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Subject: Immature Neurons From CNS Stem Cells Proliferate in Response to Platelet-Derived Growth Factor -- Erlandsson et al. 21 (10): 3483 -- Journal of Neuroscience
Date: Sun, 20 Jan 2008 15:47:18 +0100
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<P><FONT size=3D-1>The Journal of Neuroscience, May 15, 2001,=20
21(10):3483-3491</FONT><BR>
<H2>Immature Neurons From CNS Stem Cells Proliferate in Response to=20
Platelet-Derived Growth Factor </H2><STRONG><NOWRAP>Anna =
Erlandsson,<WBR> Maria=20
Enarsson, and<WBR> Karin Forsberg-Nilsson </NOWRAP></STRONG>
<P>Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala =
University,=20
SE-751 85&nbsp;Uppsala, Sweden=20
<P><A name=3Dabstract><!-- comment for mosaic --></A><BR clear=3Dall><A=20
name=3DAbstract><!-- null --></A>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"100%" bgColor=3D#e1e1e1>
  <TBODY>
  <TR>
    <TD vAlign=3Dcenter align=3Dleft width=3D"5%" =
bgColor=3D#ffffff>&nbsp;<IMG=20
      height=3D21 alt=3D"" hspace=3D5=20
      src=3D"http://www.jneurosci.org/icons/toc/rarrow.gif" =
width=3D10></TD>
    <TH vAlign=3Dcenter align=3Dleft width=3D"95%"><FONT=20
      size=3D+2>&nbsp;&nbsp;ABSTRACT</FONT></TH></TR></TBODY></TABLE>
<TABLE cellPadding=3D5 align=3Dright border=3D1 NOWRAP><R>
  <TBODY>
  <TR>
    <TH align=3Dleft><FONT size=3D-1><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Top"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>TOP</A> <BR><IMG height=3D9 hspace=3D5=20
      src=3D"http://www.jneurosci.org/icons/toc/dot.gif" width=3D11 =
border=3D0><FONT=20
      color=3D#464c53>ABSTRACT</FONT> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Introduction"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>INTRODUCTION</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC1"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>MATERIALS AND METHODS</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC2"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>RESULTS</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC3"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>DISCUSSION</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#References"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>REFERENCES</A> =
</FONT></TH></TR></TBODY></TABLE>
<P>Identifying external signals involved in the regulation of neural =
stem cell=20
proliferation and differentiation is fundamental<SUP> </SUP>to the =
understanding=20
of CNS development. In this study we show<SUP> </SUP>that =
platelet-derived=20
growth factor (PDGF) can act as a mitogen<SUP> </SUP>for neural =
precursor cells.=20
Multipotent stem cells from developing<SUP> </SUP>CNS can be maintained =
in a=20
proliferative state under serum-free<SUP> </SUP>conditions in the =
presence of=20
fibroblast growth factor-2 (FGF2)<SUP> </SUP>and induced to =
differentiate into=20
neurons, astrocytes, and oligodendrocytes<SUP> </SUP>on withdrawal of =
the=20
mitogen. PDGF has been suggested to play<SUP> </SUP>a role during the=20
differentiation into neurons. We have investigated<SUP> </SUP>the effect =
of PDGF=20
on cultured stem cells from embryonic rat cortex.<SUP> </SUP>The PDGF =
<IMG=20
alt=3D"alpha " =
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
align=3Dbottom>-receptor is constantly expressed during =
differentiation<SUP>=20
</SUP>of neural stem cells but is phosphorylated only after PDGF-AA<SUP> =

</SUP>treatment. In contrast, the PDGF <IMG alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =
align=3Dbottom>-receptor=20
is hardly detectable<SUP> </SUP>in uncommitted cells, but its expression =

increases during differentiation.<SUP> </SUP>We show that PDGF =
stimulation leads=20
to c-<I>fos</I> induction, 5'-bromo-2'deoxyuridine<SUP> =
</SUP>incorporation, and=20
an increase in the number of immature cells<SUP> </SUP>stained with =
antibodies=20
to neuronal markers. Our findings suggest<SUP> </SUP>that PDGF acts as a =
mitogen=20
in the early phase of stem cell differentiation<SUP> </SUP>to expand the =
pool of=20
immature<SUP> </SUP>neurons. </P>
<P><I>Key words: PDGF; neural stem cells; differentiation; neurogenesis; =
rat;=20
cortex; development </I></P><TXT><A name=3DSEC0><!-- comment for mosaic =
--></A><BR=20
clear=3Dall><A name=3DIntroduction><!-- null --></A>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"100%" bgColor=3D#e1e1e1>
  <TBODY>
  <TR>
    <TD vAlign=3Dcenter align=3Dleft width=3D"5%" =
bgColor=3D#ffffff>&nbsp;<IMG=20
      height=3D21 alt=3D"" hspace=3D5=20
      src=3D"http://www.jneurosci.org/icons/toc/rarrow.gif" =
width=3D10></TD>
    <TH vAlign=3Dcenter align=3Dleft width=3D"95%"><FONT=20
      =
size=3D+2>&nbsp;&nbsp;INTRODUCTION</FONT></TH></TR></TBODY></TABLE>
<TABLE cellPadding=3D5 align=3Dright border=3D1 NOWRAP><R>
  <TBODY>
  <TR>
    <TH align=3Dleft><FONT size=3D-1><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Top"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>TOP</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Abstract"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>ABSTRACT</A> <BR><IMG height=3D9 hspace=3D5=20
      src=3D"http://www.jneurosci.org/icons/toc/dot.gif" width=3D11 =
border=3D0><FONT=20
      color=3D#464c53>INTRODUCTION</FONT> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC1"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>MATERIALS AND METHODS</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC2"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>RESULTS</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC3"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>DISCUSSION</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#References"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>REFERENCES</A> =
</FONT></TH></TR></TBODY></TABLE>
<P>Neurons and glia in the mature CNS originate from precursor cells in =
the=20
ventricular zone of the fetal brain and spinal cord<SUP> </SUP>(Williams =
et al.,=20
1991<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B29"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
The division=20
of the precursor cells takes<SUP> </SUP>place close to the lumen of the=20
neuroepithelium, after which the<SUP> </SUP>postmitotic progeny migrate =
from the=20
ventricular zone to assume<SUP> </SUP>their final positions (Rakic, =
1972<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B21"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>, =
1990<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B22"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>; =
Hatten, 1990<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B10"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
</P>
<P>Many reports describe the establishment of <I>in vitro</I> culture of =

precursor cells from the embryonic rat or mouse brain (for<SUP> =
</SUP>review,=20
see Gage, 1998<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B7"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>; =
Temple and=20
Alvarez-Buylla, 1999<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B25"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>; =
Vescovi<SUP>=20
</SUP>and Snyder, 1999<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B27"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>).=20
Neuroepithelial stem cells can be expanded<SUP> </SUP>in fibroblast =
growth=20
factor-2 (FGF2)-containing serum-free medium<SUP> </SUP>and induced to=20
differentiate to neurons, astrocytes, or oligodendrocytes<SUP> </SUP>by =
the=20
withdrawal of mitogen (for review, see McKay, 1997<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B17"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
</P>
<P>Identification of external signals involved in the regulation of =
neural stem=20
cell proliferation and differentiation may allow<SUP> </SUP>for improved =

transplantation methods and augment the treatment<SUP> </SUP>of=20
neurodegenerative disorders. Several investigators report that<SUP> =
</SUP>in=20
cell cultures of neuroepithelial progenitor cells, exogenously<SUP> =
</SUP>added=20
growth factors can potentiate survival, induce proliferation,<SUP> =
</SUP>or=20
facilitate commitment or differentiation into mature phenotypes.<SUP>=20
</SUP>Besides the mitogenic effect of FGF2, it is well documented =
that<SUP>=20
</SUP>ciliary neurotrophic factor (CNTF) acts to direct multipotent<SUP> =

</SUP>stem cells to an astrocytic fate (Johe et al., 1996<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B13"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>; =
Bonni et<SUP>=20
</SUP>al., 1997<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B2"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>; =
Rajan and=20
McKay, 1998<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B20"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
The thyroid=20
hormone, triiodothyronine<SUP> </SUP>(T3), has been shown to influence=20
oligodendrocyte differentiation<SUP> </SUP>(Johe et al., 1996<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B13"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
Two previous=20
reports (Johe et al., 1996<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B13"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" =
border=3D1></A>;<SUP>=20
</SUP>Williams et al., 1997<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B30"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>) =
indicate that=20
PDGF can enhance neuronal<SUP> </SUP>differentiation. The possibility =
that PDGF=20
acts as a mitogen for<SUP> </SUP>immature neurons has been discussed but =
not=20
addressed<SUP> </SUP>experimentally. </P>
<P>Both PDGF ligands are present within the developing cortex at early =
embryonic=20
ages. Hutchins and Jefferson (1992)<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B12"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A> =
detected<SUP>=20
</SUP>PDGF-A-positive cells throughout the ventricular zone at =
mouse<SUP>=20
</SUP>embryonic day (E) 11.5,&nbsp;which is 2&nbsp;d after neural tube=20
closure<SUP> </SUP>and before the differentiation of most glial cells in =
all=20
areas<SUP> </SUP>of the nervous system. Some PDGF-B-reactive cells can =
also=20
be<SUP> </SUP>found in the subventricular zone of E14 rat embryos, which =
is<SUP>=20
</SUP>when rapid cellular proliferation and migration occur =
(Sasahara<SUP>=20
</SUP>et al., 1992<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B23"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
</P>
<P>In the present study we aimed at clarifying the effect of PDGF on =
cortical=20
neural stem cells in culture. We have found that<SUP> </SUP>PDGF-AA =
treatment=20
increases 5'-bromo-2'deoxyuridine bromodeoxyuridine<SUP> </SUP>(BrdU)=20
incorporation, and as a consequence it increases total<SUP> </SUP>cell =
number.=20
PDGF-AA was further shown to delay the differentiation<SUP> </SUP>of=20
microtubule-associated protein 2&nbsp;(MAP2)/TuJ1-positive neural<SUP>=20
</SUP>precursor cells, possibly by retaining committed but immature<SUP> =

</SUP>neurons in a proliferative state. Double staining with BrdU=20
antibodies<SUP> </SUP>showed that PDGF markedly increased the proportion =
of=20
proliferating<SUP> </SUP>cells that expressed MAP2. These results =
suggest that=20
PDGF acts<SUP> </SUP>to expand a pool of immature<SUP> </SUP>neurons. =
</P><BR=20
clear=3Dall><A name=3DSEC1><!-- null --></A>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"100%" bgColor=3D#e1e1e1>
  <TBODY>
  <TR>
    <TD vAlign=3Dcenter align=3Dleft width=3D"5%" =
bgColor=3D#ffffff>&nbsp;<IMG=20
      height=3D21 alt=3D"" hspace=3D5=20
      src=3D"http://www.jneurosci.org/icons/toc/rarrow.gif" =
width=3D10></TD>
    <TH vAlign=3Dcenter align=3Dleft width=3D"95%"><FONT=20
      size=3D+2>&nbsp;&nbsp;MATERIALS AND =
METHODS</FONT></TH></TR></TBODY></TABLE>
<TABLE cellPadding=3D5 align=3Dright border=3D1 NOWRAP><R>
  <TBODY>
  <TR>
    <TH align=3Dleft><FONT size=3D-1><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Top"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>TOP</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Abstract"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>ABSTRACT</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Introduction"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>INTRODUCTION</A> <BR><IMG height=3D9 =
hspace=3D5=20
      src=3D"http://www.jneurosci.org/icons/toc/dot.gif" width=3D11 =
border=3D0><FONT=20
      color=3D#464c53>MATERIALS AND METHODS</FONT> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC2"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>RESULTS</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC3"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>DISCUSSION</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#References"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>REFERENCES</A> =
</FONT></TH></TR></TBODY></TABLE>
<P><I>Cell culture</I>. Embryonic cortex was dissected in HBSS (Life=20
Technologies, Paisley, Scotland) from timed-pregnant Sprague Dawley<SUP> =

</SUP>rats (B&amp;K, Sollentuna, Sweden) on E15 (E1 was defined as the =
day<SUP>=20
</SUP>of copulatory plug). The tissue was gently triturated, and =
meninges<SUP>=20
</SUP>and larger cell clumps were allowed to sediment for 10&nbsp;min,=20
after<SUP> </SUP>which the cell suspension was centrifuged. The pellet =
was=20
resuspended<SUP> </SUP>in N2-medium (Bottenstein and Sato, 1979<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B3"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>) =
with=20
10&nbsp;ng/ml FGF2 (Pepro<SUP> </SUP>Technologies, London, England) and =
plated=20
at a density of 1&nbsp;=D7<SUP>&nbsp;</SUP>10<SUP><IMG alt=3D-=20
src=3D"http://www.jneurosci.org/math/12pt/normal/scr/minus.gif"=20
align=3Dbottom>6</SUP> cells per 10&nbsp;cm tissue culture dish that was =
precoated=20
with poly-<FONT size=3D-1>L</FONT>-ornithine<SUP> </SUP>(Sigma, St. =
Louis, MO) and=20
fibronectin (Life Technologies). Fresh<SUP> </SUP>FGF2 was added daily, =
and the=20
medium was changed every other day.<SUP> </SUP>When subconfluent, the =
cells were=20
passaged by scraping with a<SUP> </SUP>cell lifter (Costar, Cambridge, =
MA).=20
After the cells were gently<SUP> </SUP>triturated in HBSS, the cell =
suspension=20
was centrifuged, and the<SUP> </SUP>pellet was resuspended in N2-medium =
and=20
plated. The cells were<SUP> </SUP>used for experiments 2-6 d after the =
first=20
passage. The concentrations<SUP> </SUP>of PDGF-AA (Pepro Technologies) =
used in=20
the experiments were 10<SUP>&nbsp;</SUP>ng/ml for continuous treatment,=20
20&nbsp;ng/ml for treatment with a<SUP> </SUP>single dose, and =
100&nbsp;ng/ml=20
for shorter exposures (c-<I>fos</I> and protein<SUP> </SUP>kinase B=20
(PKB)/c-Akt<SUP> </SUP>experiments). </P>
<P><I>Immunocytochemistry</I>. Cells were fixed in ice-cold acid ethanol =
(90%=20
ethanol, 5% acetic acid). Endogenous peroxidase was quenched<SUP> =
</SUP>by 0.3%=20
H<SUB>2</SUB>O<SUB>2</SUB> in methanol for 20&nbsp;min followed by =
washing with=20
PBS<SUP> </SUP>and permeabilization and blocking in 0.1% Triton X-100 =
with=20
5%<SUP> </SUP>normal goat serum (Dako A/S, Glostrup, Denmark) in PBS for =

30<SUP>&nbsp;</SUP>min. The cells were incubated for 1-4 hr with primary =

antibody,<SUP> </SUP>washed in PBS, and incubated with biotinylated goat =

anti-mouse<SUP> </SUP>immunoglobulin (Kirkegaard and Perry Laboratories, =

Gaithersburg,<SUP> </SUP>MD) for 30&nbsp;min-1 hr. After washing with =
PBS, the=20
reaction was<SUP> </SUP>developed with diaminobenzidine (DAB SigmaFast, =
Sigma).=20
Incorporation<SUP> </SUP>of BrdU was detected using anti-BrdU antibodies =

(Amersham Pharmacia<SUP> </SUP>Biotech, Uppsala, Sweden). Cells were =
pulsed with=20
10&nbsp;m<FONT size=3D-1>M</FONT> BrdU (Amersham<SUP> </SUP>Pharmacia =
Biotech) for=20
14&nbsp;hr before fixation. Stained cells were<SUP> </SUP>counted, and =
the ratio=20
of BrdU-positive cells to total cell number<SUP> </SUP>per field at =
200=D7=20
magnification was calculated. Seven parallel<SUP> </SUP>fields were =
counted for=20
each time point, and each time point was<SUP> </SUP>performed in =
duplicate. For=20
the double staining of MAP2 and BrdU,<SUP> </SUP>a cell proliferation =
kit,=20
including a nickel-enhanced peroxidase<SUP> </SUP>reaction (Amersham =
Pharmacia=20
Biotech), was used for detection<SUP> </SUP>of BrdU-positive cells =
together with=20
the regular peroxidase reaction<SUP> </SUP>for MAP2 as described above.=20
Duplicate cultures were stained for<SUP> </SUP>MAP2, and 10&nbsp;defined =
fields=20
of each culture dish were photographed,<SUP> </SUP>using a computerized=20
microscope (Axiovert 100TV, <A=20
href=3D"http://www.jneurosci.org/cgi/inline?zeiss">Zeiss</A>). =
After<SUP>=20
</SUP>BrdU staining, the same fields were photographed again. Cells<SUP> =

</SUP>counts were determined from photographs of stained cells. =
Monoclonal<SUP>=20
</SUP>anti-MAP2 antibody (clone HM2, Sigma), dilution 1:200, and =
monoclonal<SUP>=20
</SUP>anti-Tuj1 antibody (BabCO, Nordic Biosite AB, T=E4by, =
Sweden),<SUP>=20
</SUP>dilution 1:50, were used. A polyclonal antibody to nestin was<SUP> =
</SUP>a=20
kind gift from Dr. R.&nbsp;McKay (National Institutes of Health,<SUP>=20
</SUP>Bethesda,<SUP> </SUP>MD). </P>
<P><I>Deoxynucleotidyl transferase-mediated biotinylated UTP nick end =
labeling=20
assay</I>. Cells were grown on poly-<FONT size=3D-1>L</FONT>-ornathine =
and=20
fibronectin<SUP> </SUP>precoated glass coverslips. After fixation in 2%=20
paraformaldehyde,<SUP> </SUP>pH 7.4,&nbsp;the cells were stained using a =

terminal deoxynucleotidyl<SUP> </SUP>transferase-mediated biotinylated =
UTP nick=20
end labeling (TUNEL)<SUP> </SUP>kit (Roche Diagnostics, Mannheim, =
Germany). The=20
proportion of<SUP> </SUP>cells going through apoptosis was counted in=20
10&nbsp;parallel fields,<SUP> </SUP>using a fluorescence microscope. =
Each time=20
point was performed<SUP> </SUP>in<SUP> </SUP>duplicate. </P>
<P><I>Cell counting</I>. Cell counting was performed using a Coulter Z1 =
cell=20
counter (Coulter Electronics, Herpendon, UK). The cells<SUP> </SUP>were =
removed=20
from the dishes by scraping and were then carefully<SUP> =
</SUP>dissociated and=20
counted. Each experiment was performed in<SUP> </SUP>triplicate. </P>
<P><I>Northern blot analysis</I>. Total RNA was isolated using the =
LiCl/urea=20
method (Auffrey and Rougeon, 1980<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B1"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
For Northern=20
electrophoresis,<SUP> </SUP>12&nbsp;=B5g of RNA per slot was loaded to a =
1%=20
agarose gel with 2.2<SUP>&nbsp;</SUP><FONT size=3D-1>M</FONT> =
formaldehyde. The=20
RNA was transferred to Duralon-UV membranes<SUP> </SUP>(Stratagene, La =
Jolla,=20
CA) and fixed by baking at 80=B0C under vacuum.<SUP> 32</SUP>P-labeled =
probes were=20
prepared using a kit for random priming<SUP> </SUP>(Megaprime DNA =
labeling=20
systems, Amersham Pharmacia Biotech),<SUP> </SUP>and hybridization was =
performed=20
under high stringency conditions<SUP> </SUP>(50% formamide) at 42=B0C =
using=20
QuickHyb solution (Stratagene).<SUP> </SUP>The intactness and total =
amount of=20
RNA were checked by hybridization<SUP> </SUP>to a glyceraldehyde =
phosphate=20
dehydrogenase (GAPDH) probe. After<SUP> </SUP>washing at 55-60=B0C, the =
filters=20
were exposed to Phosphor Imager<SUP> </SUP>screens (Fuji, Tokyo, Japan) =
for=20
12-14 hr and analyzed in a Phosphor<SUP> </SUP>Imager (Fuji). =
Normalization of=20
PDGF receptor RNA levels was obtained<SUP> </SUP>by scanning the =
hybridization=20
signal, and the ratio of receptor<SUP> </SUP>signal to GAPDH signal was=20
calculated. A cDNA for GAPDH was a<SUP> </SUP>gift from Dr. R.&nbsp;Wu =
(Cornell=20
University, Ithaca, NY), and murine<SUP> </SUP>cDNA for c-<I>fos</I> was =
kindly=20
provided by Dr. R.&nbsp;Wallrich (EMBL, Heidelberg,<SUP> </SUP>Germany). =
</P>
<P><I>Western blot analysis</I>: <I>total lysate</I>. Cells were lysed =
in=20
boiling 2=D7 Laemmli sample buffer (4% SDS, 20% glycerol, Tris-HCl,<SUP> =
</SUP>pH=20
6.8) and sonicated, and the cell debris was removed by =
centrifugation.<SUP>=20
</SUP>Protein samples were boiled with 5% betamercaptoethanol and =
loaded<SUP>=20
</SUP>to the wells of a 8-16% gradient Tris-glycine gel (Novex). =
For<SUP>=20
</SUP>immunoblotting, the samples were electrically transferred =
onto<SUP>=20
</SUP>a nitrocellulose membrane (Hybond ECL Nitrocellulose =
membrane,<SUP>=20
</SUP>Amersham Pharmacia Biotech) according to the protocol =
provided<SUP>=20
</SUP>by the manufacturer. Nonspecific protein binding to the =
filter<SUP>=20
</SUP>was blocked by incubation in Tris-buffered saline/Tween =
(TBS-T:<SUP>=20
</SUP>20&nbsp;m<FONT size=3D-1>M</FONT> Tris base, pH =
7.6,&nbsp;137&nbsp;m<FONT=20
size=3D-1>M</FONT> NaCl, 0.2% Tween-20) containing<SUP> </SUP>5% BSA for =
1&nbsp;hr=20
at room temperature, followed by incubation with<SUP> </SUP>primary =
antibody=20
overnight at 4=B0C. After washing with TBS-T, the<SUP> </SUP>membrane =
was=20
incubated with peroxidase-conjugated secondary antibody<SUP> =
</SUP>(Amersham=20
Pharmacia Biotech), dilution 1:5000, for 1&nbsp;hr at room<SUP>=20
</SUP>temperature. After the filter was washed in TBS-T, the blot =
was<SUP>=20
</SUP>developed using an enhanced chemiluminescence (ECL) system =
(Amersham<SUP>=20
</SUP>Pharmacia Biotech). Before rehybridization of the filters, =
the<SUP>=20
</SUP>membrane was dehybridized (20% SDS, Tris-HCl, pH =
6.8,&nbsp;340&nbsp;=B5l=20
betamercaptoethanol)<SUP> </SUP>at 55=B0C for 30&nbsp;min and washed in =
TBS-T.=20
Primary antibodies were<SUP> </SUP>anti-PKB/c-Akt antibody (Akt1 =
#06-558,=20
Upstate Biotech) and anti-phosphorylated-PKB/c-Akt<SUP> </SUP>antibody=20
(Ser-473-PhosphoAkt #9271S, New England Biolabs), diluted<SUP> =
</SUP>1:1000.=20
</P>
<P><I>Western blot analysis: immunoprecipitation</I>. Cell pellets were=20
solubilized in lysis buffer (1% Triton X-100, 20&nbsp;m<FONT =
size=3D-1>M</FONT>=20
Tris-HCl,<SUP> </SUP>pH 7.5,&nbsp;150&nbsp;m<FONT size=3D-1>M</FONT> =
NaCl,=20
2&nbsp;m<FONT size=3D-1>M</FONT> EDTA, 1% aprotinin, 2&nbsp;m<FONT=20
size=3D-1>M</FONT> phenylmethylsulfonyl<SUP> </SUP>fluoride, =
250&nbsp;=B5<FONT=20
size=3D-1>M</FONT> orthovanadate) on ice for 5&nbsp;min, after =
which<SUP>=20
</SUP>cell debris was removed by centrifugation. =
Immunoprecipitation<SUP>=20
</SUP>was performed using 150&nbsp;=B5g protein. Lysates were incubated =
with<SUP>=20
</SUP>10&nbsp;=B5l anti-PDGF <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif" =
align=3Dbottom>-receptor=20
antibody (anti-PDGF-R<IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif" =
align=3Dbottom>=20
antibody), R7,<SUP> </SUP>for 90&nbsp;min at 4=B0C and another =
45&nbsp;min on ice=20
after the addition<SUP> </SUP>of 50&nbsp;=B5l of protein-A-Sepharose =
beads=20
(Amersham Pharmacia Biotech).<SUP> </SUP>The supernatant was then =
incubated with=20
10&nbsp;=B5l of anti-PDGF <IMG alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif"=20
align=3Dbottom>-receptor<SUP> </SUP>antibody (anti-PDGF-R<IMG =
alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =
align=3Dbottom>=20
antibody), R33, and new protein-A-Sepharose<SUP> </SUP>beads. The R7 and =
R33=20
antibodies were kind gifts from Dr. C.-H.<SUP> </SUP>Heldin (Ludwig =
Institute=20
for Cancer Research, Uppsala, Sweden).<SUP> </SUP>The =
protein-A-Sepharose was=20
washed three times with lysis buffer<SUP> </SUP>and once with ice-cold =
PBS. To=20
eluate the protein, the samples<SUP> </SUP>were incubated with =
25&nbsp;=B5l of=20
SDS-sample buffer (Novex, San Diego,<SUP> </SUP>CA) with 2% =
betamercaptoethanol=20
at 95=B0C for 5&nbsp;min. The supernatant<SUP> </SUP>was loaded to the =
wells of a=20
4-12% gradient Tris-glycine gel (Novex).<SUP> </SUP>Immunoblotting was =
performed=20
as above but with a different filter<SUP> </SUP>(Immobilon transfer =
membrane;=20
Millipore, Bedford, MA). Primary<SUP> </SUP>antibodies were =
anti-phosphotyrosine=20
antibody, PY20 (diluted 1:1250;<SUP> </SUP>Transduction Laboratories, =
Exeter,=20
UK), anti-PDGF<IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
align=3Dbottom>-receptor, R7<SUP> </SUP>(diluted 1:500), or =
anti-PDGF<IMG=20
alt=3D"beta " src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =

align=3Dbottom>-receptor, M20 (diluted 1:500; Santa<SUP> </SUP>Cruz =
Biotechnology,=20
Santa Cruz,<SUP> </SUP>CA). </P>
<P><I>Statistical analysis</I>. For statistical analysis we used ANOVA =
followed=20
by Fisher's multiple comparison. Values presented are<SUP> </SUP>the=20
means&nbsp;=B1<SUP>&nbsp;</SUP>SEM. </P><BR clear=3Dall><A=20
name=3DSEC2><!-- null --></A>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"100%" bgColor=3D#e1e1e1>
  <TBODY>
  <TR>
    <TD vAlign=3Dcenter align=3Dleft width=3D"5%" =
bgColor=3D#ffffff>&nbsp;<IMG=20
      height=3D21 alt=3D"" hspace=3D5=20
      src=3D"http://www.jneurosci.org/icons/toc/rarrow.gif" =
width=3D10></TD>
    <TH vAlign=3Dcenter align=3Dleft width=3D"95%"><FONT=20
      size=3D+2>&nbsp;&nbsp;RESULTS</FONT></TH></TR></TBODY></TABLE>
<TABLE cellPadding=3D5 align=3Dright border=3D1 NOWRAP><R>
  <TBODY>
  <TR>
    <TH align=3Dleft><FONT size=3D-1><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Top"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>TOP</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Abstract"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>ABSTRACT</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Introduction"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>INTRODUCTION</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC1"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>MATERIALS AND METHODS</A> <BR><IMG =
height=3D9 hspace=3D5=20
      src=3D"http://www.jneurosci.org/icons/toc/dot.gif" width=3D11 =
border=3D0><FONT=20
      color=3D#464c53>RESULTS</FONT> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC3"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>DISCUSSION</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#References"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>REFERENCES</A> =
</FONT></TH></TR></TBODY></TABLE>
<P align=3Dleft><B>Expression of PDGF receptors during stem=20
cell&nbsp;differentiation</B> </P>
<P>The cell culture system used in this study has been described in =
detail=20
elsewhere (Johe et al., 1996<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B13"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
Briefly,=20
multipotential<SUP> </SUP>neural stem cells can be maintained in the =
presence of=20
FGF2 and<SUP> </SUP>induced to differentiate on withdrawal of the =
mitogen to=20
form<SUP> </SUP>neurons, astrocytes, and oligodendrocytes. During the =
first=20
few<SUP> </SUP>days after FGF2 withdrawal, addition of polypeptide =
growth=20
factors,<SUP> </SUP>cytokines, or hormones can change the proportion of =
cell=20
lineages<SUP> </SUP>that are generated. PDGF has been suggested to =
augment=20
neuronal<SUP> </SUP>differentiation in two studies (Johe et al., 1996<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B13"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>; =
Williams=20
et<SUP> </SUP>al., 1997<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B30"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
In this study=20
we aimed to define the action of PDGF<SUP> </SUP>on neuronal stem cells =
isolated=20
from embryonic rat cortex. We<SUP> </SUP>have shown previously that =
these cells=20
express PDGF <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
align=3Dbottom>-receptor<SUP> </SUP>RNA and can respond to PDGF by =
chemotaxis=20
(Forsberg-Nilsson et<SUP> </SUP>al., 1998<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B6"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
</P>
<P>Western blot analysis was used to investigate the expression and=20
phosphorylation of PDGF receptors in cultured neuroepithelial<SUP> =
</SUP>cells=20
during differentiation (Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F1">1</A>).=20
Proliferating cells were<SUP> </SUP>compared with cells differentiating =
after=20
withdrawal of FGF2.<SUP> </SUP>Cell lysates were prepared from =
proliferating=20
stem cells (FGF2)<SUP> </SUP>and cortical stem cells at=20
1,&nbsp;2,&nbsp;4,&nbsp;and 6&nbsp;d after FGF2 withdrawal<SUP> =
</SUP>(Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F1">1</A>,=20
<I>No add</I>.). Each cell lysate was first immunoprecipitated<SUP> =
</SUP>with=20
antibodies to PDGF <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif" =
align=3Dbottom>-receptor=20
(R7), after which the supernatant<SUP> </SUP>was subjected to a second =
round of=20
immunoprecipitation with anti-PDGF<SUP> </SUP><IMG alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =
align=3Dbottom>-receptor=20
antibodies (R33). The membranes were then hybridized<SUP> </SUP>with =
antibodies=20
to the PDGF <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif" =
align=3Dbottom>-receptor=20
or the PDGF <IMG alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif"=20
align=3Dbottom>-receptor.<SUP> </SUP>Anti-phosphotyrosine antibody was =
used to=20
detect phosphorylation<SUP> </SUP>of the receptors. Figure <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F1">1</A>=20
shows that although the PDGF <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
align=3Dbottom>-receptor<SUP> </SUP>is expressed on cortical stem cells, =
no=20
phosphorylation occurred<SUP> </SUP>in the absence of exogenous ligand.=20
Proliferating stem cells treated<SUP> </SUP>with 20&nbsp;ng/ml of =
PDGF-AA were=20
used as a positive control for phosphorylated<SUP> </SUP><IMG =
alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
align=3Dbottom>-receptor. <A name=3DF1><!-- FIG 1 --></A>
<P><BR clear=3Dall>
<CENTER>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"95%" border=3D1>
  <TBODY>
  <TR bgColor=3D#e1e1e1>
    <TD>
      <TABLE cellSpacing=3D2 cellPadding=3D2>
        <TBODY>
        <TR bgColor=3D#e1e1e1>
          <TD vAlign=3Dtop align=3Dmiddle bgColor=3D#ffffff><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F1"><IMG=20
            height=3D132 hspace=3D10=20
            =
src=3D"http://www.jneurosci.org/content/vol21/issue10/images/small/ns1015=
209001.gif"=20
            width=3D200 vspace=3D5 border=3D2></A><BR><STRONG>View =
larger=20
            version</STRONG> (27K): <BR><NOBR><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F1">[in=20
            this window]</A> <BR><A=20
            onmouseover=3D"window.status=3D'View figure in a separate =
window'; return true"=20
            onclick=3D"startTarget('F1', 590, 490); =
this.href=3D'/cgi/content-nw/full/21/10/3483/F1'"=20
            =
href=3D"http://www.jneurosci.org/cgi/content-nw/full/21/10/3483/F1"=20
            target=3DF1>[in a new window]</A><BR>&nbsp; </NOBR></TD>
          <TD vAlign=3Dtop align=3Dleft><STRONG>Figure 1. =
&nbsp;</STRONG> Western=20
            blot analysis was used to study PDGF receptor expression and =

            phosphorylation in CNS stem cells during differentiation.=20
            Proliferating cells (<I>FGF2</I>) were compared with =
differentiating=20
            cells after FGF2 withdrawal (<I>No add</I>.). All cell =
lysates were=20
            immunoprecipitated with a PDGF <IMG alt=3D"alpha "=20
            src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
            align=3Dbottom>-receptor antibody. For immunoprecipitation =
of the PDGF=20
            <IMG alt=3D"beta "=20
            src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif"=20
            align=3Dbottom>-receptor, the supernatant from the <IMG =
alt=3D"alpha "=20
            src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
            align=3Dbottom>-receptor precipitation was further incubated =
with a=20
            PDGF <IMG alt=3D"beta "=20
            src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif"=20
            align=3Dbottom>-receptor antibody. Anti-PDGF <IMG =
alt=3D"alpha "=20
            src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
            align=3Dbottom>-receptor antibody, anti-PDGF <IMG =
alt=3D"beta "=20
            src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif"=20
            align=3Dbottom>-receptor antibody, or anti-phosphotyrosine =
antibody=20
            was used for immunoblotting. Lysates were made from cortical =
stem=20
            cells grown in the presence of FGF2, at the start of the =
experiment,=20
            and from cells grown 1,&nbsp;2,&nbsp;4,&nbsp;and 6&nbsp;d =
after FGF2=20
            withdrawal. Cells treated with PDGF-AA for 24&nbsp;hr were =
included=20
            as a positive control for phosphorylated PDGF <IMG =
alt=3D"alpha "=20
            src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
            align=3Dbottom>-receptor. <BR=20
  =
clear=3Dleft></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER=
><!-- /FIG 1 -->
<P></P>
<P>PDGF <IMG alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =
align=3Dbottom>-receptor=20
expression in cortical stem cells was hardly detectable in uncommitted =
cells but=20
increased as the cells differentiated<SUP> </SUP>after FGF2 withdrawal. =
The=20
phosphorylation of the <IMG alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =
align=3Dbottom>-receptor=20
followed<SUP> </SUP>the same pattern, with no or very little =
phosphorylation the=20
first<SUP> </SUP>2&nbsp;d of differentiation and a distinct =
phosphorylation at=20
day 4<SUP>&nbsp;</SUP>that increased further until day 6&nbsp;after FGF2 =

withdrawal. On the<SUP> </SUP>basis of these and previous results from =
our=20
laboratory we concluded<SUP> </SUP>that the PDGF <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif" =
align=3Dbottom>-receptor=20
is the predominant receptor isoform during<SUP> </SUP>early stem =
cell<SUP>=20
</SUP>differentiation. </P>
<P align=3Dleft><B>Expression of PDGF receptors after =
PDGF-AA&nbsp;stimulation</B>=20
</P>
<P>Because PDGF has been implicated in neuronal differentiation of CNS =
stem=20
cells, we next investigated how receptor levels were<SUP> =
</SUP>influenced by=20
differentiation in the presence of PDGF. Western<SUP> </SUP>blot =
analysis was=20
used to study expression and phosphorylation<SUP> </SUP>of PDGF =
receptors in=20
neuroepithelial cells after PDGF-AA stimulation.<SUP> </SUP>Subconfluent =

cortical stem cells were grown for 1,&nbsp;2,&nbsp;4,&nbsp;and=20
6<SUP>&nbsp;</SUP>d after withdrawal of FGF2, in the presence of =
10&nbsp;ng/ml=20
of PDGF-AA,<SUP> </SUP>and compared with proliferating stem cells =
(FGF2).=20
Because previous<SUP> </SUP>investigators (Williams et al., 1997<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B30"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>) =
suggest that a=20
short exposure<SUP> </SUP>to PDGF suffices to stimulate neuronal=20
differentiation, parallel<SUP> </SUP>cultures received a single dose of=20
20&nbsp;ng/ml PDGF-AA on day 0,<SUP>&nbsp;</SUP>after which they were =
kept in=20
plain N2-medium. </P>
<P>Figure <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F2">2</A><I>A</I>=20
shows that the PDGF <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif" =
align=3Dbottom>-receptor=20
expression level remained constant throughout the experiment, in which =
cortical=20
stem<SUP> </SUP>cells were treated continuously with PDGF-AA. In =
cultures=20
treated<SUP> </SUP>with a single dose of PDGF-AA, the PDGF <IMG =
alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif" =
align=3Dbottom>-receptor=20
expression<SUP> </SUP>level decreased 4&nbsp;d after withdrawal of FGF2 =
(Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F2">2</A><I>B</I>).=20
<A name=3DF2><!-- FIG 2 --></A>
<P><BR clear=3Dall>
<CENTER>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"95%" border=3D1>
  <TBODY>
  <TR bgColor=3D#e1e1e1>
    <TD>
      <TABLE cellSpacing=3D2 cellPadding=3D2>
        <TBODY>
        <TR bgColor=3D#e1e1e1>
          <TD vAlign=3Dtop align=3Dmiddle bgColor=3D#ffffff><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F2"><IMG=20
            height=3D200 hspace=3D10=20
            =
src=3D"http://www.jneurosci.org/content/vol21/issue10/images/small/ns1015=
209002.gif"=20
            width=3D149 vspace=3D5 border=3D2></A><BR><STRONG>View =
larger=20
            version</STRONG> (28K): <BR><NOBR><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F2">[in=20
            this window]</A> <BR><A=20
            onmouseover=3D"window.status=3D'View figure in a separate =
window'; return true"=20
            onclick=3D"startTarget('F2', 477, 640); =
this.href=3D'/cgi/content-nw/full/21/10/3483/F2'"=20
            =
href=3D"http://www.jneurosci.org/cgi/content-nw/full/21/10/3483/F2"=20
            target=3DF2>[in a new window]</A><BR>&nbsp; </NOBR></TD>
          <TD vAlign=3Dtop align=3Dleft><STRONG>Figure 2. =
&nbsp;</STRONG> Western=20
            blot analysis of PDGF receptor expression and =
phosphorylation in CNS=20
            stem cells after PDGF-AA stimulation. Immunoprecipitation of =
cell=20
            lysates was performed using antibodies to PDGF <IMG =
alt=3D"alpha "=20
            src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
            align=3Dbottom>-receptor and PDGF <IMG alt=3D"beta "=20
            src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif"=20
            align=3Dbottom>-receptor. Anti-PDGF <IMG alt=3D"alpha "=20
            src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
            align=3Dbottom>-receptor antibody, anti-PDGF <IMG =
alt=3D"beta "=20
            src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif"=20
            align=3Dbottom>-receptor antibody, or anti-phosphotyrosine =
antibody=20
            was used for immunoblotting. Lysates were made from cortical =
stem=20
            cells grown in the presence of FGF2 (at the start of the=20
            experiment), from cells treated continuously with PDGF-AA =
after the=20
            withdrawal of FGF2 (<I>A</I>), and from cells stimulated =
with a=20
            single dose of PDGF-AA at the time of FGF2 withdrawal =
(<I>B</I>).=20
            <BR =
clear=3Dleft></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER=
><!-- /FIG 2 -->
<P></P>
<P>After PDGF-AA stimulation, phosphorylation of the PDGF <IMG =
alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif" =
align=3Dbottom>-receptor=20
in cortical stem cell cultures reached a maximum the first<SUP> =
</SUP>day after=20
stimulation. Thereafter, the level of receptor phosphorylation<SUP>=20
</SUP>decreased but was maintained at a level higher than that of =
control<SUP>=20
</SUP>cells (Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F2">2</A><I>A</I>).=20
The phosphorylation level in cells that received<SUP> </SUP>a single =
dose of=20
PDGF also decreased after a peak on day 1&nbsp;and<SUP> </SUP>reached =
control=20
levels at 4&nbsp;d after stimulation (Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F2">2</A><I>B</I>).=20
</P>
<P>Similarly to cortical stem cells grown in the absence of PDGF, the =
PDGF <IMG=20
alt=3D"beta " src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =

align=3Dbottom>-receptor level for PDGF-treated cells was very low<SUP> =
</SUP>at=20
the beginning of the experiment, but the expression increased<SUP> =
</SUP>as the=20
cells differentiated. In contrast to cultures differentiating<SUP> =
</SUP>in=20
plain N2-medium, in which a distinct phosphorylation of the<SUP> =
</SUP>PDGF <IMG=20
alt=3D"beta " src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =

align=3Dbottom>-receptor was seen at days 4&nbsp;and 6,&nbsp;no =
phosphorylation=20
of<SUP> </SUP>the <IMG alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =
align=3Dbottom>-receptor=20
was detected in cultures treated with PDGF-AA (data<SUP> </SUP>not<SUP>=20
</SUP>shown). </P>
<P align=3Dleft><B>PDGF induces c-fos RNA&nbsp;expression</B> </P>
<P>In our examination of cellular events initiated by activation of PDGF =

receptors, we next studied expression of the immediate-early<SUP> =
</SUP>gene=20
c-<I>fos</I> in response to PDGF-AA. We first incubated cortical<SUP> =
</SUP>stem=20
cell cultures for 3&nbsp;hr in N2-medium without FGF2 to minimize<SUP>=20
</SUP>possible c-<I>fos</I> background expression. Cells were then=20
stimulated<SUP> </SUP>with PDGF-AA and harvested for RNA preparation =
after=20
10&nbsp;min, 30<SUP>&nbsp;</SUP>min, 1&nbsp;hr, and 2&nbsp;hr. Control =
cells=20
were incubated for 3&nbsp;hr without<SUP> </SUP>FGF, after which RNA was =

prepared. In Figure <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F3">3</A>=20
the ratio of c-<I>fos</I><SUP> </SUP>RNA to GAPDH RNA is shown. The=20
PDGF-AA-stimulated c-<I>fos</I> expression<SUP> </SUP>displayed a =
maximal=20
induction of 50-fold the control level at<SUP> </SUP>1&nbsp;hr. At =
2&nbsp;hr the=20
amount of c-<I>fos</I> RNA had declined but had not<SUP> </SUP>yet =
reached that=20
of control cells. <A name=3DF3><!-- FIG 3 --></A>
<P><BR clear=3Dall>
<CENTER>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"95%" border=3D1>
  <TBODY>
  <TR bgColor=3D#e1e1e1>
    <TD>
      <TABLE cellSpacing=3D2 cellPadding=3D2>
        <TBODY>
        <TR bgColor=3D#e1e1e1>
          <TD vAlign=3Dtop align=3Dmiddle bgColor=3D#ffffff><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F3"><IMG=20
            height=3D200 hspace=3D10=20
            =
src=3D"http://www.jneurosci.org/content/vol21/issue10/images/small/ns1015=
209003.gif"=20
            width=3D171 vspace=3D5 border=3D2></A><BR><STRONG>View =
larger=20
            version</STRONG> (17K): <BR><NOBR><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F3">[in=20
            this window]</A> <BR><A=20
            onmouseover=3D"window.status=3D'View figure in a separate =
window'; return true"=20
            onclick=3D"startTarget('F3', 526, 640); =
this.href=3D'/cgi/content-nw/full/21/10/3483/F3'"=20
            =
href=3D"http://www.jneurosci.org/cgi/content-nw/full/21/10/3483/F3"=20
            target=3DF3>[in a new window]</A><BR>&nbsp; </NOBR></TD>
          <TD vAlign=3Dtop align=3Dleft><STRONG>Figure 3. =
&nbsp;</STRONG> Northern=20
            blot analysis of c-<I>fos</I> expression in cortical stem =
cells=20
            after PDGF-AA stimulation. Cell cultures were incubated =
without FGF2=20
            for 3&nbsp;hr, stimulated with PDGF-AA, and harvested for =
RNA=20
            preparation after 10&nbsp;min, 30&nbsp;min, 1&nbsp;hr, and=20
            2&nbsp;hr. The levels of RNA expression are given as the =
ratios of=20
            c-<I>fos</I> to GAPDH pixels. <BR=20
  =
clear=3Dleft></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER=
><!-- /FIG 3 -->
<P></P>
<P align=3Dleft><B>PDGF-AA stimulation of BrdU&nbsp;incorporation</B> =
</P>
<P>To investigate whether PDGF-AA can stimulate DNA synthesis in =
cortical stem=20
cells, cultures were pulse labeled with BrdU for<SUP> </SUP>14&nbsp;hr =
and=20
stained with an anti-BrdU antibody. The BrdU incorporation<SUP> </SUP>in =

FGF2-cultured cells were 86&nbsp;=B1&nbsp;2.9% (data not shown). =
Cells<SUP>=20
</SUP>grown in the absence of FGF2 were compared with cells treated<SUP> =

</SUP>once with PDGF-AA, at the time of FGF2 withdrawal, and cells =
treated<SUP>=20
</SUP>continuously with PDGF-AA (Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F4">4</A>).=20
Counting of BrdU-positive<SUP> </SUP>cells revealed that treatment with =
PDGF-AA=20
gives a fourfold increase<SUP> </SUP>in BrdU incorporation 2&nbsp;d =
after=20
stimulation, compared with control<SUP> </SUP>cultures that received no =
PDGF. In=20
cultures receiving a single<SUP> </SUP>dose of PDGF, the amount of cells =

incorporating BrdU had declined<SUP> </SUP>to that of control cells =
after=20
4&nbsp;d. In cells treated continuously<SUP> </SUP>with PDGF-AA, the =
number of=20
BrdU-positive cells remained significantly<SUP> </SUP>higher after =
4&nbsp;d. In=20
all PDGF-treated cells, BrdU incorporation<SUP> </SUP>levels had =
declined to=20
that of control cells after 6&nbsp;d. <A name=3DF4><!-- FIG 4 --></A>
<P><BR clear=3Dall>
<CENTER>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"95%" border=3D1>
  <TBODY>
  <TR bgColor=3D#e1e1e1>
    <TD>
      <TABLE cellSpacing=3D2 cellPadding=3D2>
        <TBODY>
        <TR bgColor=3D#e1e1e1>
          <TD vAlign=3Dtop align=3Dmiddle bgColor=3D#ffffff><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F4"><IMG=20
            height=3D200 hspace=3D10=20
            =
src=3D"http://www.jneurosci.org/content/vol21/issue10/images/small/ns1015=
209004.gif"=20
            width=3D185 vspace=3D5 border=3D2></A><BR><STRONG>View =
larger=20
            version</STRONG> (29K): <BR><NOBR><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F4">[in=20
            this window]</A> <BR><A=20
            onmouseover=3D"window.status=3D'View figure in a separate =
window'; return true"=20
            onclick=3D"startTarget('F4', 557, 640); =
this.href=3D'/cgi/content-nw/full/21/10/3483/F4'"=20
            =
href=3D"http://www.jneurosci.org/cgi/content-nw/full/21/10/3483/F4"=20
            target=3DF4>[in a new window]</A><BR>&nbsp; </NOBR></TD>
          <TD vAlign=3Dtop align=3Dleft><STRONG>Figure 4. =
&nbsp;</STRONG> BrdU=20
            labeling of PDGF-AA-stimulated cortical stem cells. Parallel =
stem=20
            cell cultures were untreated, treated once with PDGF-AA, or =
treated=20
            continuously with PDGF-AA for 2,&nbsp;4,&nbsp;and 6&nbsp;d. =
Before=20
            fixation the cells were exposed to BrdU for 14&nbsp;hr.=20
            Incorporation of BrdU was detected using anti-BrdU =
antibodies.=20
            Stained cells (duplicate dishes) were counted (in seven =
parallel=20
            fields; 200=D7 magnification) and plotted as the ratio of=20
            BrdU-positive cells to the total cell number. =
***&nbsp;denotes=20
            <I>p</I>&nbsp;&lt;&nbsp;0.001. <BR=20
  =
clear=3Dleft></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER=
><!-- /FIG 4 -->
<P></P>
<P align=3Dleft><B>PDGF-AA treatment increases the total =
cell&nbsp;number</B> </P>
<P>To further clarify the mitogenic effect of PDGF-AA on cortical stem =
cells, we=20
performed cell counting experiments. FGF2-treated<SUP> </SUP>cells were=20
harvested and counted at the start of the experiment.<SUP> </SUP>After =
FGF2=20
withdrawal, cultures were left untreated, treated once<SUP> </SUP>with =
PDGF-AA,=20
or treated continuously with PDGF-AA. The total<SUP> </SUP>cell number =
was=20
measured using a Coulter Z1 cell counter, on days<SUP>=20
</SUP>2,&nbsp;4,&nbsp;6,&nbsp;and 8&nbsp;(Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F5">5</A>).=20
A fourfold increase in total cell number<SUP> </SUP>was observed in =
cortical=20
cultures treated continuously with PDGF-AA,<SUP> </SUP>compared with =
untreated=20
cultures, at day 8.&nbsp;The increase in total<SUP> </SUP>cell number =
was lower,=20
but significant, in cultures treated with<SUP> </SUP>a single dose of =
PDGF-AA.=20
<A name=3DF5><!-- FIG 5 --></A>
<P><BR clear=3Dall>
<CENTER>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"95%" border=3D1>
  <TBODY>
  <TR bgColor=3D#e1e1e1>
    <TD>
      <TABLE cellSpacing=3D2 cellPadding=3D2>
        <TBODY>
        <TR bgColor=3D#e1e1e1>
          <TD vAlign=3Dtop align=3Dmiddle bgColor=3D#ffffff><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F5"><IMG=20
            height=3D200 hspace=3D10=20
            =
src=3D"http://www.jneurosci.org/content/vol21/issue10/images/small/ns1015=
209005.gif"=20
            width=3D189 vspace=3D5 border=3D2></A><BR><STRONG>View =
larger=20
            version</STRONG> (31K): <BR><NOBR><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F5">[in=20
            this window]</A> <BR><A=20
            onmouseover=3D"window.status=3D'View figure in a separate =
window'; return true"=20
            onclick=3D"startTarget('F5', 566, 640); =
this.href=3D'/cgi/content-nw/full/21/10/3483/F5'"=20
            =
href=3D"http://www.jneurosci.org/cgi/content-nw/full/21/10/3483/F5"=20
            target=3DF5>[in a new window]</A><BR>&nbsp; </NOBR></TD>
          <TD vAlign=3Dtop align=3Dleft><STRONG>Figure 5. =
&nbsp;</STRONG> Total=20
            cell number in cortical cultures after PDGF-AA treatment.=20
            FGF2-treated cells were harvested and counted at the start =
of the=20
            experiment. After FGF2 withdrawal, parallel stem cell =
cultures were=20
            untreated, treated with a single dose of PDGF-AA, or treated =

            continuously with PDGF-AA for 2,&nbsp;4,&nbsp;6,&nbsp;and =
8&nbsp;d.=20
            The total cell number was measured using a Coulter Z1 cell =
counter.=20
            *&nbsp;denotes <I>p</I>&nbsp;&lt;&nbsp;0.05,&nbsp;**=20
            <I>p</I>&nbsp;&lt;&nbsp;0.01,&nbsp;and ***=20
            <I>p</I>&nbsp;&lt;&nbsp;0.001. <BR=20
  =
clear=3Dleft></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER=
><!-- /FIG 5 -->
<P></P>
<P align=3Dleft><B>PDGF treatment retains an immature phenotype that can =
be=20
stained with neuronal&nbsp;markers</B> </P>
<P>To further investigate the effect of PDGF on cortical stem cell=20
differentiation, cells were stained with antibodies specific<SUP> =
</SUP>to the=20
neuronal marker, MAP2. Cells were grown for 2,&nbsp;4,&nbsp;or=20
6<SUP>&nbsp;</SUP>d without FGF2 in the absence or presence of PDGF. =
Parallel=20
cultures<SUP> </SUP>were untreated, received a single dose of PDGF-AA, =
or=20
received<SUP> </SUP>a daily dose of PDGF-AA (Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F6">6</A>).=20
After fixation, the cells were<SUP> </SUP>stained with MAP2 antibodies =
to reveal=20
neuronal differentiation.<SUP> </SUP>Parallel cultures were stained with =

antibodies to nestin. At the<SUP> </SUP>time of FGF2 withdrawal, close =
to 100%=20
of the cells were nestin<SUP> </SUP>positive, and this number declined =
during=20
the course of the experiment,<SUP> </SUP>as the cells differentiated =
(data not=20
shown). In cultures grown<SUP> </SUP>in the presence of FGF2, very few=20
MAP2-positive cells were seen<SUP> </SUP>(Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F6">6</A><I>A</I>).=20
In untreated cultures, MAP2-positive cells started<SUP> </SUP>to appear =
2&nbsp;d=20
after FGF withdrawal, indicative of neuronal differentiation<SUP> =
</SUP>(Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F6">6</A><I>B</I>).=20
The intensity of MAP2 staining in control cultures<SUP> </SUP>increased =
with=20
time (Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F6">6</A><I>E</I>,<I>H</I>),=20
and 6&nbsp;d after FGF2 withdrawal<SUP> </SUP>(<I>H</I>) most the cells =
had=20
differentiated. The MAP2-positive neurons<SUP> </SUP>now had a more =
mature=20
phenotype with extended processes (Fig.<SUP> </SUP><A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F6">6</A><I>H</I>).=20
<A name=3DF6><!-- FIG 6 --></A>
<P><BR clear=3Dall>
<CENTER>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"95%" border=3D1>
  <TBODY>
  <TR bgColor=3D#e1e1e1>
    <TD>
      <TABLE cellSpacing=3D2 cellPadding=3D2>
        <TBODY>
        <TR bgColor=3D#e1e1e1>
          <TD vAlign=3Dtop align=3Dmiddle bgColor=3D#ffffff><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F6"><IMG=20
            height=3D200 hspace=3D10=20
            =
src=3D"http://www.jneurosci.org/content/vol21/issue10/images/small/ns1015=
209006.gif"=20
            width=3D150 vspace=3D5 border=3D2></A><BR><STRONG>View =
larger=20
            version</STRONG> (109K): <BR><NOBR><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F6">[in=20
            this window]</A> <BR><A=20
            onmouseover=3D"window.status=3D'View figure in a separate =
window'; return true"=20
            onclick=3D"startTarget('F6', 481, 640); =
this.href=3D'/cgi/content-nw/full/21/10/3483/F6'"=20
            =
href=3D"http://www.jneurosci.org/cgi/content-nw/full/21/10/3483/F6"=20
            target=3DF6>[in a new window]</A><BR>&nbsp; </NOBR></TD>
          <TD vAlign=3Dtop align=3Dleft><STRONG>Figure 6. =
&nbsp;</STRONG> Staining=20
            of PDGF-AA-treated cortical stem cells with neuronal =
markers. After=20
            fixation, the cells were stained with MAP2 antibodies. =
Control=20
            cells, grown in the presence of FGF2, were fixed at the =
start of the=20
            experiment (<I>A</I>). Cells were grown for 2&nbsp;d=20
            (<I>B</I>-<I>D</I>), 4&nbsp;d (<I>E</I>-<I>G</I>), or =
6&nbsp;d=20
            (<I>H</I>-<I>J</I>) without FGF2 in the absence or presence =
of PDGF.=20
            Parallel cultures were untreated (<I>B</I>, <I>E</I>, =
<I>H</I>),=20
            received a single dose of PDGF-AA (<I>C</I>, <I>F</I>, =
<I>I</I>), or=20
            PDGF-AA was added daily (<I>D</I>, <I>G</I>, <I>J</I>). The =
cells=20
            were photographed at 200=D7 magnification. <BR=20
        =
clear=3Dleft></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER=
><!-- /FIG 6 -->
<P></P>
<P>Cells treated with a single dose of PDGF-AA showed the same type of=20
differentiation pattern (Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F6">6</A><I>C</I>,<I>F</I>,<I>I</I>)=
,=20
but the differentiation<SUP> </SUP>process was slightly slower, with =
more=20
immature MAP2-positive<SUP> </SUP>cells at days 2&nbsp;and 4&nbsp;than =
in the=20
untreated cultures. In contrast,<SUP> </SUP>the MAP2-positive cells in =
cultures=20
treated continuously with<SUP> </SUP>PDGF-AA did not differentiate into=20
process-extending neurons (Fig.<SUP> </SUP><A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F6">6</A><I>D</I>,<I>G</I>,<I>J</I>)=
=20
during the course of 6&nbsp;d. Still, at the end of the experiment<SUP>=20
</SUP>(<I>J</I>) only a few cells showed morphological maturation. The=20
MAP2-positive<SUP> </SUP>cells rather display a rounded morphology, and =
many=20
cells appear<SUP> </SUP>in clusters. Together with the data from the =
BrdU=20
incorporation<SUP> </SUP>and cell counting experiments, these results =
manifest a=20
mitogenic<SUP> </SUP>effect of PDGF-AA on cortical stem cells during the =
early=20
stage<SUP> </SUP>of neuronal differentiation. Parallel experiments were=20
stained<SUP> </SUP>with an antibody to Tuj1, another commonly used =
neuronal=20
marker,<SUP> </SUP>and an identical staining pattern was observed (data =
not<SUP>=20
</SUP>shown). </P>
<P align=3Dleft><B>MAP2-positive progenitor cells incorporate BrdU in=20
PDGF-AA-treated&nbsp;cultures</B> </P>
<P>To further investigate whether dividing cells are neuronal =
precursors, we=20
used double staining of cortical stem cells with<SUP> </SUP>antibodies =
to MAP2=20
and BrdU (Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F7">7</A>).=20
Cells were grown for 2&nbsp;or<SUP> </SUP>4&nbsp;d without FGF2 in the =
absence=20
or presence of 10&nbsp;ng/ml PDGF-AA.<SUP> </SUP>Before fixation, the =
cells were=20
pulse labeled with BrdU for 14<SUP>&nbsp;</SUP>hr. The cells were first =
stained=20
for MAP2 and photographed, using<SUP> </SUP>a computerized microscope. =
After=20
subsequent staining with BrdU<SUP> </SUP>antibodies, the same fields of =
the=20
culture dish were photographed<SUP> </SUP>again. A high proportion of =
cells=20
grown in the absence of PDGF<SUP> </SUP>for 2&nbsp;d were MAP2 positive =
(Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F7">7</A><I>A</I>)=20
and BrdU negative (Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F7">7</A><I>B</I>,<SUP>=20
</SUP>Table <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#T1">1</A>)=20
and extended short processes. Cells grown in the presence<SUP> </SUP>of =
PDGF-AA=20
showed an undifferentiated phenotype 2&nbsp;d after FGF2<SUP> =
</SUP>withdrawal=20
(Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F7">7</A><I>C</I>).=20
Staining with antibodies to BrdU indicated<SUP> </SUP>that a population =
of the=20
cells treated with PDGF-AA for 2&nbsp;d were<SUP> </SUP>positive for =
both MAP2=20
and BrdU (Fig. <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F7">7</A><I>D</I>,=20
Table <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#T1">1</A>).=20
After 4&nbsp;d<SUP> </SUP>of differentiation, the PDGF-treated cells =
still=20
showed an immature<SUP> </SUP>phenotype, but the amount of =
double-positive cells=20
had decreased<SUP> </SUP>to control levels (Table <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#T1">1</A>).=20
Untreated cells had a more differentiated<SUP> </SUP>phenotype with long =

processes (data not shown). <A name=3DF7><!-- FIG 7 --></A>
<P><BR clear=3Dall>
<CENTER>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"95%" border=3D1>
  <TBODY>
  <TR bgColor=3D#e1e1e1>
    <TD>
      <TABLE cellSpacing=3D2 cellPadding=3D2>
        <TBODY>
        <TR bgColor=3D#e1e1e1>
          <TD vAlign=3Dtop align=3Dmiddle bgColor=3D#ffffff><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F7"><IMG=20
            height=3D200 hspace=3D10=20
            =
src=3D"http://www.jneurosci.org/content/vol21/issue10/images/small/ns1015=
209007.gif"=20
            width=3D196 vspace=3D5 border=3D2></A><BR><STRONG>View =
larger=20
            version</STRONG> (172K): <BR><NOBR><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F7">[in=20
            this window]</A> <BR><A=20
            onmouseover=3D"window.status=3D'View figure in a separate =
window'; return true"=20
            onclick=3D"startTarget('F7', 581, 640); =
this.href=3D'/cgi/content-nw/full/21/10/3483/F7'"=20
            =
href=3D"http://www.jneurosci.org/cgi/content-nw/full/21/10/3483/F7"=20
            target=3DF7>[in a new window]</A><BR>&nbsp; </NOBR></TD>
          <TD vAlign=3Dtop align=3Dleft><STRONG>Figure 7. =
&nbsp;</STRONG> MAP2 and=20
            BrdU double staining of PDGF-AA-treated cortical cells. Stem =
cell=20
            cultures were untreated (<I>A</I>, <I>B</I>) or treated with =
PDGF-AA=20
            (<I>C</I>, <I>D</I>) for 2&nbsp;d after FGF2 withdrawal. The =
cells=20
            were exposed to BrdU for 14&nbsp;hr, fixed, and stained with =
MAP2=20
            antibodies (<I>A</I>, <I>C</I>). Defined fields of the =
culture dish=20
            were photographed using a computerized microscope. The =
cultures were=20
            subsequently stained for BrdU, and the same fields were =
photographed=20
            again (<I>B</I>, <I>D</I>). <BR=20
  =
clear=3Dleft></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER=
><!-- /FIG 7 -->
<P><BR clear=3Dall>
<CENTER>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"95%" border=3D1>
  <TBODY>
  <TR bgColor=3D#e1e1e1>
    <TD>
      <TABLE cellSpacing=3D2 cellPadding=3D2>
        <TBODY>
        <TR bgColor=3D#e1e1e1>
          <TD vAlign=3Dtop align=3Dmiddle=20
            =
bgColor=3D#ffffff><NOBR>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&=
nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&n=
bsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</NOBR><BR><NOB=
R><STRONG>View=20
            this table:</STRONG></NOBR> <BR><NOBR><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/T1">[in=20
            this window]</A></NOBR><BR><NOBR><A=20
            onmouseover=3D"window.status=3D'View table in a separate =
window';&#10;&#9;&#9;return true"=20
            onclick=3D"startTarget('T1', 500, 400); =
this.href=3D'/cgi/content-nw/full/21/10/3483/T1'"=20
            =
href=3D"http://www.jneurosci.org/cgi/content-nw/full/21/10/3483/T1"=20
            target=3DT1>[in a new window]</A><BR>&nbsp;</NOBR> </TD>
          <TD vAlign=3Dtop align=3Dleft><A name=3DT1><!-- TABLE 1 =
--></A><B>Table=20
            1.</B> &nbsp; <STRONG>Percentage of MAP2 and BrdU =
double-positive=20
            cells=20
            =
<P></P></STRONG></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CEN=
TER>
<P></P>
<P align=3Dleft><B>PDGF-AA is involved in control of stem =
cell&nbsp;survival</B>=20
</P>
<P>We next investigated whether the increase in cell number, seen after =
addition=20
of PDGF-AA, results from increased cell survival<SUP> </SUP>in addition =
to the=20
effect on proliferation. Staining for apoptotic<SUP> </SUP>nuclei using =
a TUNEL=20
assay revealed no differences between PDGF-AA<SUP> </SUP>and untreated =
cultures=20
at 2&nbsp;d of differentiation, but at day 4<SUP>&nbsp;</SUP>we detected =
a=20
twofold decrease in TUNEL staining in PDGF-AA-treated<SUP> =
</SUP>cultures (Fig.=20
<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F8">8</A><I>A</I>).=20
<A name=3DF8><!-- FIG 8 --></A>
<P><BR clear=3Dall>
<CENTER>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"95%" border=3D1>
  <TBODY>
  <TR bgColor=3D#e1e1e1>
    <TD>
      <TABLE cellSpacing=3D2 cellPadding=3D2>
        <TBODY>
        <TR bgColor=3D#e1e1e1>
          <TD vAlign=3Dtop align=3Dmiddle bgColor=3D#ffffff><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F8"><IMG=20
            height=3D200 hspace=3D10=20
            =
src=3D"http://www.jneurosci.org/content/vol21/issue10/images/small/ns1015=
209008.gif"=20
            width=3D95 vspace=3D5 border=3D2></A><BR><STRONG>View larger =

            version</STRONG> (17K): <BR><NOBR><A=20
            =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483/F8">[in=20
            this window]</A> <BR><A=20
            onmouseover=3D"window.status=3D'View figure in a separate =
window'; return true"=20
            onclick=3D"startTarget('F8', 360, 640); =
this.href=3D'/cgi/content-nw/full/21/10/3483/F8'"=20
            =
href=3D"http://www.jneurosci.org/cgi/content-nw/full/21/10/3483/F8"=20
            target=3DF8>[in a new window]</A><BR>&nbsp; </NOBR></TD>
          <TD vAlign=3Dtop align=3Dleft><STRONG>Figure 8. =
&nbsp;</STRONG> Cell=20
            survival studies of PDGF-AA-treated cortical stem cells. A =
TUNEL=20
            assay was performed on cells grown for 2&nbsp;and 4&nbsp;d =
in the=20
            presence or absence (no addition, <I>no add.</I>) of =
PDGF-AA. Cells=20
            grown in the presence of FGF2 were included as a control. =
Cells=20
            going through apoptosis were counted using a fluorescence =
microscope=20
            and plotted as the ratio to total number of cells =
(<I>A</I>). ***=20
            denotes <I>p</I>&nbsp;&lt;&nbsp;0.001.&nbsp;Western blot =
analysis=20
            was used to study PKB/c-Akt phosphorylation in CNS stem =
cells after=20
            FGF2 and PDGF-AA stimulation compared with untreated cells =
(<I>no=20
            add.</I>) (<I>B</I>). Total cell lysates were used for=20
            immunoblotting with anti-PKB/c-Akt antibody (<I>Akt</I>) and =

            anti-phosphorylated PKB/c-Akt antibody (<I>p-Akt</I>). <BR=20
            =
clear=3Dleft></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER=
><!-- /FIG 8 -->
<P></P>
<P>The serine threonine kinase PKB/c-Akt has been linked to increased =
cell=20
survival. In addition to the TUNEL assay, we studied<SUP> </SUP>the=20
phosphorylation of PKB/c-Akt as a measure of increased cell<SUP> =
</SUP>survival.=20
Proliferating stem cell cultures were stimulated for<SUP> =
</SUP>5&nbsp;min with=20
100&nbsp;ng/ml PDGF-AA or 100&nbsp;ng/ml FGF2, and total protein<SUP>=20
</SUP>lysates were performed. Cell lysates from parallel cultures,=20
receiving<SUP> </SUP>only medium, were used as a control. Figure <A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#F8">8</A><I>B</I>=20
shows that PDGF-AA<SUP> </SUP>stimulated a moderate PKB/c-Akt =
phosphorylation.=20
FGF2 treatment<SUP> </SUP>also resulted in stimulation of PKB/c-Akt=20
phosphorylation, but<SUP> </SUP>the level was lower than in PDGF-treated =

cultures. No phosphorylated<SUP> </SUP>PKB/c-Akt was detected in =
untreated=20
control cells. Total PKB/c-Akt<SUP> </SUP>levels were not found to be =
affected=20
by PDGF-AA or FGF2<SUP> </SUP>stimulation. </P><BR clear=3Dall><A =
name=3DSEC3><!-- null --></A>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"100%" bgColor=3D#e1e1e1>
  <TBODY>
  <TR>
    <TD vAlign=3Dcenter align=3Dleft width=3D"5%" =
bgColor=3D#ffffff>&nbsp;<IMG=20
      height=3D21 alt=3D"" hspace=3D5=20
      src=3D"http://www.jneurosci.org/icons/toc/rarrow.gif" =
width=3D10></TD>
    <TH vAlign=3Dcenter align=3Dleft width=3D"95%"><FONT=20
      size=3D+2>&nbsp;&nbsp;DISCUSSION</FONT></TH></TR></TBODY></TABLE>
<TABLE cellPadding=3D5 align=3Dright border=3D1 NOWRAP><R>
  <TBODY>
  <TR>
    <TH align=3Dleft><FONT size=3D-1><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Top"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>TOP</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Abstract"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>ABSTRACT</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Introduction"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>INTRODUCTION</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC1"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>MATERIALS AND METHODS</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC2"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>RESULTS</A> <BR><IMG height=3D9 hspace=3D5=20
      src=3D"http://www.jneurosci.org/icons/toc/dot.gif" width=3D11 =
border=3D0><FONT=20
      color=3D#464c53>DISCUSSION</FONT> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#References"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/darrow.gif"=20
      width=3D11 border=3D0>REFERENCES</A> =
</FONT></TH></TR></TBODY></TABLE>
<P>Multipotent neural stem cells can be maintained in monolayer cultures =
in the=20
presence of FGF2, and on withdrawal of the mitogen<SUP> </SUP>give rise =
to=20
neurons, astrocytes, and oligodendrocytes (for review,<SUP> </SUP>see =
McKay,=20
1997<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B17"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
The=20
experiments described in this study were<SUP> </SUP>performed on stem =
cell=20
cultures from the E15 rat cortex in their<SUP> </SUP>first passage. This =

provides a culture system with homogeneous<SUP> </SUP>cells, with =
respect to=20
morphological criteria and staining for<SUP> </SUP>the intermediate=20
filament<SUP> </SUP>nestin. </P>
<P>The influence of exogenous factors on differentiation of CNS stem =
cells has=20
been addressed in several studies. Although the<SUP> </SUP>pathway of =
astrocytic=20
differentiation induced by CNTF has been<SUP> </SUP>studied in detail =
(Bonni et=20
al., 1997<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B2"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>; =
Rajan and=20
McKay, 1998<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B20"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" =
border=3D1></A>),<SUP>=20
</SUP>less is known about the development of the neuronal lineage =
in<SUP>=20
</SUP>stem cells. Two reports (Johe et al., 1996<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B13"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>; =
Williams et=20
al., 1997<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B30"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" =
border=3D1></A>)<SUP>=20
</SUP>have suggested that PDGF-treatment of cortical progenitor =
cells<SUP>=20
</SUP>leads to an increase in the number of neurons. In the study =
by<SUP>=20
</SUP>Williams et al. (1997)<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B30"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>, a =
different=20
cell culture protocol was<SUP> </SUP>used, and cortical cells were =
treated with=20
PDGF-AA without previous<SUP> </SUP>expansion of stem cells. Therefore, =
their=20
data cannot be directly<SUP> </SUP>compared with our results. The =
present=20
investigation uses the<SUP> </SUP>method described in Johe et al. =
(1996)<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B13"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>. =
These authors=20
raise the<SUP> </SUP>possibility that PDGF does not act as an =
instructive factor=20
like<SUP> </SUP>CNTF, a statement that fits with our<SUP> </SUP>data. =
</P>
<P>PDGF is active either as a homodimer (AA, BB) or heterodimer (AB). =
Activation=20
of PDGF receptors is brought about by ligand<SUP> </SUP>binding to =
dimerizing=20
receptor subunits <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif" =
align=3Dbottom>&nbsp;or=20
<IMG alt=3D"beta " =
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif"=20
align=3Dbottom>. The BB homodimer<SUP> </SUP>can only bind to <IMG =
alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =
align=3Dbottom>-receptor=20
subunits, whereas PDGF-AA can activate<SUP> </SUP>both <IMG alt=3D"alpha =
"=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif" =
align=3Dbottom>- and=20
<IMG alt=3D"beta " =
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif"=20
align=3Dbottom>-receptors (Westermark and Sorg, 1993<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B28"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
The AA=20
homodimer,<SUP> </SUP>however, has a slightly lower affinity for the =
<IMG=20
alt=3D"beta " src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =

align=3Dbottom>-receptor. In<SUP> </SUP>accordance with our previous =
data, we=20
detected PDGF <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
align=3Dbottom>-receptor<SUP> </SUP>expression already on dissociated =
cortices at=20
E15. In contrast,<SUP> </SUP>the <IMG alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =
align=3Dbottom>-receptors=20
were hardly detected in uncommitted cells, suggesting<SUP> </SUP>that in =

cortical stem cells the <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif" =
align=3Dbottom>-receptor=20
is the predominant<SUP> </SUP>receptor isoform. Therefore we chose to =
use=20
PDGF-AA as the ligand<SUP> </SUP>throughout this<SUP> </SUP>study. </P>
<P>Tyrosine phosphorylation of the PDGF <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif" =
align=3Dbottom>-receptor=20
was not seen in dissociated cortices, in analogy to previous =
investigations<SUP>=20
</SUP>(Williams et al., 1997<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B30"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>), =
and during=20
differentiation, induced by<SUP> </SUP>discontinued addition of FGF2, we =
could=20
not detect PDGF <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
align=3Dbottom>-receptor<SUP> </SUP>phosphorylation using =
phosphotyrosine=20
antibodies. The cortical<SUP> </SUP>stem cells responded to addition of =
PDGF-AA=20
by receptor phosphorylation,<SUP> </SUP>such that a prolonged exposure =
to the=20
ligand gave rise to a sustained<SUP> </SUP>receptor activation. This =
prolonged=20
tyrosine phosphorylation,<SUP> </SUP>however, never reached the level =
seen=20
during the first day after<SUP> </SUP>stimulation, suggesting receptor=20
downregulation in response to<SUP> </SUP>the ligand. In lysates prepared =
from=20
cells that were given a single<SUP> </SUP>dose of PDGF, phosphorylation =
of the=20
<IMG alt=3D"alpha " =
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
align=3Dbottom>-receptor returned to background<SUP> </SUP>levels more =
quickly=20
than in cells exposed continuously to PDGF-AA. </P>
<P>In contrast to the PDGF <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
align=3Dbottom>-receptor, the <IMG alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =
align=3Dbottom>-receptor=20
is barely detectable during the first days of differentiation in =
cortical<SUP>=20
</SUP>stem cells, and we could see no phosphorylation on tyrosine =
residues<SUP>=20
</SUP>in response to PDGF-AA. Therefore, we conclude that the =
increased<SUP>=20
</SUP>BrdU uptake and total cell number after PDGF-AA stimulation =
are<SUP>=20
</SUP>mediated through PDGF <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
align=3Dbottom>-receptors. During differentiation induced<SUP> </SUP>by=20
withdrawing FGF2, <IMG alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =
align=3Dbottom>-receptor=20
expression can be visualized by<SUP> </SUP>Western blot analysis.=20
Phosphorylation of the PDGF <IMG alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif"=20
align=3Dbottom>-receptor<SUP> </SUP>was also seen when cortical stem =
cells=20
differentiated in the absence<SUP> </SUP>of PDGF-AA. Immunohistochemical =

analysis of rat brain has shown<SUP> </SUP>that the PDGF <IMG =
alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =
align=3Dbottom>-receptor=20
is expressed in neurons in many areas<SUP> </SUP>of the CNS (for review, =
see=20
Valenzuela et al., 1997<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B26"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>), =
and it=20
has<SUP> </SUP>been reported that PDGF-BB exerts neurotrophic activity =
on=20
neurons<SUP> </SUP>from newborn rats (Smits et al., 1991<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B24"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
Additional=20
studies support<SUP> </SUP>the notion that PDGF-BB and the <IMG =
alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =
align=3Dbottom>-receptor=20
are involved in survival<SUP> </SUP>and proliferation of neurons =
(Giacobini et=20
al., 1993<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B8"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>; =
Nikkhah<SUP>=20
</SUP>et al., 1993<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B19"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
In view of=20
these findings it is likely that the<SUP> </SUP><IMG alt=3D"beta "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =
align=3Dbottom>-receptor,=20
detected after several days of differentiation, reflects<SUP> =
</SUP>expression=20
on neurons. However, the exact stage of maturation<SUP> </SUP>at which =
the <IMG=20
alt=3D"beta " src=3D"http://www.jneurosci.org/math/12pt/normal/beta.gif" =

align=3Dbottom>-receptor starts to be expressed remains to be<SUP>=20
</SUP>determined. </P>
<P>Expression of immediate-early genes is induced in various tissues in =
response=20
to a wide range of extracellular cues, for example,<SUP> </SUP>PDGF =
(Cochran and=20
Weissman, 1984<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B4"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>; =
Greenberg and=20
Ziff, 1984<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B9"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>; =
Kruijer<SUP>=20
</SUP>et al., 1984<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B16"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>; =
M=FCller et al.,=20
1984<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B18"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
Among the=20
immediate-early<SUP> </SUP>genes are common transcription factors, but =
also=20
genes that are<SUP> </SUP>more restricted to differentiation of a =
specific cell=20
lineage<SUP> </SUP>(for review, see Herschman, 1991<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B11"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
Core=20
transcription factors<SUP> </SUP>include c-Myc and c-Fos, both of which =
are=20
activated by PDGF in<SUP> </SUP>many cell types. PDGF-AA-stimulated =
c-<I>fos</I>=20
RNA expression in cortical<SUP> </SUP>stem cells peaked at 1&nbsp;hr, =
suggesting=20
that it is mediated by signaling<SUP> </SUP>through PDGF <IMG =
alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif"=20
align=3Dbottom>-receptors. The possibility that c-<I>fos</I> =
activation<SUP>=20
</SUP>is a secondary phenomenon is less likely, because of the time<SUP> =

</SUP>frame of<SUP> </SUP>induction. </P>
<P>From MAP2 (and Tuj1) staining of differentiating cortical cultures, =
it is=20
evident that exogenous PDGF has a profound effect<SUP> </SUP>on cell =
morphology.=20
The presence of PDGF-AA after withdrawal of<SUP> </SUP>FGF2 almost =
completely=20
abolished morphological neuronal maturation.<SUP> </SUP>Instead of =
extending=20
elaborate processes as in control cultures,<SUP> </SUP>PDGF-AA-treated =
cells=20
retained a rounded immature phenotype even<SUP> </SUP>after 6&nbsp;d. It =
is=20
interesting to note that a single dose of PDGF,<SUP> </SUP>at the time =
of FGF2=20
withdrawal, is not sufficient to obtain the<SUP> </SUP>same effect on=20
differentiation. Cell culture medium is changed<SUP> </SUP>every second =
day, and=20
any residual PDGF is washed out from the<SUP> </SUP>culture at that =
time. Thus,=20
the presence of PDGF over several<SUP> </SUP>days of differentiation is =
needed=20
for the effect reported in this<SUP> </SUP>investigation. </P>
<P>The immature morphology of PDGF-AA-treated stem cell cultures could =
be caused=20
by a block in differentiation, a continuous<SUP> </SUP>proliferation, or =
an=20
increase in cell survival, or a combination<SUP> </SUP>of these. =
Previous=20
investigators (Johe et al., 1996<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B13"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>) =
and our=20
unpublished<SUP> </SUP>observations have shown that PDGF cannot =
substitute FGF2=20
as a<SUP> </SUP>major mitogen for cortical stem cells. When FGF2 is not=20
present,<SUP> </SUP>however, our data from BrdU incorporation and cell =
counting=20
experiments<SUP> </SUP>show that PDGF is able to stimulate division of =
stem=20
cells. Both<SUP> </SUP>a single dose and renewed addition of PDGF-AA =
gave a=20
fourfold<SUP> </SUP>increase in BrdU-positive cells at 2&nbsp;d, whereas =
at=20
4&nbsp;d a continuous<SUP> </SUP>presence of PDGF was needed to yield a =
higher=20
percentage compared<SUP> </SUP>with control cells. Double staining for =
MAP2 and=20
BrdU reveals<SUP> </SUP>a population of cells that express a neuronal =
marker=20
while still<SUP> </SUP>dividing. </P>
<P>The possible effect of PDGF-AA on cell survival was investigated. =
TUNEL=20
staining revealed that the amount of cells going through<SUP> =
</SUP>apoptosis=20
during the earliest phase of differentiation (2&nbsp;d) is<SUP> =
</SUP>similar=20
between control and PDGF-treated cultures. At 4&nbsp;d of =
differentiation,<SUP>=20
</SUP>however, the number of apoptotic nuclei was reduced by half =
in<SUP>=20
</SUP>the presence of PDGF. Activation of PKB/c-Akt has been linked<SUP> =

</SUP>to reduced apoptosis (Kandel and Hay, 1999<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B14"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>) =
and was shown=20
to act<SUP> </SUP>as a survival factor for cerebellar neurons (Dudek et =
al.,=20
1997<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B5"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" =
border=3D1></A>).<SUP> </SUP>A=20
modest phosphorylation of PKB/c-Akt was also seen in response<SUP> =
</SUP>to=20
PDGF-AA. Thus, in addition to stimulating stem cell proliferation,<SUP>=20
</SUP>PDGF also exerts a cell survival<SUP> </SUP>effect. </P>
<P>Our results fit with the notion that PDGF stimulates a neuronal fate =
of stem=20
cells, although not as an instructive agent but<SUP> </SUP>rather to =
expand a=20
pool of immature neurons before their morphological<SUP> =
</SUP>maturation<SUP>=20
</SUP>occurs. </P>
<P>PDGF-AA and the <IMG alt=3D"alpha "=20
src=3D"http://www.jneurosci.org/math/12pt/normal/alpha.gif" =
align=3Dbottom>-receptor=20
play an important role in the development of the oligodendrocyte =
lineage. A=20
recent report points<SUP> </SUP>at an unexpected plasticity of early=20
oligodendrocyte progenitors<SUP> </SUP>(Kondo and Raff, 2000<A=20
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#B15"><IMG=20
src=3D"http://www.jneurosci.org/icons/ref-arrow.gif" border=3D1></A>). =
To evaluate=20
the differentiation capacity<SUP> </SUP>of the MAP<SUP>2+</SUP>/BrdU+=20
progenitors in this study on treatment with agents that<SUP> =
</SUP>promote=20
oligodendrocyte development, such as T3, is therefore<SUP> =
</SUP>greatly<SUP>=20
</SUP>warranted. </P></TXT><!-- Stray figures and tables --><!-- =
Acknowledgements --><!-- Notes --><!-- Footnotes --><A=20
name=3Dfoo><!-- comment for mosaic --></A><BR clear=3Dall>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"100%" bgColor=3D#e1e1e1>
  <TBODY>
  <TR>
    <TD vAlign=3Dcenter align=3Dleft width=3D"5%" =
bgColor=3D#ffffff>&nbsp;<IMG=20
      height=3D21 alt=3D"" hspace=3D5=20
      src=3D"http://www.jneurosci.org/icons/toc/rarrow.gif" =
width=3D10></TD>
    <TH vAlign=3Dcenter align=3Dleft width=3D"95%"><FONT=20
      =
size=3D+2>&nbsp;&nbsp;FOOTNOTES</FONT></TH></TR></TBODY></TABLE><!-- =
Received & Accepted -->
<P>Received June 12,&nbsp;2000; revised Feb. 12,&nbsp;2001; accepted =
Feb.=20
23,&nbsp;2001. </P><!-- Footnotes -->
<P>This study was supported by grants from the Swedish Cancer =
Foundation, the=20
Children Cancer Foundation of Sweden, Magnus Bergwall's<SUP> =
</SUP>Foundation,=20
Wiberg's Foundation, and Selander's Foundation. We<SUP> </SUP>thank=20
M.&nbsp;Lindstr=F6m for excellent technical<SUP> </SUP>assistance.=20
<P>Correspondence should be addressed to Karin Forsberg-Nilsson, =
Department of=20
Genetics and Pathology, Rudbeck Laboratory, Uppsala<SUP> =
</SUP>University,=20
SE-751 85&nbsp;Uppsala, Sweden. E-mail: <SPAN=20
id=3Dem0>karin.nilsson{at}genpat.uu.se</SPAN>
<SCRIPT type=3Dtext/javascript><!--=0A=
 var u =3D "karin.nilsson", d =3D "genpat.uu.se"; =
document.getElementById("em0").innerHTML =3D '<a href=3D"mailto:' + u + =
'@' + d + '">' + u + '@' + d + '<\/a>'//--></SCRIPT>
.</P>
<P><!-- Appendices --><!-- Bibliography --><BR clear=3Dall><A =
name=3DReferences><!-- null --></A>
<TABLE cellSpacing=3D0 cellPadding=3D0 width=3D"100%" bgColor=3D#e1e1e1>
  <TBODY>
  <TR>
    <TD vAlign=3Dcenter align=3Dleft width=3D"5%" =
bgColor=3D#ffffff>&nbsp;<IMG=20
      height=3D21 alt=3D"" hspace=3D5=20
      src=3D"http://www.jneurosci.org/icons/toc/rarrow.gif" =
width=3D10></TD>
    <TH vAlign=3Dcenter align=3Dleft width=3D"95%"><FONT=20
      size=3D+2>&nbsp;&nbsp;REFERENCES</FONT></TH></TR></TBODY></TABLE>
<TABLE cellPadding=3D5 align=3Dright border=3D1 NOWRAP><R>
  <TBODY>
  <TR>
    <TH align=3Dleft><FONT size=3D-1><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Top"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>TOP</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Abstract"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>ABSTRACT</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#Introduction"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>INTRODUCTION</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC1"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>MATERIALS AND METHODS</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC2"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>RESULTS</A> <BR><A=20
      =
href=3D"http://www.jneurosci.org/cgi/content/full/21/10/3483?ijkey=3De83a=
eb6016e0bfe3b6e4be7c741221c7bf01543b#SEC3"><IMG=20
      height=3D9 hspace=3D5 =
src=3D"http://www.jneurosci.org/icons/toc/uarrow.gif"=20
      width=3D11 border=3D0>DISCUSSION</A> <BR><IMG height=3D9 =
hspace=3D5=20
      src=3D"http://www.jneurosci.org/icons/toc/dot.gif" width=3D11 =
border=3D0><FONT=20
      color=3D#464c53>REFERENCES</FONT> =
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------=_NextPart_000_0241_01C85B7B.BC962280
Content-Type: application/octet-stream
Content-Transfer-Encoding: quoted-printable
Content-Location: http://www.jneurosci.org/javascript/ajax/xmlhttprequest.js

/*=0A=
=0A=
Cross-Browser XMLHttpRequest v1.2=0A=
=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=3D=3D=3D=3D=3D=3D=0A=
=0A=
Emulate Gecko 'XMLHttpRequest()' functionality in IE and Opera. Opera =
requires=0A=
the Sun Java Runtime Environment <http://www.java.com/>.=0A=
=0A=
by Andrew Gregory=0A=
http://www.scss.com.au/family/andrew/webdesign/xmlhttprequest/=0A=
=0A=
This work is licensed under the Creative Commons Attribution License. To =
view a=0A=
copy of this license, visit =
http://creativecommons.org/licenses/by-sa/2.5/ or=0A=
send a letter to Creative Commons, 559 Nathan Abbott Way, Stanford, =
California=0A=
94305, USA.=0A=
=0A=
Attribution: Leave my name and web address in this script intact.=0A=
=0A=
Not Supported in Opera=0A=
----------------------=0A=
* user/password authentication=0A=
* responseXML data member=0A=
=0A=
Not Fully Supported in Opera=0A=
----------------------------=0A=
* async requests=0A=
* abort()=0A=
* getAllResponseHeaders(), getAllResponseHeader(header)=0A=
=0A=
*/=0A=
// IE support=0A=
if (window.ActiveXObject && !window.XMLHttpRequest) {=0A=
  window.XMLHttpRequest =3D function() {=0A=
    var msxmls =3D new Array(=0A=
      'Msxml2.XMLHTTP.5.0',=0A=
      'Msxml2.XMLHTTP.4.0',=0A=
      'Msxml2.XMLHTTP.3.0',=0A=
      'Msxml2.XMLHTTP',=0A=
      'Microsoft.XMLHTTP');=0A=
    for (var i =3D 0; i < msxmls.length; i++) {=0A=
      try {=0A=
        return new ActiveXObject(msxmls[i]);=0A=
      } catch (e) {=0A=
      }=0A=
    }=0A=
    return null;=0A=
  };=0A=
}=0A=
// Gecko support=0A=
/* ;-) */=0A=
// Opera support=0A=
if (window.opera && !window.XMLHttpRequest) {=0A=
  window.XMLHttpRequest =3D function() {=0A=
    this.readyState =3D 0; // =
0=3Duninitialized,1=3Dloading,2=3Dloaded,3=3Dinteractive,4=3Dcomplete=0A=
    this.status =3D 0; // HTTP status codes=0A=
    this.statusText =3D '';=0A=
    this._headers =3D [];=0A=
    this._aborted =3D false;=0A=
    this._async =3D true;=0A=
    this._defaultCharset =3D 'ISO-8859-1';=0A=
    this._getCharset =3D function() {=0A=
      var charset =3D _defaultCharset;=0A=
      var contentType =3D =
this.getResponseHeader('Content-type').toUpperCase();=0A=
      val =3D contentType.indexOf('CHARSET=3D');=0A=
      if (val !=3D -1) {=0A=
        charset =3D contentType.substring(val);=0A=
      }=0A=
      val =3D charset.indexOf(';');=0A=
      if (val !=3D -1) {=0A=
        charset =3D charset.substring(0, val);=0A=
      }=0A=
      val =3D charset.indexOf(',');=0A=
      if (val !=3D -1) {=0A=
        charset =3D charset.substring(0, val);=0A=
      }=0A=
      return charset;=0A=
    };=0A=
    this.abort =3D function() {=0A=
      this._aborted =3D true;=0A=
    };=0A=
    this.getAllResponseHeaders =3D function() {=0A=
      return this.getAllResponseHeader('*');=0A=
    };=0A=
    this.getAllResponseHeader =3D function(header) {=0A=
      var ret =3D '';=0A=
      for (var i =3D 0; i < this._headers.length; i++) {=0A=
        if (header =3D=3D '*' || this._headers[i].h =3D=3D header) {=0A=
          ret +=3D this._headers[i].h + ': ' + this._headers[i].v + '\n';=0A=
        }=0A=
      }=0A=
      return ret;=0A=
    };=0A=
    this.getResponseHeader =3D function(header) {=0A=
      var ret =3D getAllResponseHeader(header);=0A=
      var i =3D ret.indexOf('\n');=0A=
      if (i !=3D -1) {=0A=
        ret =3D ret.substring(0, i);=0A=
      }=0A=
      return ret;=0A=
    };=0A=
    this.setRequestHeader =3D function(header, value) {=0A=
      this._headers[this._headers.length] =3D {h:header, v:value};=0A=
    };=0A=
    this.open =3D function(method, url, async, user, password) {=0A=
      this.method =3D method;=0A=
      this.url =3D url;=0A=
      this._async =3D true;=0A=
      this._aborted =3D false;=0A=
      this._headers =3D [];=0A=
      if (arguments.length >=3D 3) {=0A=
        this._async =3D async;=0A=
      }=0A=
      if (arguments.length > 3) {=0A=
        opera.postError('XMLHttpRequest.open() - user/password not =
supported');=0A=
      }=0A=
      this.readyState =3D 1;=0A=
      if (this.onreadystatechange) {=0A=
        this.onreadystatechange();=0A=
      }=0A=
    };=0A=
    this.send =3D function(data) {=0A=
      if (!navigator.javaEnabled()) {=0A=
        alert("XMLHttpRequest.send() - Java must be installed and =
enabled.");=0A=
        return;=0A=
      }=0A=
      if (this._async) {=0A=
        setTimeout(this._sendasync, 0, this, data);=0A=
        // this is not really asynchronous and won't execute until the =
current=0A=
        // execution context ends=0A=
      } else {=0A=
        this._sendsync(data);=0A=
      }=0A=
    }=0A=
    this._sendasync =3D function(req, data) {=0A=
      if (!req._aborted) {=0A=
        req._sendsync(data);=0A=
      }=0A=
    };=0A=
    this._sendsync =3D function(data) {=0A=
      this.readyState =3D 2;=0A=
      if (this.onreadystatechange) {=0A=
        this.onreadystatechange();=0A=
      }=0A=
      // open connection=0A=
      var url =3D new java.net.URL(new =
java.net.URL(window.location.href), this.url);=0A=
      var conn =3D url.openConnection();=0A=
      for (var i =3D 0; i < this._headers.length; i++) {=0A=
        conn.setRequestProperty(this._headers[i].h, this._headers[i].v);=0A=
      }=0A=
      this._headers =3D [];=0A=
      if (this.method =3D=3D 'POST') {=0A=
        // POST data=0A=
        conn.setDoOutput(true);=0A=
        var wr =3D new =
java.io.OutputStreamWriter(conn.getOutputStream(), this._getCharset());=0A=
        wr.write(data);=0A=
        wr.flush();=0A=
        wr.close();=0A=
      }=0A=
      // read response headers=0A=
      // NOTE: the getHeaderField() methods always return nulls for me :(=0A=
      var gotContentEncoding =3D false;=0A=
      var gotContentLength =3D false;=0A=
      var gotContentType =3D false;=0A=
      var gotDate =3D false;=0A=
      var gotExpiration =3D false;=0A=
      var gotLastModified =3D false;=0A=
      for (var i =3D 0; ; i++) {=0A=
        var hdrName =3D conn.getHeaderFieldKey(i);=0A=
        var hdrValue =3D conn.getHeaderField(i);=0A=
        if (hdrName =3D=3D null && hdrValue =3D=3D null) {=0A=
          break;=0A=
        }=0A=
        if (hdrName !=3D null) {=0A=
          this._headers[this._headers.length] =3D {h:hdrName, =
v:hdrValue};=0A=
          switch (hdrName.toLowerCase()) {=0A=
            case 'content-encoding': gotContentEncoding =3D true; break;=0A=
            case 'content-length'  : gotContentLength   =3D true; break;=0A=
            case 'content-type'    : gotContentType     =3D true; break;=0A=
            case 'date'            : gotDate            =3D true; break;=0A=
            case 'expires'         : gotExpiration      =3D true; break;=0A=
            case 'last-modified'   : gotLastModified    =3D true; break;=0A=
          }=0A=
        }=0A=
      }=0A=
      // try to fill in any missing header information=0A=
      var val;=0A=
      val =3D conn.getContentEncoding();=0A=
      if (val !=3D null && !gotContentEncoding) =
this._headers[this._headers.length] =3D {h:'Content-encoding', v:val};=0A=
      val =3D conn.getContentLength();=0A=
      if (val !=3D -1 && !gotContentLength) =
this._headers[this._headers.length] =3D {h:'Content-length', v:val};=0A=
      val =3D conn.getContentType();=0A=
      if (val !=3D null && !gotContentType) =
this._headers[this._headers.length] =3D {h:'Content-type', v:val};=0A=
      val =3D conn.getDate();=0A=
      if (val !=3D 0 && !gotDate) this._headers[this._headers.length] =
=3D {h:'Date', v:(new Date(val)).toUTCString()};=0A=
      val =3D conn.getExpiration();=0A=
      if (val !=3D 0 && !gotExpiration) =
this._headers[this._headers.length] =3D {h:'Expires', v:(new =
Date(val)).toUTCString()};=0A=
      val =3D conn.getLastModified();=0A=
      if (val !=3D 0 && !gotLastModified) =
this._headers[this._headers.length] =3D {h:'Last-modified', v:(new =
Date(val)).toUTCString()};=0A=
      // read response data=0A=
      var reqdata =3D '';=0A=
      var stream =3D conn.getInputStream();=0A=
      if (stream) {=0A=
        var reader =3D new java.io.BufferedReader(new =
java.io.InputStreamReader(stream, this._getCharset()));=0A=
        var line;=0A=
        while ((line =3D reader.readLine()) !=3D null) {=0A=
          if (this.readyState =3D=3D 2) {=0A=
            this.readyState =3D 3;=0A=
            if (this.onreadystatechange) {=0A=
              this.onreadystatechange();=0A=
            }=0A=
          }=0A=
          reqdata +=3D line + '\n';=0A=
        }=0A=
        reader.close();=0A=
        this.status =3D 200;=0A=
        this.statusText =3D 'OK';=0A=
        this.responseText =3D reqdata;=0A=
        this.readyState =3D 4;=0A=
        if (this.onreadystatechange) {=0A=
          this.onreadystatechange();=0A=
        }=0A=
        if (this.onload) {=0A=
          this.onload();=0A=
        }=0A=
      } else {=0A=
        // error=0A=
        this.status =3D 404;=0A=
        this.statusText =3D 'Not Found';=0A=
        this.responseText =3D '';=0A=
        this.readyState =3D 4;=0A=
        if (this.onreadystatechange) {=0A=
          this.onreadystatechange();=0A=
        }=0A=
        if (this.onerror) {=0A=
          this.onerror();=0A=
        }=0A=
      }=0A=
    };=0A=
  };=0A=
}=0A=
// ActiveXObject emulation=0A=
if (!window.ActiveXObject && window.XMLHttpRequest) {=0A=
  window.ActiveXObject =3D function(type) {=0A=
    switch (type.toLowerCase()) {=0A=
      case 'microsoft.xmlhttp':=0A=
      case 'msxml2.xmlhttp':=0A=
      case 'msxml2.xmlhttp.3.0':=0A=
      case 'msxml2.xmlhttp.4.0':=0A=
      case 'msxml2.xmlhttp.5.0':=0A=
        return new XMLHttpRequest();=0A=
    }=0A=
    return null;=0A=
  };=0A=
}=0A=

------=_NextPart_000_0241_01C85B7B.BC962280
Content-Type: application/octet-stream
Content-Transfer-Encoding: quoted-printable
Content-Location: http://www.jneurosci.org/javascript/ajax/utility.js

/************************************************************************=
*****=0A=
 * javascript/ajax/utility.js=0A=
 *=0A=
 * Utility functions for working with XMLHttpRequest data.=0A=
 *=0A=
 * Copyright 2006 Board of Trustees of the Leland Stanford Junior =
University.=0A=
 =
*************************************************************************=
***/=0A=
=0A=
/*=0A=
 * Copy XML nodes into an HTMLElement. This effectively=0A=
 * clones XML markup which uses XHTML naming conventions=0A=
 * into an HTML DOM.=0A=
 */=0A=
function copy_xml_to_html(src, dst) {=0A=
  if (src.nodeType =3D=3D 1) { /* Node.ELEMENT_NODE */=0A=
    var e =3D document.createElement(src.nodeName);=0A=
    for (var i =3D 0; i < src.childNodes.length; i++) {=0A=
	  copy_xml_to_html(src.childNodes[i], e);=0A=
    }=0A=
    for (var i =3D 0; i < src.attributes.length; i++) {=0A=
      var n =3D src.attributes[i].name;=0A=
      var v =3D unescape_xml_string(src.attributes[i].value);      =0A=
      e.setAttribute(n, v);=0A=
      if (n =3D=3D "class") {=0A=
        e.className =3D v;=0A=
      }=0A=
      else if (n =3D=3D "style") {=0A=
        set_css_style(v, e, "");=0A=
      }=0A=
    }=0A=
    dst.appendChild(e);=0A=
  }=0A=
  else if (src.nodeType =3D=3D 3) { /* Node.TEXT_NODE */=0A=
    dst.appendChild(document.createTextNode(src.nodeValue));=0A=
  }=0A=
}=0A=
=0A=
/* =0A=
 * It is unclear that this is the right thing to be calling=0A=
 * from copy_xml_to_html, but it appears that Safari decides=0A=
 * to convert &amp; to the NCR &#35;, and then encodes that=0A=
 * NCR to &%26%2338;.  So, I'm going to treat the DOM Attr=0A=
 * value as a plain string, and run our XML string input=0A=
 * through the decoding routine below.=0A=
 */=0A=
function unescape_xml_string(s) {=0A=
  return s.replace(/&apos;/g, "'")=0A=
          .replace(/&#39;/g,  "'")=0A=
          .replace(/&quot;/g, "\"")=0A=
          .replace(/&#34;/g,  "\"")=0A=
          .replace(/&gt;/g,   ">")=0A=
          .replace(/&#62;/g,  ">")=0A=
          .replace(/&lt;/g,   "<")=0A=
          .replace(/&#60;/g,  "<")=0A=
          .replace(/&amp;/g,  "&")=0A=
          .replace(/&#38;/g,  "&");=0A=
}=0A=
=0A=
/*=0A=
 * Parse set of CSS rules and apply them to an element.=0A=
 * This is quite horrifying, but I'm unable to determine=0A=
 * how else to handle this with IE 6.  FireFox and other=0A=
 * sane browsers let you simply set the style attribute=0A=
 * or use e.style.setProperty(rule, value, priority),=0A=
 * IE 6 appears to have neither of these capabilities..=0A=
 */=0A=
function set_css_style(css, e, priority) {=0A=
  var rules =3D css.split(";");=0A=
  for (var i =3D 0; i < rules.length; i++) {=0A=
    var nvpair =3D rules[i].split(":");=0A=
    if (nvpair.length =3D=3D 2) {=0A=
      try {=0A=
        var name  =3D nvpair[0]; /* style attribute */=0A=
        var value =3D nvpair[1]; /* attribute value */=0A=
  =0A=
        /*=0A=
         * For each possible style attribute, set the=0A=
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          e.style.font =3D value;=0A=
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          e.style.fontVariant =3D value;=0A=
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          e.style.height =3D value;=0A=
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          e.style.marginTop =3D value;=0A=
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          e.style.minHeight =3D value;=0A=
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          e.style.paddingTop =3D value;=0A=
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          e.style.pageBreakAfter =3D value;=0A=
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          e.style.pixelHeight =3D value;=0A=
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        }=0A=
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          e.style.textOverflow =3D value;=0A=
        }=0A=
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          e.style.textTransform =3D value;=0A=
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          e.style.textUnderlinePosition =3D value;=0A=
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        else if (name =3D=3D "top") {=0A=
          e.style.top =3D value;=0A=
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          e.style.unicodeBidi =3D value;=0A=
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          e.style.verticalAlign =3D value;=0A=
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          e.style.visibility =3D value;=0A=
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          e.style.whiteSpace =3D value;=0A=
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          e.style.width =3D value;=0A=
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          e.style.wordBreak =3D value;=0A=
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          e.style.wordSpacing =3D value;=0A=
        }=0A=
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          e.style.wordWrap =3D value;=0A=
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          e.style.writingMode =3D value;=0A=
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          e.style.zIndex =3D value;=0A=
        }=0A=
        else if (name =3D=3D "zoom") {=0A=
          e.style.zoom =3D value;=0A=
        }=0A=
      }=0A=
      catch (e) {=0A=
        /* ignore error on attempt to set e.style.[property] */=0A=
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    }=0A=
  }=0A=
}=0A=

------=_NextPart_000_0241_01C85B7B.BC962280
Content-Type: application/octet-stream
Content-Transfer-Encoding: quoted-printable
Content-Location: http://www.jneurosci.org/javascript/entrez/callback.js

/************************************************************************=
*****=0A=
 * javascript/entrez/callback.js=0A=
 *=0A=
 * Entrez Linking callback to populate content box.=0A=
 *=0A=
 * Copyright 2006 Board of Trustees of the Leland Stanford Junior =
University.=0A=
 =
*************************************************************************=
***/=0A=
=0A=
/*=0A=
 * Execute callback to fill content box with Entrez Linking information.=0A=
 */=0A=
function entrez_callback(pmid, callback_url) {=0A=
  /*=0A=
   * MSIE 5.5 and below have issues with the JavaScript=0A=
   * used for Entrez Linking. For now we have to disable=0A=
   * the callback until we can track down a proper fix=0A=
   * (or everybody sanely upgrades to version 6 or 7!).=0A=
   */=0A=
  if (navigator) {=0A=
    var appname =3D navigator.appName;=0A=
    if (appname =3D=3D "Microsoft Internet Explorer") {=0A=
      var userAgent =3D navigator["userAgent"];=0A=
      var s =3D "MSIE ";=0A=
      var n =3D -1;      =0A=
      if ((n =3D userAgent.indexOf(s)) !=3D -1) {=0A=
        var v =3D parseFloat(userAgent.substring(n+s.length));=0A=
        if (v < 6) {=0A=
          return;=0A=
        }=0A=
      }=0A=
    }=0A=
  }=0A=
=0A=
  /*=0A=
   * Acquire table row element to update, initiate callback=0A=
   * to update table with Entrez Links.=0A=
   */=0A=
  var tr =3D document.getElementById('entrez_callback_'+pmid);=0A=
  if (!tr) {=0A=
    return;=0A=
  }=0A=
  var req =3D new XMLHttpRequest();=0A=
  if (!req) {=0A=
    return;=0A=
  }=0A=
  req.onreadystatechange =3D function() {=0A=
    if (req.readyState =3D=3D 4 && (req.status =3D=3D 200 || req.status =
=3D=3D 304)) {=0A=
      var src =3D req.responseXML.documentElement;=0A=
      var dst =3D document.createDocumentFragment();=0A=
      for (var i =3D 0; i < src.childNodes.length; i++) {=0A=
      	copy_xml_to_html(src.childNodes[i], dst);=0A=
      }=0A=
      var tbl =3D tr.parentNode;=0A=
      tbl.replaceChild(dst, tr);=0A=
    }=0A=
  }=0A=
  req.open('GET', callback_url, true);=0A=
  req.send(null);=0A=
}=0A=

------=_NextPart_000_0241_01C85B7B.BC962280
Content-Type: application/octet-stream
Content-Transfer-Encoding: quoted-printable
Content-Location: http://www.google-analytics.com/urchin.js

