Combined Immunochemistry and Live Imaging of Fluorescent Protein Expressing Neurons in Mouse Brain

  • Ruth M. Empson
  • Malinda L. S. Tantirigama
  • Manfred J. Oswald
  • Stephanie M. Hughes
  • Thomas KnöpfelEmail author
Part of the Neuromethods book series (NM, volume 101)


The use of transgenic mice expressing fluorescent proteins to report a specific protein or to identify specific groups of neurons in the brain is revolutionizing many different aspects of neuroscience. Here we use an example of a GFP-expressing reporter mouse from the GENSAT project that allows identification of a specific group of neurons in the mouse cortex. Live GFP detection facilitates identification of the neurons for whole-cell patch clamp electrophysiological recording to probe their functional properties. Post hoc immunohistochemistry allows specific reconstruction of the shape of the recorded neuron; this together with the detection of other co-expressed proteins helps confirm the functional identity of specific neuron types. Approaches such as these are beginning to progress the major task of untangling the complexity of a variety of brain circuits.

Key words

GENSAT Motor cortex Whole-cell electrophysiology E-GFP reporter Layer 5 



Supported by the Marsden Fund Council from Government funding, administered by the Royal Society of New Zealand. M.T. is the recipient of a Department of Physiology, University of Otago PhD scholarship.


  1. 1.
    Miller MN, Okaty BW, Nelson SB (2008) Region-specific spike-frequency acceleration in layer 5 pyramidal neurons mediated by Kv1 subunits. J Neurosci 28:13716–13726CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Yu J, Anderson CT, Kiritani T et al (2008) Local-circuit phenotypes of layer 5 neurons in motor-frontal cortex of YFP-H mice. Front Neural Circuits 2:6CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Porrero C, Rubio-Garrido P, Avendaño C et al (2010) Mapping of fluorescent protein- expressing neurons and axon pathways in adult and developing Thy-eYFP-H transgenic mice. Brain Res 1345:59–72CrossRefPubMedGoogle Scholar
  4. 4.
    Akemann W, Zhong Y-M, Ichinohe N et al (2004) Transgenic mice expressing a fluorescent in vivo label in a distinct subpopulation of neocortical layer 5 pyramidal cells. J Comp Neurol 480:72–88CrossRefPubMedGoogle Scholar
  5. 5.
    Uusisaari M, Knöpfel T (2010) GlyT2+ neurons in the lateral cerebellar nucleus. Cerebellum 9:42–55CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Groh A, Meyer HS, Schmidt EF et al (2009) Cell-type specific properties of pyramidal neurons in neocortex underlying a layout that is modifiable depending on the cortical area. Cereb Cortex 20:826–836CrossRefPubMedGoogle Scholar
  7. 7.
    Heintz N (2004) Gene expression nervous system atlas (GENSAT). Nat Neurosci 7:483CrossRefPubMedGoogle Scholar
  8. 8.
    Arlotta P, Molyneaux BJ, Chen J et al (2005) Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45:207–221CrossRefPubMedGoogle Scholar
  9. 9.
    Chen B, Schaevitz LR, McConnell SK (2005) Fezl regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex. Proc Natl Acad Sci U S A 102:17184–17189CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Chen J-G, Rašin M-R, Kwan KY et al (2005) Zfp312 is required for subcortical axonal projections and dendritic morphology of deep-layer pyramidal neurons of the cerebral cortex. Proc Natl Acad Sci U S A 102:17792–17797CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Molyneaux BJ, Arlotta P, Hirata T et al (2005) Fezl is required for the birth and specification of corticospinal motor neurons. Neuron 47:817–831CrossRefPubMedGoogle Scholar
  12. 12.
    Molyneaux BJ, Arlotta P, Menezes JRL et al (2007) Neuronal subtype specification in the cerebral cortex. Nat Rev Neurosci 8:427–437CrossRefPubMedGoogle Scholar
  13. 13.
    Leone DP, Srinivasan K, Chen B et al (2008) The determination of projection neuron identity in the developing cerebral cortex. Curr Opin Neurobiol 18:28–35CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Nelson SB, Sugino K, Hempel CM (2006) The problem of neuronal cell types: a physiological genomics approach. Trends Neurosci 29:339–345CrossRefPubMedGoogle Scholar
  15. 15.
    Rouaux C, Bhai S, Arlotta P (2012) Programming and reprogramming neuronal subtypes in the central nervous system. Dev Neurobiol 72:1085–1098CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Özdinler PH, Benn S, Yamamoto TH et al (2011) Corticospinal motor neurons and related subcerebral projection neurons undergo early and specific neurodegeneration in hSOD1G93A transgenic ALS mice. J Neurosci 31:4166–4177CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Franklin K, Paxinos G (2001) The mouse brain in stereotaxic coordinates, 2nd edn. London Academic Press, San DiegoGoogle Scholar
  18. 18.
    Anderson CT, Sheets PL, Kiritani T et al (2010) Sublayer-specific microcircuits of corticospinal and corticostriatal neurons in motor cortex. Nat Neurosci 13:739–744CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Meijering E, Jacob M, Sarria JCF et al (2004) Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. Cytometry A 58A:167–176CrossRefGoogle Scholar
  20. 20.
    Chen B, Wang SS, Hattox AM et al (2008) The Fezf2-Ctip2 genetic pathway regulates the fate choice of subcortical projection neurons in the developing cerebral cortex. Proc Natl Acad Sci U S A 105:11382–11387CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Alcamo EA, Chirivella L, Dautzenberg M et al (2008) Satb2 regulates callosal projection neuron identity in the developing cerebral cortex. Neuron 57:364–377CrossRefPubMedGoogle Scholar
  22. 22.
    Britanova O, de Juan Romero C, Cheung A et al (2008) Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex. Neuron 57:378–392CrossRefPubMedGoogle Scholar
  23. 23.
    Han W, Kwan KY, Shim S (2011) TBR1 directly represses Fezf2 to control the laminar origin and development of the corticospinal tract. Proc Natl Acad Sci U S A 108:3041–3046CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    McKenna WL, Betancourt J, Larkin KA et al (2011) Tbr1 and Fezf2 regulate alternate corticofugal neuronal identities during neocortical development. J Neurosci 31:549–564CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Tantirigama MLS, Oswald MJ, Duynstee C et al (2014) Expression of the developmental transcription factor Fezf2 identifies a distinct subpopulation of layer 5 intratelencephalic-projection neurons in mature mouse motor cortex. J Neurosci 34(12):4303–4308CrossRefPubMedGoogle Scholar
  26. 26.
    Dombeck DA, Harvey CD, Tian L et al (2010) Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nat Neurosci 13:1433–1440CrossRefPubMedCentralPubMedGoogle Scholar
  27. 27.
    Muto A, Ohkura M, Kotani T et al (2011) Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish. Proc Natl Acad Sci U S A 108:5425–5430CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Akemann W, Mutoh H, Perron A (2010) Imaging brain electric signals with genetically targeted voltage-sensitive fluorescent proteins. Nat Methods 7:643–649CrossRefPubMedGoogle Scholar
  29. 29.
    Akemann W, Mutoh H, Perron A (2012) Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein. J Neurophysiol 108:2323–2337CrossRefPubMedGoogle Scholar
  30. 30.
    Knöpfel T (2012) Genetically encoded optical indicators for the analysis of neuronal circuits. Nat Rev Neurosci 13:687–700PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Ruth M. Empson
    • 1
  • Malinda L. S. Tantirigama
    • 1
  • Manfred J. Oswald
    • 1
  • Stephanie M. Hughes
    • 2
  • Thomas Knöpfel
    • 3
    Email author
  1. 1.Department of Physiology, Brain Health Research Centre, Otago School of Medical SciencesUniversity of OtagoDunedinNew Zealand
  2. 2.Department of Biochemistry, Brain Health Research Centre, Otago School of Medical SciencesUniversity of OtagoDunedinNew Zealand
  3. 3.Optogenetics and Circuit Neurosciences, Division of Brain SciencesImperial College LondonLondonUK

Personalised recommendations