Protocol | Published:

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

Nature Protocols volume 3, pages 3440 (2008) | Download Citation

Subjects

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , , & The septohippocampal pathway: structure and function of a central cholinergic system. Physiol. Rev. 75, 393–427 (1995).

  2. 2.

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

  3. 3.

    , , & 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).

  4. 4.

    , , , & Induction and maintenance of the neuronal cholinergic phenotype in the central nervous system by BMP-9. Science 289, 313–316 (2000).

  5. 5.

    , , & Impaired spatial learning in aged rats is associated with loss of p75-positive neurons in the basal forebrain. Neuroscience 100, 363–373 (2000).

  6. 6.

    , , & 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).

  7. 7.

    , & Coexistence of choline acetyltransferase and nerve growth factor receptors in the rat basal forebrain. Neurosci. Lett. 94, 138–144 (1988).

  8. 8.

    , & Nerve growth factor receptor is associated with cholinergic neurons of the basal forebrain but not the pontomesencephalon. Neuroscience 30, 143–152 (1989).

  9. 9.

    , , & 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).

  10. 10.

    & 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).

  11. 11.

    , , & 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).

  12. 12.

    & IGF-I specifically enhances axon outgrowth of corticospinal motor neurons. Nat. Neurosci. 9, 1371–1381 (2006).

  13. 13.

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

  14. 14.

    , & Simultaneous release of glutamate and acetylcholine from single magnocellular 'cholinergic' basal forebrain neurons. J. Neurosci. 26, 1588–1595 (2006).

  15. 15.

    , & 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).

  16. 16.

    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).

  17. 17.

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

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

Affiliations

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

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

Authors

  1. Search for Aletta C Schnitzler in:

  2. Search for Ignacio Lopez-Coviella in:

  3. Search for Jan Krzysztof Blusztajn in:

Corresponding author

Correspondence to Jan Krzysztof Blusztajn.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Specificity of the ATS anti-p75 antibody

Videos

  1. 1.

    Supplementary Video

    Dissection of embryonic forebrain septa

About this article

Publication history

Published

DOI

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

Further reading

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.