Skip to main content
SpringerLink
Log in
Book cover

Immunocytochemistry and Related Techniques pp 313–328Cite as

Neuronal Circuitry Dissected by Immunocytochemistry Combined with Retrograde Tracing and Electrophysiology

  • Protocol
  • First Online:

  • 1555 Accesses

Part of the book series: Neuromethods ((NM,volume 101))

Abstract

Several substances, once injected in a given central structure, are taken up by axon terminals and transported retrogradely over long distances, to the cell bodies of neurons projecting to the injection area. They are then visualized by means of histochemical (or immunohistochemical ) reactions, making it possible to trace neural pathways .

Here we describe a method to label retrogradely neocortical pyramidal neurons in a Golgi-like fashion. This method allows reconstructing the entire dendritic tree of retrogradely labeled cells and can be easily combined with immunohistochemical techniques. Methods suitable to establish relationships between morphological and functional features of projecting neurons are also discussed.

This is a preview of subscription content, 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   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Glickstein M (2006) Golgi and Cajal: the neuron doctrine and the 100th anniversary of the 1906 Nobel Prize. Curr Biol 16:R147–R151

    Article  CAS  PubMed  Google Scholar 

  2. Bentivoglio M, Jones EG, Mazzarello P et al (2011) Camillo Golgi and modern neuroscience. Brain Res Rev 66:1–4

    Article  PubMed  Google Scholar 

  3. Vercelli A, Repici M, Garbossa D et al (2000) Recent techniques for tracing pathways in the central nervous system of developing and adult mammals. Brain Res Bull 51:11–28

    Article  CAS  PubMed  Google Scholar 

  4. Kristensson K, Olsson Y (1971) Retrograde axonal transport of protein. Brain Res 29:363–365

    Article  CAS  PubMed  Google Scholar 

  5. Goldberg S, Kotani M (1967) The projection of optic nerve fibers in the frog Rana catesbeiana as studied by radioautography. Anat Rec 158:325–331

    Article  CAS  PubMed  Google Scholar 

  6. Cowan WM, Gottlieb DI, Hendrickson AE et al (1972) The autoradiographic demonstration of axonal connections in the central nervous system. Brain Res 37:21–51

    Article  CAS  PubMed  Google Scholar 

  7. Kuypers HG, Bentivoglio M, van der Kooy D et al (1979) Retrograde transport of bisbenzimide and propidium iodide through axons to their parent cell bodies. Neurosci Lett 12:1–7

    Article  CAS  PubMed  Google Scholar 

  8. Bentivoglio M, Kuypers HG, Catsman-Berrevoets CE et al (1979) Fuorescent retrograde neuronal labeling in rat by means of substances binding specifically to adenine-thymine rich DNA. Neurosci Lett 12:235–240

    Article  CAS  PubMed  Google Scholar 

  9. Kuypers HG, Bentivoglio M, Catsman-Berrevoets CE et al (1980) Double retrograde neuronal labeling through divergent axon collaterals, using two fluorescent tracers with the same excitation wavelength which label different features of the cell. Exp Brain Res 40:383–392

    Article  CAS  PubMed  Google Scholar 

  10. Bentivoglio M, Kuypers HG, Catsman-Berrevoets CE et al (1980) Two new fluorescent retrograde neuronal tracers which are transported over long distances. Neurosci Lett 18:25–30

    Article  CAS  PubMed  Google Scholar 

  11. Keizer K, Kuypers HG, Huisman AM et al (1983) Diamidino yellow dihydrochloride (DY.2HCl); a new fluorescent retrograde neuronal tracer, which migrates only very slowly out of the cell. Exp Brain Res 51:179–191

    Article  CAS  PubMed  Google Scholar 

  12. Bentivoglio M, Molinari M (1984) Fluorescent retrograde triple labeling of brainstem reticular neurons. Neurosci Lett 46:121–126

    Article  CAS  PubMed  Google Scholar 

  13. Bentivoglio M, Molinari M (1981) Axonal branches of the same cerebellar neurons terminate bilaterally in the thalamus. Neurosci Lett 23:291–296

    Article  CAS  PubMed  Google Scholar 

  14. Minciacchi D, Molinari M, Bentivoglio M et al (1985) The organization of the ipsi- and contralateral claustrocortical system in rat with notes on the bilateral claustrocortical projections in cat. Neuroscience 16:557–576

    Article  CAS  PubMed  Google Scholar 

  15. Granato A, Santarelli M, Minciacchi D (1991) Bihemispheric organization of amygdalo-cortical projections in the rat. Neurosci Lett 127:53–56

    Article  CAS  PubMed  Google Scholar 

  16. Minciacchi D, Granato A, Barbaresi P (1991) Organization of claustro-cortical projections to the primary somatosensory area of primates. Brain Res 553:309–312

    Article  CAS  PubMed  Google Scholar 

  17. Minciacchi D, Granato A, Antonini A et al (1995) Mapping subcortical extrarelay afferents onto primary somatosensory and visual areas in cats. J Comp Neurol 362:46–70

    Article  CAS  PubMed  Google Scholar 

  18. Stanfield BB, O’Leary DD, Fricks C (1982) Selective collateral elimination in early postnatal development restricts cortical distribution of rat pyramidal tract neurones. Nature 298:371–373

    Article  CAS  PubMed  Google Scholar 

  19. O’Leary DD, Stanfield BB (1986) A transient pyramidal tract projection from the visual cortex in the hamster and its removal by selective collateral elimination. Brain Res 392:87–99

    Article  PubMed  Google Scholar 

  20. Innocenti GM (1981) Growth and reshaping of axons in the establishment of visual callosal connections. Science 212:824–827

    Article  CAS  PubMed  Google Scholar 

  21. Innocenti GM, Clarke S, Kraftsik R (1986) Interchange of callosal and association projections in the developing visual cortex. J Neurosci 6:1384–1409

    CAS  PubMed  Google Scholar 

  22. Rende M, Granato A, Lo Monaco M et al (1991) Accuracy of reinnervation by peripheral nerve axons regenerating across a 10-mm gap within an impermeable chamber. Exp Neurol 111:332–339

    Article  CAS  PubMed  Google Scholar 

  23. Weinberg RJ, Bentivoglio M, Phend K et al (1985) A new double-labeling method demonstrates transmitter-specific projections. Neurosci Lett 55:349–353

    Article  CAS  PubMed  Google Scholar 

  24. Skirboll L, Hökfelt T, Norell G et al (1984) A method for specific transmitter identification of retrogradely labeled neurons: immunofluorescence combined with fluorescence tracing. Brain Res 320:99–127

    Article  CAS  PubMed  Google Scholar 

  25. Airan RD, Hu ES, Vijaykumar R et al (2007) Integration of light-controlled neuronal firing and fast circuit imaging. Curr Opin Neurobiol 17:587–592

    Article  CAS  PubMed  Google Scholar 

  26. Tye KM, Deisseroth K (2012) Optogenetic investigation of neural circuits underlying brain disease in animal models. Nat Rev Neurosci 13:251–266

    Article  CAS  PubMed  Google Scholar 

  27. Rein ML, Deussing JM (2012) The optogenetic (r)evolution. Mol Genet Genomics 287:95–109

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Aston-Jones G, Deisseroth K (2013) Recent advances in optogenetics and pharmacogenetics. Brain Res 1511:1–5

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Cardin JA, Carlén M, Meletis K et al (2009) Driving fast-spiking cells induces gamma rhythm and controls sensory responses. Nature 459:663–667

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Rothermel M, Brunert D, Zabawa C et al (2013) Transgene expression in target-defined neuron populations mediated by retrograde infection with adeno-associated viral vectors. J Neurosci 33:15195–15206

