat frontal cortex. J Neurophysiol 69:416–431

    CAS  PubMed  Google Scholar 

  30. Kawaguchi Y (1995) Physiological subgroups of nonpyramidal cells with specific morphological characteristics in layer II/III of rat frontal cortex. J Neurosci 15:2638–2655

    CAS  PubMed  Google Scholar 

  31. Puig MV, Watakabe A, Ushimaru M, Yamamori T, Kawaguchi Y (2010) Serotonin modulates fast-spiking interneuron and synchronous activity in the rat prefrontal cortex through 5-HT1A and 5-HT2A receptors. J Neurosci 30:2211–2222

    Article  CAS  PubMed  Google Scholar 

  32. Ushimaru M, Ueta Y, Kawaguchi Y (2012) Differentiated participation of thalamocortical subnetworks in slow/spindle waves and desynchronization. J Neurosci 32:1730–1746

    Article  CAS  PubMed  Google Scholar 

  33. Karube F, Kubota Y, Kawaguchi Y (2004) Axon branching and synaptic bouton phenotypes in GABAergic nonpyramidal cell subtypes. J Neurosci 24:2853–2865

    Article  CAS  PubMed  Google Scholar 

  34. Kubota Y, Hatada SN, Kawaguchi Y (2009) Important factors for the three-dimensional reconstruction of neuronal structures from serial ultrathin sections. Front Neural Circuits 3:4

    Article  PubMed Central  PubMed  Google Scholar 

  35. Kubota Y, Karube F, Nomura M, Gulledge AT, Mochizuki A, Schertel A, Kawaguchi Y (2011) Conserved properties of dendritic trees in four cortical interneuron subtypes. Sci Rep 1:89

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Mikula S, Binding J, Denk W (2012) Staining and embedding the whole mouse brain for electron microscopy. Nat Methods 9:1198–1201

    Article  CAS  PubMed  Google Scholar 

  37. Willingham MC, Rutherford AV (1984) The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells. J Histochem Cytochem 32:455–460

    Article  CAS  PubMed  Google Scholar 

  38. Naoki Shigematsu, Yoshifumi Ueta, Alsayed A. Mohamed, Sayuri Hatada, Takaichi Fukuda, Yoshiyuki Kubota, Yasuo Kawaguchi (2015)Selective thalamic innervation of rat frontal cortical neuronsCerebral Cortex, (Advanced online publication) doi: 10.1093/cercor/bhv124http://cercor.oxfordjournals.org/content/early/2015/06/03/cercor.bhv124.abstract?sid=c935d413-2c23-4747-b336-aa30cc60c216

  39. Hayat MA (2000) Electron microscopy biological applications, 4th edn. Cambridge University Press, Cambridge

    Google Scholar 

  40. Kubota Y (2015) New developments in electron microscopy for serial image acquisition of neuronal profiles. Microscopy (Oxf) 64:27–36

    Article  Google Scholar 

  41. Fiala JC, Kirov SA, Feinberg MD, Petrak LJ, George P, Goddard CA, Harris KM (2003) Timing of neuronal and glial ultrastructure disruption during brain slice preparation and recovery in vitro. J Comp Neurol 465:90–103

    Article  PubMed  Google Scholar 

  42. Kirov SA, Petrak LJ, Fiala JC, Harris KM (2004) Dendritic spines disappear with chilling but proliferate excessively upon rewarming of mature hippocampus. Neuroscience 127:69–80

    Article  CAS  PubMed  Google Scholar 

  43. Kubota Y, Hatada S, Kondo S, Karube F, Kawaguchi Y (2007)Neocortical Inhibitory Terminals Innervate Dendritic Spines Targeted by Thalamocortical AfferentsJ. Neurosci 27: 1139-1150.

    Google Scholar 

Download references

Acknowledgments

We thank Drs. Yasuo Kawaguchi, Satoru Kondo, Mieko Morishima, Fuyuki Karube, and Yasuharu Hirai for neuron drawings, photos, and micrographs in the figures and Drs. Steven R. Vincent, Richard Miles and Fuyuki Karube for valuable comments. This work was supported by Grant-in-Aid for Scientific Research (B) (25290012), Grant-in-Aid for Scientific Research on Innovative Areas “Neural creativity for communication (No. 4103)” (24120718) and “Adaptive circuit shift (No. 3603)” 26112006 from the MEXT of Japan; The Imaging Science Program of National Institutes of Natural Sciences (NINS); Toyoaki Scholarship Foundation; and The Uehara Memorial Foundation.

Author information

Authors and Affiliations

  1. Division of Cerebral Circuitry, National Institute for Physiological Sciences, 5-1 Myodaiji-Higashiyama, Okazaki, Aichi, 444-8787, Japan

    Yoshiyuki Kubota PhD

  2. Department of Physiological Sciences, The Graduate University for Advanced Studies (SOKENDAI), Okazaki, Aichi, 444-8787, Japan

    Yoshiyuki Kubota PhD

Authors
  1. Yoshiyuki Kubota PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yoshiyuki Kubota PhD .

Editor information

Editors and Affiliations

  1. Research Institute on Neurological Disabilities (IDINE), Department of Medical Sciences, School of Medicine, University of Castilla- La Mancha, Albacete, Spain

    Rafael Luján

  2. Pharmacology Unit, Department of Pathology and Experimental Therapeutics, School of Medicine, IDIBELL, University of Barcelona, Barcelona, Spain

    Francisco Ciruela

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Kubota, Y. (2016). Morphological and Neurochemical Characterization of Electrophysiologically Identified Cells. In: Luján, R., Ciruela, F. (eds) Receptor and Ion Channel Detection in the Brain. Neuromethods, vol 110. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3064-7_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3064-7_20

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3063-0

  • Online ISBN: 978-1-4939-3064-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation