Validation of procedures for neural progenitor isolation.

<p>Neocortex was dissected from fetal rat brains, followed by preparation of lower cell layer fractions and subsequent culture with EGF for 12 days under floating conditions. Cells were then dispersed and cultured without EGF for 6 days under adherent conditions. Cells were fixed for immunocytochemistry analysis on the progenitor marker nestin, the neuronal marker lMAP2 and astroglial marker GFAP.</p

Expression of nAChR subunits in undifferentiated rat neural progenitors.

<p>Neocortex was dissected from fetal rat brains, followed by preparation of the lower cell layer fractions. (A) Total RNA was extracted from cells before or after the culture in the presence of EGF for 12 consecutive days for RT-PCR analysis. Adult rat whole brain was used as a positive control. Typical pictures are shown with similar results in three independent sets of experiments. Undifferentiated progenitors were cultured with EGF in the presence of nicotine at different concentrations for determination of (B) the total size during culture, (C) MTT reduction, (D) LDH release and (E) the ratio of PI-positive cells cultured for 12 days. *P<0.05, **P<0.01, significantly different from each control value obtained in cells not exposed to nicotine.</p

Effects of nicotine on differentiation of mouse progenitors.

<p>(A) Cells were cultured with EGF in either the presence or absence of 10 µM nicotine, 10 µM DHβE, 10 µM TC2559 and 10 µM MLA for 10 days, followed by dispersion after removal of EGF and subsequent double immunocytochemistry analysis along with Hechst33342 staining for counting the number of immunoreactive cells. (B) Cells were cultured with EGF for 10 days in either the presence or absence of 10 µM nicotine, followed by extraction of total RNA and subsequent RT-PCR analysis on different bHLH genes. (C) Cells were cultured with EGF in either the presence or absence of 10 µM nicotine and 10 µM DHβE, followed by extraction of total RNA and subsequent RT-PCR analysis on <i>Math1</i> gene. *P<0.05, **P<0.01, significantly different from each control value obtained in cells not exposed to nicotine. ^##P<0.01, significantly different from the control value obtained in cells exposed to nicotine alone.</p

Neural progenitors prepared from NMDAR1-null mice.

<p>Neocortex was dissected from WT (NR1^+/+) and NMDAR1-null (NR1^−/−) mice, followed by culture with EGF for 10 days and subsequent determination of (A) the total size of neurospheres and (B) the number of individual immunoreactive cells on double immunocytochemistry analysis along with Hechst33342 staining. *P<0.05, **P<0.01, significantly different from the value obtained cells from WT mice. Cells from WT and NMDAR1-null mice were also cultured with EGF in either the presence or absence of 10 µM nicotine, 10 µM DHβE and 10 µM MLA for 10 days, followed by determination of (C) MTT reduction, (D) neurosphere size and (E) PI-positive dead cells. *P<0.05, **P<0.01, significantly different from each control value obtained in cells not exposed to nicotine. ^##P<0.01, significantly different from the control value obtained in cells exposed to nicotine alone.</p

Effects of nicotine on differentiation of rat progenitors.

<p>Cells were dispersed after the culture with EGF in either the presence or absence of 10 µM nicotine for 12 days, followed by further culture in (A) the absence and (B) presence of differentiation inducers such as ATRA and CNTF for an additional 6 days. Cells were then fixed for double immunocytochemical detection of both MAP2 and GFAP, followed by counting of the number of individual immunoreactive cells. *P<0.05, **P<0.01, significantly different from each control value obtained in cells not exposed to nicotine. ^##P<0.01, significantly different from the value obtained in cells exposed to nicotine alone.</p

Effects of mecamylamine on cellular viability in rat progenitors.

<p>Undifferentiated progenitors were cultured with EGF in either the presence or absence of 10 µM nicotine and 10 µM mecamylamine for a period up to 12 days for determination of (A) neurosphere size, (B) MTT reduction, (C) LDH release and (BD) the ratio of PI-positive cells. *P<0.05, **P<0.01, significantly different from each control value obtained in cells not exposed to nicotine. ^#P<0.05, significantly different from the value obtained in cells exposed to nicotine alone.</p

Expression and phosphorylation of particular proteins in undifferentiated mouse progenitors exposed to nicotine.

<p>Undifferentiated mouse progenitors were cultured with EGF for 10 days and subjected to (A) Western blotting and (B) immunocytochemistry for α4 and β2 subunits of nAChRs. Immunocytochemical images are also shown with cells not treated with the primary antibody. (C) Undifferentiated cells were also exposed to 10 µM nicotine for 5 to 10 min for subsequent Western blotting. Typical pictures are shown with similar results in three independent sets of experiments.</p

Differentiation of rat progenitors.

<p>Cells were dispersed after the culture with EGF in either the presence or absence of 10 µM nicotine for 12 days, followed by further culture for an additional 6 days. Cells were then fixed for double immunocytochemical detection of both MAP2 and GFAP together with Hoechst33342 staining.</p