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Reiner A, Veenman CL, Medina L et al (2000) Pathway tracing using biotinylated dextran amines. J Neurosci Methods 103:23–37

    Article  CAS  PubMed  Google Scholar 

  32. Schmued L, Kyriakidis K, Heimer L (1990) In vivo anterograde and retrograde axonal transport of the fluorescent rhodamine-dextran-amine, Fluoro-Ruby, within the CNS. Brain Res 526:127–134

    Article  CAS  PubMed  Google Scholar 

  33. Giannetti S, Gaglini PD, Rocco F et al (2000) Organization of cortico-cortical associative projections in a rat model of microgyria. Neuroreport 11:2185–2189

    Article  CAS  PubMed  Google Scholar 

  34. Granato A, Di Rocco F, Zumbo A et al (2003) Organization of cortico-cortical associative projections in rats exposed to ethanol during early postnatal life. Brain Res Bull 60:339–344

    Article  CAS  PubMed  Google Scholar 

  35. Minciacchi D, Del Tongo C, Carretta D et al (2010) Alterations of the cortico-cortical network in sensori-motor areas of dystrophin deficient mice. Neuroscience 166:1129–1139

    Article  CAS  PubMed  Google Scholar 

  36. Di Rocco F, Giannetti S, Gaglini P et al (2002) Dendritic architecture of corticothalamic neurons in a rat model of microgyria. Childs Nerv Syst 18:690–693

    Article  PubMed  Google Scholar 

  37. Jiang X, Johnson RR, Burkhalter A (1993) Visualization of dendritic morphology of cortical projection neurons by retrograde axonal tracing. J Neurosci Methods 50:45–60

    Article  CAS  PubMed  Google Scholar 

  38. Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates. Academic, San Diego, CA

    Google Scholar 

  39. DeFelipe J (1997) Types of neurons, synaptic connections and chemical characteristics of cells immunoreactive for calbindin-D28K, parvalbumin and calretinin in the neocortex. J Chem Neuroanat 14:1–19

    Article  CAS  PubMed  Google Scholar 

  40. Markram H, Toledo-Rodriguez M, Wang Y et al (2004) Interneurons of the neocortical inhibitory system. Nat Rev Neurosci 5:793–807

    Article  CAS  PubMed  Google Scholar 

  41. Moore CI, Carlen M, Knoblich U et al (2010) Neocortical interneurons: from diversity, strength. Cell 142:189–193

    Article  PubMed Central  PubMed  Google Scholar 

  42. Elston GN, De Felipe J, Arellano JI et al (1999) Variation in the spatial relationship between parvalbumin immunoreactive interneurons and pyramidal neurones in rat somatosensory cortex. Neuroreport 10:2975–2979

    Article  CAS  PubMed  Google Scholar 

  43. Glaser JR, Glaser EM (1990) Neuron imaging with Neurolucida: a PC-based system for image combining microscopy. Comput Med Imaging Graph 14:307–317

    Article  CAS  PubMed  Google Scholar 

  44. Anderson K, Yamamoto E, Kaplan J et al (2010) Neurolucida Lucivid versus Neurolucida camera: a quantitative and qualitative comparison of three-dimensional neuronal reconstructions. J Neurosci Methods 186:209–214

    Article  PubMed  Google Scholar 

  45. Verwer RWH, van Pelt J (1986) Descriptive and comparative analysis of geometrical properties of neuronal tree structures. J Neurosci Methods 18:179–206

    Article  CAS  PubMed  Google Scholar 

  46. Mainen ZF, Sejnowski TJ (1996) Influence of dendritic structure on firing pattern in model neocortical neurons. Nature 382:363–366

    Article  CAS  PubMed  Google Scholar 

  47. Schaefer AT, Larkum ME, Sakmann B et al (2003) Coincidence detection in pyramidal neurons is tuned by their dendritic branching pattern. J Neurophysiol 89:3143–3154

