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

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



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.

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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.

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  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


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Correspondence to Jan Krzysztof Blusztajn.

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    Supplementary Fig. 1

    Specificity of the ATS anti-p75 antibody


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    Supplementary Video

    Dissection of embryonic forebrain septa

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