Protocols

Adult Neural Stem Cell Culture
Identification of functionally integrated adult-born neurons
Cell Sorting with anti-Notch 1 Antiserum and magnetic beads
Eph receptor studies:   Adhesion, Chemotaxis, Aggregation and Co-Cultivation assays
Generalised potential of Adult Neural Stem Cells:   Chick assay & Micro injection
Immunohistochemistry and primary antibodies
Intraventricular injection:   DiI labeling, Adenoviral infection and Retroviral infection
Proliferation studies

1. The spinal cord or the lateral wall of the lateral ventricles is prepared.
2. The tissue is enzymatically dissociated in HBSS with 2 mM glucose at 37°C for 30 min.
(0.7 mg/ml hyaluronic acid, 0.2 mg/ml kynurenic acid, and 1.33 mg/ml trypsin)
3. The cells are centrifuged at 200 g for 5 min and resuspended in 0.9 M sucrose in HBSS
4. The cells are centrifuged for 10 min at 750 g and resuspended in 2 ml of culture medium
5. The cell suspension is placed on top of 10 ml 4% BSA in EBSS solution and centrifuged at 200 g for 7 min
6. The cells are washed in DMEM/F12.

The culture medium consist of 20 ng/ml EGF, 20 ng/ml bFGF, B27 supplement, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin in DMEM-F12 medium.

For single cell culture, cells are cultured in 96-well dishes in 50% neurosphere-conditioned medium and 50% fresh medium.

Download a detailed protocol in a WORD 6.0/95 format: adultnsc.doc

Pseudorabies virus encoding the green fluorescent protein (GFP) gene
To analyze whether adult-born neurons integrate in synaptic circuitry, we created a green fluorescent protein (GFP)-expressing pseudorabies virus (PRV GS518). Directly injected into the brain, this neurotropic virus selectively infects and replicates in neurons. The virus is actively transported along axons and dendrites to synapses, where it passes on to infect higher order neurons within a circuit, in a manner strictly dependent on synaptic contact [Enquist L.W., et al., Adv. Virus Res. 51 (1998), pp. 237-347; P. Card and L.W. Enquist, Transneuronal circuit analysis with pseudorabies viruses., Current Protocols in Neuroscience, Wiley & Sons, New York (2001) 1.5.1-1.5.28.pp.].


Administration of BrdU and pseudorabies virus to identify adult-born neurons
To label dividing neuronal progenitor cells and their progeny, adult C57BL/6 mice received BrdU (1 mg/ml) in their drinking water for 4 weeks followed by a 3-week chase period to allow for cell migration, neuronal differentiation, and potential synaptic integration. This procedure results in extensive labeling of newborn granule cells and periglomerular neurons in the OB. At the end of the chase period, we injected 7.5 x 10^4 particles of PRV GS518 into the piriform cortex. Cholera toxin B (CTB), a retrograde neuronal tracer that is unable to cross synapses, was coinjected with PRV GS518 into the piriform cortex. Four days later, we analyzed OB neurons for BrdU incorporation and viral infection. Since CTB is unable to cross synapses, labeling of a neuron with PRV GS518 and CTB reveals a direct infection, whereas labeling of a neuron with PRV GS518 alone indicates that the virus has infected the neuron transsynaptically.


c-Fos analysis to assess funtional integration of adult-born neurons
To assess whether neurons generated in the adult brain can respond to a physiological stimulus, we exposed mice to a cocktail of odorants and examined the induction of neural activity as indicated by the upregulation of c-Fos expression. The ability of odorants to activate adult-generated neurons was revealed by the colocalization of c-Fos induction with BrdU incorporation and expression of the neuronal marker NeuN within single periglomerular neurons of activated glomeruli.

Dissociation solution: 0.075% collagenase type 1, 0.075% hyaluronidase, and 500 U/ml DNaseI in 0.2 M PIPES.

1. The cells are resuspended in 1:100 anti-Notch 1 antiserum (Mitsiadis et al., 1995 ) and incubated for 20 min at 4°C.
2. Rinse in a large volume of DMEM/F12.
3. The cells are resuspended in 100 µl of culture medium containing magnetic beadconjugated antiserum.

• Goat anti-rabbit antiserum from Dynal: 1.8 - 2.1 * 10^7 beads / 100 µl culture medium
4. Incubate for 20 min at 4°C.
5. Subsequently, 2 ml of culture medium is added to the tube which is placed in a magnetic separator for 2 min.
6. The supernatant is then collected and 2 ml culture medium is added to the tube.
7. The magnet is then removed and the cells that have bound magnetic beads are collected.

For the adhesion assays we dissected the edges of the cranial neural folds from 8.5-day-old embryos from ephrin-A5+/- intercrosses. Equal numbers of cells from individual embryos were dissociated in Ca2+- and Mg 2+-free PBS, and cells from single embryos were plated in EphA7-Fc-coated (5 µg/ml) wells on a rotary shaker (100 r.p.m. at 37 °C) and allowed to adhere for 1 h. The wells were rinsed to remove non-adherent cells; the remaining cells were fixed and the number of EphA7-FL-immunoreactive cells (used here as a marker for cells from the edge of the neural folds) in each well was quantified. The adhesion assays were carried out blindly of genotype, which was established by PCR of DNA from the individual embryos.

The chemotaxis assay was performed essentially as described [Wang, et.al., Neuron 18, 383-396 (1997)]. Briefly, 27µl of medium or medium containing EphA7-Fc fusion protein (5 µg/ml) clustered with anti-human immunoglobulin- (IgG) antibodies (50 µg/ml) was added to the lower wells. The upper wells were then filled with 2x10^5 293 cells per ml.

For the aggregation assay, cells were labelled by addition of DiI (0.33 ng/ml) or DiO (1.7 ng/ml) to the tissue culture medium 48 h before the assay or by stable transfection with pEGFP-1. Cells (107) were transferred to polystyrene tubes in 1 ml of buffer (137 mM NaCl, 4.7 mM KCl, 0.6 mM MgSO4, 1.2 mM CaCl2, 10 mM Hepes, pH 7.4) with 1 mg/ml glucose and 20 U/ml Dnase [Wikström et.al., Exp. Cell Res. 227, 360-366 (1996)]. The tubes were placed on a rotary shaker (200 r.p.m. for 1 h at 37 °C) and aggregate formation was analysed in a FACScalibur flow cytometer (Becton Dickson) equipped with Cellquest 3.1 software.

For the co-cultivation experiments, we mixed and plated labelled cells (DiI 1 ng/ml, DiA 5 ng/ml, DiO 5 ng/ml or stably pEGFP-1 transfected) together in tissue culture plates and allowed them to grow together for 12-24 h. In other co-cultivation experiments, wild-type 293 cells (labelled with DiA) and pEGFP-1 transfected cells expressing ep