    Article  PubMed  Google Scholar 

  48. Freedman LJ, Maddox MT (2001) A comparison of anti-biotin and biotinylated anti-avidin double-bridge and biotinylated tyramide immunohistochemical amplification. J Neurosci Methods 112:43–49

    Article  CAS  PubMed  Google Scholar 

  49. Larkman A, Mason A (1990) Correlations between morphology and electrophysiology of pyramidal neurons in slices of rat visual cortex. I. Establishment of cell classes. J Neurosci 10:1407–1414

    CAS  PubMed  Google Scholar 

  50. Petersen CC, Grinvald A, Sakmann B (2003) Spatiotemporal dynamics of sensory responses in layer 2/3 of rat barrel cortex measured in vivo by voltage-sensitive dye imaging combined with whole-cell voltage recordings and neuron reconstructions. J Neurosci 23:1298–1309

    CAS  PubMed  Google Scholar 

  51. Granato A, Palmer LM, De Giorgio A et al (2012) Early exposure to alcohol leads to permanent impairment of dendritic excitability in neocortical pyramidal neurons. J Neurosci 32:1377–1382

    Article  CAS  PubMed  Google Scholar 

  52. 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 

  53. Song S, Sjöström PJ, Reigl M et al (2005) Highly nonrandom features of synaptic connectivity in local cortical circuits. PLoS Biol 3:e68

    Article  PubMed Central  PubMed  Google Scholar 

  54. Sakmann B (2006) Patch pipettes are more useful than initially thought: simultaneous pre- and postsynaptic recording from mammalian CNS synapses in vitro and in vivo. Pflugers Arch 453:249–259

    Article  CAS  PubMed  Google Scholar 

  55. Frick A, Feldmeyer D, Helmstaedter M et al (2008) Monosynaptic connections between pairs of L5A pyramidal neurons in columns of juvenile rat somatosensory cortex. Cereb Cortex 18:397–406

    Article  PubMed  Google Scholar 

  56. Schubert D, Kötter R, Luhmann HJ et al (2006) Morphology, electrophysiology and functional input connectivity of pyramidal neurons characterizes a genuine layer Va in the primary somatosensory cortex. Cereb Cortex 16:223–236

    Article  CAS  PubMed  Google Scholar 

  57. Davie JT, Kole MH, Letzkus JJ et al (2006) Dendritic patch-clamp recording. Nat Protoc 1:1235–1247

    Article  CAS  PubMed  Google Scholar 

  58. Nevian T, Larkum ME, Polsky A et al (2007) Properties of basal dendrites of layer 5 pyramidal neurons: a direct patch-clamp recording study. Nat Neurosci 10:206–214

    Article  CAS  PubMed  Google Scholar 

  59. Larkum ME, Nevian T, Sandler M et al (2009) Synaptic integration in tuft dendrites of layer 5 pyramidal neurons: a new unifying principle. Science 325:756–760

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Psychology, Catholic University, Largo A. Gemelli 1, 20123, Milan, Italy

    Alberto Granato & Andrea De Giorgio

Authors
  1. Alberto Granato
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrea De Giorgio
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alberto Granato .

Editor information

Editors and Affiliations

  1. Department of Veterinary Sciences, University of Torino, Grugliasco, Torino, Torino, Italy

    Adalberto Merighi

  2. Department of Veterinary Sciences, University of Torino, Grugliasco, Torino, Torino, Italy

    Laura Lossi

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Granato, A., De Giorgio, A. (2015). Neuronal Circuitry Dissected by Immunocytochemistry Combined with Retrograde Tracing and Electrophysiology. In: Merighi, A., Lossi, L. (eds) Immunocytochemistry and Related Techniques. Neuromethods, vol 101. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2313-7_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2313-7_17

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2312-0

  • Online ISBN: 978-1-4939-2313-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation