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Differential effects of chemical differentiation on voltage-gated inward and outward currents in foetal rat forebrain neural stem cells Reliable sources of proliferating neural stem cells will be required for use in cell replacement therapies for neural repair (Minger et al, 1996). Understanding the culture conditions that promote the development of functional, mature neuronal phenotypes from neural stem and progenitor cells is essential for the development of tissue that is suitable for transplantation. This investigation aimed to assess the effect of chemical treatments that promote neural differentiation on the expression of voltage-gated membrane currents in voltage-clamped neural stem cells (NSCs) in E14 Fischer rat forebrain explant cultures, isolated from males and females of unspecified weight. NSCs were cultured in basal medium (BM) containing 1 µM FGF-2 for 2 days (condition A) or up to 10 days (condition A′). NSCs were in the presence of FGF-2 for 6 days followed by a further 2 - 4 days with either BM alone (condition B), BM plus 1 mM dibutyryl cAMP (DcAMP) (condition C), BM plus 1 µM retinoic acid (RA) (condition D) or BM plus both DcAMP and RA (condition E). NSCs were whole-cell voltage-clamped after 2 days in vitro (condition A) or after 8-10 days in vitro (conditions A’-E). Patch clamp electrodes were filled with (mM) 133 KCl, 0.1 CaCl2, 1 MgCl2, 10 HEPES and 10 EGTA, pH 7.4, having resistances of 3 - 5 MΩ. Extracellular solution contained (mM) 132 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, 5 HEPES and 5 glucose, pH 7∙4. NSCs were held at -70 mV and voltage-gated currents were evoked during 50 or 500 ms steps to membrane potentials between -80 to +50 mV, increasing in 10 mV increments. NSCs expressed transient inward (TI), transient outward (TO) and persistent outward ( PO) currents under all culture conditions.
Table 1: Data are currents in conditions A to E (mean ± SEM; *P<0.05, **P<0.01, Analysis of variance with Dunnett’s post-test with Condition A’ as the control group.) TI current amplitudes increased significantly following FGF-2 withdrawal but DcAMP and RA addition failed to augment this increase. By contrast, TO current was unaltered by withdrawal of FGF-2, however increased significantly with combined DcAMP and RA treatment. PO current expression was not significantly increased following any of the differentiation tretments. Data in Table 1 show NSCs in explant cultures express TI, TO and PO currents in the presence of FGF-2. Furthermore TI and TO currents are influenced differently by chemical differentiation treatments, TI currents are sensitive to FGF-2 in the culture medium, TO current expression is not. However, TO current expression is enhanced by DcAMP and RA, although expression of TI current is not.
Minger et al, (1996): Exp Neurol 141(1): 12-24. Supported by the BBSRC. |
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