Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Purification and culture of nerve growth factor receptor (p75)-expressing basal forebrain cholinergic neurons

Nature Protocols volume 3, pages 34–40 (2008)Cite this article

Abstract

The activity of the basal forebrain cholinergic neurons (BFCNs) that innervate the cerebral cortex and hippocampus is essential for normal learning and memory. Here, we present a method to isolate and culture BFCNs from the embryonic murine septum that takes advantage of their restricted expression of the nerve growth factor receptor (p75) in conjunction with fluorescence-activated cell sorting. The septal region dissection, cell dissociation and staining process, and cell sorting parameters are described in detail. Sufficient cell yield and optimized cell culture conditions make this protocol suitable for multiple assays including immunocytochemistry, reverse transcriptase PCR, microarray profiling, acetylcholine measurements and electrophysiological assessment. The study of these neurons as a purified population will greatly advance our understanding of factors that influence their development and maintenance.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Fire-polished glass pipettes.
Figure 2: Dissection of the late embryonic septum.
Figure 3: Live, single-cell sorting gates for septal cells.
Figure 4: Isolation of p75+ BFCNs via FACS.
Figure 5: Sorted p75− and p75+ septal cells persist in culture.

Similar content being viewed by others

References

  1. Dutar, P., Bassant, M.H., Senut, M.C. & Lamour, Y. The septohippocampal pathway: structure and function of a central cholinergic system. Physiol. Rev. 75, 393–427 (1995).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Mesulam, M.M., Mufson, E.J., Levey, A.I. & Wainer, B.H. Cholinergic innervation of cortex by the basal forebrain: cytochemistry and cortical connections of the septal area, diagonal band nuclei, nucleus basalis (substantia innominata), and hypothalamus in the rhesus monkey. J. Comp. Neurol. 214, 170–197 (1983).

    Article  CAS  Google Scholar 

  4. Lopez-Coviella, I., Berse, B., Krauss, R., Thies, R.S. & Blusztajn, J.K. Induction and maintenance of the neuronal cholinergic phenotype in the central nervous system by BMP-9. Science 289, 313–316 (2000).

    Article  CAS  Google Scholar 

  5. Greferath, U., Bennie, A., Kourakis, A. & Barrett, G.L. Impaired spatial learning in aged rats is associated with loss of p75-positive neurons in the basal forebrain. Neuroscience 100, 363–373 (2000).

    Article  CAS  Google Scholar 

  6. Sweeney, J.E., Hohmann, C.F., Oster-Granite, M.L. & Coyle, J.T. Neurogenesis of the basal forebrain in euploid and trisomy 16 mice: an animal model for developmental disorders in Down syndrome. Neuroscience 31, 413–425 (1989).

    Article  CAS  Google Scholar 

  7. Dawbarn, D., Allen, S.J. & Semenenko, F.M. Coexistence of choline acetyltransferase and nerve growth factor receptors in the rat basal forebrain. Neurosci. Lett. 94, 138–144 (1988).

    Article  CAS  Google Scholar 

  8. Woolf, N.J., Gould, E. & Butcher, L.L. Nerve growth factor receptor is associated with cholinergic neurons of the basal forebrain but not the pontomesencephalon. Neuroscience 30, 143–152 (1989).

    Article  CAS  Google Scholar 

  9. Batchelor, P.E., Armstrong, D.M., Blaker, S.N. & Gage, F.H. Nerve growth factor receptor and choline acetyltransferase colocalization in neurons within the rat forebrain: response to fimbria–fornix transection. J. Comp. Neurol. 284, 187–204 (1989).

    Article  CAS  Google Scholar 

  10. Koh, S. & Loy, R. Localization and development of nerve growth factor-sensitive rat basal forebrain neurons and their afferent projections to hippocampus and neocortex. J. Neurosci. 9, 2999–0318 (1989).

    Article  CAS  Google Scholar 

  11. Sergent-Tanguy, S., Chagneau, C., Neveu, I. & Naveilhan, P. Fluorescent activated cell sorting (FACS): a rapid and reliable method to estimate the number of neurons in a mixed population. J. Neurosci. Methods 129, 73–79 (2003).

    Article  Google Scholar 

  12. Ozdinler, P.H. & Macklis, J.D. IGF-I specifically enhances axon outgrowth of corticospinal motor neurons. Nat. Neurosci. 9, 1371–1381 (2006).

    Article  Google Scholar 

  13. Lopez-Coviella, I. et al. Bone morphogenetic protein 9 induces the transcriptome of basal forebrain cholinergic neurons. Proc. Natl. Acad. Sci. USA 102, 6984–6989 (2005).

    Article  CAS  Google Scholar 

  14. Allen, T.G., Abogadie, F.C. & Brown, D.A. Simultaneous release of glutamate and acetylcholine from single magnocellular 'cholinergic' basal forebrain neurons. J. Neurosci. 26, 1588–1595 (2006).

    Article  CAS  Google Scholar 

  15. Hefti, F., Hartikka, J. & Sanchez-Ramos, J. Dissociated cholinergic neurons of the basal forebrain in culture. in A Dissection and Tissue Culture Manual of the Nervous System, 172–182 (eds: Shahar, A., de Vellis, J., Vernadakis, A. & Haken, B. Wiley-Liss Inc., New York, 1989).

    Google Scholar 

  16. Naumann, T. et al. Complete deletion of the neurotrophin receptor p75NTR leads to long-lasting increases in the number of basal forebrain cholinergic neurons. J. Neurosci. 22, 2409–2418 (2002).

    Article  CAS  Google Scholar 

  17. Tallini, Y.N. et al. BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons. Physiol. Genomics 27, 391–397 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Yanhui Deng and Stephen Kwok of the Boston University Medical Center Flow Cytometry Core Facility and the Tufts University Flow Cytometry Core in Pathology, respectively, for their expertise in FACS. p75 knockout Po mice were kindly provided by Dr Susan Birren. We thank Dr Michael Kotlikoff for providing ChAT-eGFP founder males for colony establishment. This work was supported by NIA grant AG 009525.

Author information

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, 02118, Massachusetts, USA

    Aletta C Schnitzler, Ignacio Lopez-Coviella & Jan Krzysztof Blusztajn

Authors
  1. Aletta C Schnitzler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ignacio Lopez-Coviella
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jan Krzysztof Blusztajn
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jan Krzysztof Blusztajn.

Supplementary information

Supplementary Fig. 1

Specificity of the ATS anti-p75 antibody (PDF 133 kb)

Supplementary Video

Dissection of embryonic forebrain septa (MOV 25823 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schnitzler, A., Lopez-Coviella, I. & Blusztajn, J. Purification and culture of nerve growth factor receptor (p75)-expressing basal forebrain cholinergic neurons. Nat Protoc 3, 34–40 (2008). https://doi.org/10.1038/nprot.2007.477

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.477

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing