Lectin-based isolation and culture of mouse embryonic motoneurons.

Rebecca Conrad, Sibylle Jablonka, Teresa Sczepan, Michael Sendtner, Stefan Wiese, Alice Klausmeyer

Index: J. Vis. Exp. 55 , 3200, (2011)

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Abstract

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons(1). Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract(2,3). More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)(4). Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75(NTR). The p75(NTR) can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75(NTR) is exclusively expressed by the spinal motoneurons(5). This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells(6). Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75(NTR) has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75(NTR) as well(7). The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75(NTR) antibody. The lectin is an extremely inexpensive alternative to the p75(NTR) antibody and the purification grades using lectin are comparable to that of the p75(NTR) antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.