//-- Google Analytics Urchin Module=0A=
//-- Copyright 2007 Google, All Rights Reserved.=0A=
=0A=
//-- Urchin On Demand Settings ONLY=0A=
var _uacct=3D"";			// set up the Urchin Account=0A=
var _userv=3D1;			// service mode (0=3Dlocal,1=3Dremote,2=3Dboth)=0A=
=0A=
//-- UTM User Settings=0A=
var _ufsc=3D1;			// set client info flag (1=3Don|0=3Doff)=0A=
var _udn=3D"auto";		// (auto|none|domain) set the domain name for cookies=0A=
var _uhash=3D"on";		// (on|off) unique domain hash for cookies=0A=
var _utimeout=3D"1800";   	// set the inactive session timeout in seconds=0A=
var _ugifpath=3D"/__utm.gif";	// set the web path to the __utm.gif file=0A=
var _utsp=3D"|";			// transaction field separator=0A=
var _uflash=3D1;			// set flash version detect option (1=3Don|0=3Doff)=0A=
var _utitle=3D1;			// set the document title detect option =
(1=3Don|0=3Doff)=0A=
var _ulink=3D0;			// enable linker functionality (1=3Don|0=3Doff)=0A=
var _uanchor=3D0;			// enable use of anchors for campaign =
(1=3Don|0=3Doff)=0A=
var _utcp=3D"/";			// the cookie path for tracking=0A=
var _usample=3D100;		// The sampling % of visitors to track (1-100).=0A=
=0A=
//-- UTM Campaign Tracking Settings=0A=
var _uctm=3D1;			// set campaign tracking module (1=3Don|0=3Doff)=0A=
var _ucto=3D"15768000";		// set timeout in seconds (6 month default)=0A=
var _uccn=3D"utm_campaign";	// name=0A=
var _ucmd=3D"utm_medium";		// medium (cpc|cpm|link|email|organic)=0A=
var _ucsr=3D"utm_source";		// source=0A=
var _uctr=3D"utm_term";		// term/keyword=0A=
var _ucct=3D"utm_content";	// content=0A=
var _ucid=3D"utm_id";		// id number=0A=
var _ucno=3D"utm_nooverride";	// don't override=0A=
=0A=
//-- Auto/Organic Sources and Keywords=0A=
var _uOsr=3Dnew Array();=0A=
var _uOkw=3Dnew Array();=0A=
_uOsr[0]=3D"google";	_uOkw[0]=3D"q";=0A=
_uOsr[1]=3D"yahoo";	_uOkw[1]=3D"p";=0A=
_uOsr[2]=3D"msn";		_uOkw[2]=3D"q";=0A=
_uOsr[3]=3D"aol";		_uOkw[3]=3D"query";=0A=
_uOsr[4]=3D"aol";		_uOkw[4]=3D"encquery";=0A=
_uOsr[5]=3D"lycos";	_uOkw[5]=3D"query";=0A=
_uOsr[6]=3D"ask";		_uOkw[6]=3D"q";=0A=
_uOsr[7]=3D"altavista";	_uOkw[7]=3D"q";=0A=
_uOsr[8]=3D"netscape";	_uOkw[8]=3D"query";=0A=
_uOsr[9]=3D"cnn";	_uOkw[9]=3D"query";=0A=
_uOsr[10]=3D"looksmart";	_uOkw[10]=3D"qt";=0A=
_uOsr[11]=3D"about";	_uOkw[11]=3D"terms";=0A=
_uOsr[12]=3D"mamma";	_uOkw[12]=3D"query";=0A=
_uOsr[13]=3D"alltheweb";	_uOkw[13]=3D"q";=0A=
_uOsr[14]=3D"gigablast";	_uOkw[14]=3D"q";=0A=
_uOsr[15]=3D"voila";	_uOkw[15]=3D"rdata";=0A=
_uOsr[16]=3D"virgilio";	_uOkw[16]=3D"qs";=0A=
_uOsr[17]=3D"live";	_uOkw[17]=3D"q";=0A=
_uOsr[18]=3D"baidu";	_uOkw[18]=3D"wd";=0A=
_uOsr[19]=3D"alice";	_uOkw[19]=3D"qs";=0A=
_uOsr[20]=3D"yandex";	_uOkw[20]=3D"text";=0A=
_uOsr[21]=3D"najdi";	_uOkw[21]=3D"q";=0A=
_uOsr[22]=3D"aol";	_uOkw[22]=3D"q";=0A=
_uOsr[23]=3D"club-internet"; _uOkw[23]=3D"q";=0A=
_uOsr[24]=3D"mama";	_uOkw[24]=3D"query";=0A=
_uOsr[25]=3D"seznam";	_uOkw[25]=3D"q";=0A=
_uOsr[26]=3D"search";	_uOkw[26]=3D"q";=0A=
_uOsr[27]=3D"szukaj";	_uOkw[27]=3D"szukaj";=0A=
_uOsr[28]=3D"szukaj";	_uOkw[28]=3D"qt";=0A=
_uOsr[29]=3D"netsprint";	_uOkw[29]=3D"q";=0A=
_uOsr[30]=3D"google.interia";	_uOkw[30]=3D"q";=0A=
_uOsr[31]=3D"szukacz";	_uOkw[31]=3D"q";=0A=
_uOsr[32]=3D"yam";	_uOkw[32]=3D"k";=0A=
_uOsr[33]=3D"pchome";	_uOkw[33]=3D"q";=0A=
=0A=
=0A=
//-- Auto/Organic Keywords to Ignore=0A=
var _uOno=3Dnew Array();=0A=
//_uOno[0]=3D"urchin";=0A=
//_uOno[1]=3D"urchin.com";=0A=
//_uOno[2]=3D"www.urchin.com";=0A=
=0A=
//-- Referral domains to Ignore=0A=
var _uRno=3Dnew Array();=0A=
//_uRno[0]=3D".urchin.com";=0A=
=0A=
//-- **** Don't modify below this point ***=0A=
var =
_uff,_udh,_udt,_ubl=3D0,_udo=3D"",_uu,_ufns=3D0,_uns=3D0,_ur=3D"-",_ufno=3D=
0,_ust=3D0,_ubd=3Ddocument,_udl=3D_ubd.location,_udlh=3D"",_uwv=3D"1";=0A=
var _ugifpath2=3D"http://www.google-analytics.com/__utm.gif";=0A=
if (_udl.hash) _udlh=3D_udl.href.substring(_udl.href.indexOf('#'));=0A=
if (_udl.protocol=3D=3D"https:") =
_ugifpath2=3D"https://ssl.google-analytics.com/__utm.gif";=0A=
if (!_utcp || _utcp=3D=3D"") _utcp=3D"/";=0A=
function urchinTracker(page) {=0A=
 if (_udl.protocol=3D=3D"file:") return;=0A=
 if (_uff && (!page || page=3D=3D"")) return;=0A=
 var a,b,c,xx,v,z,k,x=3D"",s=3D"",f=3D0;=0A=
 var nx=3D" expires=3D"+_uNx()+";";=0A=
 var dc=3D_ubd.cookie;=0A=
 _udh=3D_uDomain();=0A=
 if (!_uVG()) return;=0A=
 _uu=3DMath.round(Math.random()*2147483647);=0A=
 _udt=3Dnew Date();=0A=
 _ust=3DMath.round(_udt.getTime()/1000);=0A=
 a=3Ddc.indexOf("__utma=3D"+_udh);=0A=
 b=3Ddc.indexOf("__utmb=3D"+_udh);=0A=
 c=3Ddc.indexOf("__utmc=3D"+_udh);=0A=
 if (_udn && _udn!=3D"") { _udo=3D" domain=3D"+_udn+";"; }=0A=
 if (_utimeout && _utimeout!=3D"") {=0A=
  x=3Dnew Date(_udt.getTime()+(_utimeout*1000));=0A=
  x=3D" expires=3D"+x.toGMTString()+";";=0A=
 }=0A=
 if (_ulink) {=0A=
  if (_uanchor && _udlh && _udlh!=3D"") s=3D_udlh+"&";=0A=
  s+=3D_udl.search;=0A=
  if(s && s!=3D"" && s.indexOf("__utma=3D")>=3D0) {=0A=
   if (!(_uIN(a=3D_uGC(s,"__utma=3D","&")))) a=3D"-";=0A=
   if (!(_uIN(b=3D_uGC(s,"__utmb=3D","&")))) b=3D"-";=0A=
   if (!(_uIN(c=3D_uGC(s,"__utmc=3D","&")))) c=3D"-";=0A=
   v=3D_uGC(s,"__utmv=3D","&");=0A=
   z=3D_uGC(s,"__utmz=3D","&");=0A=
   k=3D_uGC(s,"__utmk=3D","&");=0A=
   xx=3D_uGC(s,"__utmx=3D","&");=0A=
   if ((k*1) !=3D ((_uHash(a+b+c+xx+z+v)*1)+(_udh*1))) =
{_ubl=3D1;a=3D"-";b=3D"-";c=3D"-";xx=3D"-";z=3D"-";v=3D"-";}=0A=
   if (a!=3D"-" && b!=3D"-" && c!=3D"-") f=3D1;=0A=
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  }=0A=
 }=0A=
 if(f=3D=3D1) {=0A=
  _ubd.cookie=3D"__utma=3D"+a+"; path=3D"+_utcp+";"+nx+_udo;=0A=
  _ubd.cookie=3D"__utmb=3D"+b+"; path=3D"+_utcp+";"+x+_udo;=0A=
  _ubd.cookie=3D"__utmc=3D"+c+"; path=3D"+_utcp+";"+_udo;=0A=
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  a=3D_uFixA(s,"&",_ust);=0A=
  _ubd.cookie=3D"__utma=3D"+a+"; path=3D"+_utcp+";"+nx+_udo;=0A=
  _ubd.cookie=3D"__utmb=3D"+_udh+"; path=3D"+_utcp+";"+x+_udo;=0A=
  _ubd.cookie=3D"__utmc=3D"+_udh+"; path=3D"+_utcp+";"+_udo;=0A=
  _ufns=3D1;=0A=
 } else if (a>=3D0 && b>=3D0 && c>=3D0) {=0A=
  _ubd.cookie=3D"__utmb=3D"+_udh+"; path=3D"+_utcp+";"+x+_udo;=0A=
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  if (a>=3D0) a=3D_uFixA(_ubd.cookie,";",_ust);=0A=
  else a=3D_udh+"."+_uu+"."+_ust+"."+_ust+"."+_ust+".1";=0A=
  _ubd.cookie=3D"__utma=3D"+a+"; path=3D"+_utcp+";"+nx+_udo;=0A=
  _ubd.cookie=3D"__utmb=3D"+_udh+"; path=3D"+_utcp+";"+x+_udo;=0A=
  _ubd.cookie=3D"__utmc=3D"+_udh+"; path=3D"+_utcp+";"+_udo;=0A=
  _ufns=3D1;=0A=
 }=0A=
 if (_ulink && xx && xx!=3D"" && xx!=3D"-") {=0A=
   xx=3D_uUES(xx);=0A=
   if (xx.indexOf(";")=3D=3D-1) _ubd.cookie=3D"__utmx=3D"+xx+"; =
path=3D"+_utcp+";"+nx+_udo;=0A=
 }=0A=
 if (_ulink && v && v!=3D"" && v!=3D"-") {=0A=
  v=3D_uUES(v);=0A=
  if (v.indexOf(";")=3D=3D-1) _ubd.cookie=3D"__utmv=3D"+v+"; =
path=3D"+_utcp+";"+nx+_udo;=0A=
 }=0A=
 _uInfo(page);=0A=
 _ufns=3D0;=0A=
 _ufno=3D0;=0A=
 if (!page || page=3D=3D"") _uff=3D1;=0A=
}=0A=
function _uInfo(page) {=0A=
 var p,s=3D"",dm=3D"",pg=3D_udl.pathname+_udl.search;=0A=
 if (page && page!=3D"") pg=3D_uES(page,1);=0A=
 _ur=3D_ubd.referrer;=0A=
 if (!_ur || _ur=3D=3D"") { _ur=3D"-"; }=0A=
 else {=0A=
  dm=3D_ubd.domain;=0A=
  if(_utcp && _utcp!=3D"/") dm+=3D_utcp;=0A=
  p=3D_ur.indexOf(dm);=0A=
  if ((p>=3D0) && (p<=3D8)) { _ur=3D"0"; }=0A=
  if (_ur.indexOf("[")=3D=3D0 && =
_ur.lastIndexOf("]")=3D=3D(_ur.length-1)) { _ur=3D"-"; }=0A=
 }=0A=
 s+=3D"&utmn=3D"+_uu;=0A=
 if (_ufsc) s+=3D_uBInfo();=0A=
 if (_uctm) s+=3D_uCInfo();=0A=
 if (_utitle && _ubd.title && _ubd.title!=3D"") =
s+=3D"&utmdt=3D"+_uES(_ubd.title);=0A=
 if (_udl.hostname && _udl.hostname!=3D"") =
s+=3D"&utmhn=3D"+_uES(_udl.hostname);=0A=
 s+=3D"&utmr=3D"+_ur;=0A=
 s+=3D"&utmp=3D"+pg;=0A=
 if ((_userv=3D=3D0 || _userv=3D=3D2) && _uSP()) {=0A=
  var i=3Dnew Image(1,1);=0A=
  i.src=3D_ugifpath+"?"+"utmwv=3D"+_uwv+s;=0A=
  i.onload=3Dfunction() {_uVoid();}=0A=
 }=0A=
 if ((_userv=3D=3D1 || _userv=3D=3D2) && _uSP()) {=0A=
  var i2=3Dnew Image(1,1);=0A=
  =
i2.src=3D_ugifpath2+"?"+"utmwv=3D"+_uwv+s+"&utmac=3D"+_uacct+"&utmcc=3D"+=
_uGCS();=0A=
  i2.onload=3Dfunction() { _uVoid(); }=0A=
 }=0A=
 return;=0A=
}=0A=
function _uVoid() { return; }=0A=
function _uCInfo() {=0A=
 if (!_ucto || _ucto=3D=3D"") { _ucto=3D"15768000"; }=0A=
 if (!_uVG()) return;=0A=
 var =
c=3D"",t=3D"-",t2=3D"-",t3=3D"-",o=3D0,cs=3D0,cn=3D0,i=3D0,z=3D"-",s=3D""=
;=0A=
 if (_uanchor && _udlh && _udlh!=3D"") s=3D_udlh+"&";=0A=
 s+=3D_udl.search;=0A=
 var x=3Dnew Date(_udt.getTime()+(_ucto*1000));=0A=
 var dc=3D_ubd.cookie;=0A=
 x=3D" expires=3D"+x.toGMTString()+";";=0A=
 if (_ulink && !_ubl) {=0A=
  z=3D_uUES(_uGC(s,"__utmz=3D","&"));=0A=
  if (z!=3D"-" && z.indexOf(";")=3D=3D-1) { =
_ubd.cookie=3D"__utmz=3D"+z+"; path=3D"+_utcp+";"+x+_udo; return ""; }=0A=
 }=0A=
 z=3Ddc.indexOf("__utmz=3D"+_udh);=0A=
 if (z>-1) { z=3D_uGC(dc,"__utmz=3D"+_udh,";"); }=0A=
 else { z=3D"-"; }=0A=
 t=3D_uGC(s,_ucid+"=3D","&");=0A=
 t2=3D_uGC(s,_ucsr+"=3D","&");=0A=
 t3=3D_uGC(s,"gclid=3D","&");=0A=
 if ((t!=3D"-" && t!=3D"") || (t2!=3D"-" && t2!=3D"") || (t3!=3D"-" && =
t3!=3D"")) {=0A=
  if (t!=3D"-" && t!=3D"") c+=3D"utmcid=3D"+_uEC(t);=0A=
  if (t2!=3D"-" && t2!=3D"") { if (c !=3D "") c+=3D"|"; =
c+=3D"utmcsr=3D"+_uEC(t2); }=0A=
  if (t3!=3D"-" && t3!=3D"") { if (c !=3D "") c+=3D"|"; =
c+=3D"utmgclid=3D"+_uEC(t3); }=0A=
  t=3D_uGC(s,_uccn+"=3D","&");=0A=
  if (t!=3D"-" && t!=3D"") c+=3D"|utmccn=3D"+_uEC(t);=0A=
  else c+=3D"|utmccn=3D(not+set)";=0A=
  t=3D_uGC(s,_ucmd+"=3D","&");=0A=
  if (t!=3D"-" && t!=3D"") c+=3D"|utmcmd=3D"+_uEC(t);=0A=
  else  c+=3D"|utmcmd=3D(not+set)";=0A=
  t=3D_uGC(s,_uctr+"=3D","&");=0A=
  if (t!=3D"-" && t!=3D"") c+=3D"|utmctr=3D"+_uEC(t);=0A=
  else { t=3D_uOrg(1); if (t!=3D"-" && t!=3D"") =
c+=3D"|utmctr=3D"+_uEC(t); }=0A=
  t=3D_uGC(s,_ucct+"=3D","&");=0A=
  if (t!=3D"-" && t!=3D"") c+=3D"|utmcct=3D"+_uEC(t);=0A=
  t=3D_uGC(s,_ucno+"=3D","&");=0A=
  if (t=3D=3D"1") o=3D1;=0A=
  if (z!=3D"-" && o=3D=3D1) return "";=0A=
 }=0A=
 if (c=3D=3D"-" || c=3D=3D"") { c=3D_uOrg(); if (z!=3D"-" && =
_ufno=3D=3D1)  return ""; }=0A=
 if (c=3D=3D"-" || c=3D=3D"") { if (_ufns=3D=3D1)  c=3D_uRef(); if =
(z!=3D"-" && _ufno=3D=3D1)  return ""; }=0A=
 if (c=3D=3D"-" || c=3D=3D"") {=0A=
  if (z=3D=3D"-" && _ufns=3D=3D1) { =
c=3D"utmccn=3D(direct)|utmcsr=3D(direct)|utmcmd=3D(none)"; }=0A=
  if (c=3D=3D"-" || c=3D=3D"") return "";=0A=
 }=0A=
 if (z!=3D"-") {=0A=
  i=3Dz.indexOf(".");=0A=
  if (i>-1) i=3Dz.indexOf(".",i+1);=0A=
  if (i>-1) i=3Dz.indexOf(".",i+1);=0A=
  if (i>-1) i=3Dz.indexOf(".",i+1);=0A=
  t=3Dz.substring(i+1,z.length);=0A=
  if (t.toLowerCase()=3D=3Dc.toLowerCase()) cs=3D1;=0A=
  t=3Dz.substring(0,i);=0A=
  if ((i=3Dt.lastIndexOf(".")) > -1) {=0A=
   t=3Dt.substring(i+1,t.length);=0A=
   cn=3D(t*1);=0A=
  }=0A=
 }=0A=
 if (cs=3D=3D0 || _ufns=3D=3D1) {=0A=
  t=3D_uGC(dc,"__utma=3D"+_udh,";");=0A=
  if ((i=3Dt.lastIndexOf(".")) > 9) {=0A=
   _uns=3Dt.substring(i+1,t.length);=0A=
   _uns=3D(_uns*1);=0A=
  }=0A=
  cn++;=0A=
  if (_uns=3D=3D0) _uns=3D1;=0A=
  _ubd.cookie=3D"__utmz=3D"+_udh+"."+_ust+"."+_uns+"."+cn+"."+c+"; =
path=3D"+_utcp+"; "+x+_udo;=0A=
 }=0A=
 if (cs=3D=3D0 || _ufns=3D=3D1) return "&utmcn=3D1";=0A=
 else return "&utmcr=3D1";=0A=
}=0A=
function _uRef() {=0A=
 if (_ur=3D=3D"0" || _ur=3D=3D"" || _ur=3D=3D"-") return "";=0A=
 var i=3D0,h,k,n;=0A=
 if ((i=3D_ur.indexOf("://"))<0) return "";=0A=
 h=3D_ur.substring(i+3,_ur.length);=0A=
 if (h.indexOf("/") > -1) {=0A=
  k=3Dh.substring(h.indexOf("/"),h.length);=0A=
  if (k.indexOf("?") > -1) k=3Dk.substring(0,k.indexOf("?"));=0A=
  h=3Dh.substring(0,h.indexOf("/"));=0A=
 }=0A=
 h=3Dh.toLowerCase();=0A=
 n=3Dh;=0A=
 if ((i=3Dn.indexOf(":")) > -1) n=3Dn.substring(0,i);=0A=
 for (var ii=3D0;ii<_uRno.length;ii++) {=0A=
  if ((i=3Dn.indexOf(_uRno[ii].toLowerCase())) > -1 && =
n.length=3D=3D(i+_uRno[ii].length)) { _ufno=3D1; break; }=0A=
 }=0A=
 if (h.indexOf("www.")=3D=3D0) h=3Dh.substring(4,h.length);=0A=
 return =
"utmccn=3D(referral)|utmcsr=3D"+_uEC(h)+"|"+"utmcct=3D"+_uEC(k)+"|utmcmd=3D=
referral";=0A=
}=0A=
function _uOrg(t) {=0A=
 if (_ur=3D=3D"0" || _ur=3D=3D"" || _ur=3D=3D"-") return "";=0A=
 var i=3D0,h,k;=0A=
 if ((i=3D_ur.indexOf("://")) < 0) return "";=0A=
 h=3D_ur.substring(i+3,_ur.length);=0A=
 if (h.indexOf("/") > -1) {=0A=
  h=3Dh.substring(0,h.indexOf("/"));=0A=
 }=0A=
 for (var ii=3D0;ii<_uOsr.length;ii++) {=0A=
  if (h.toLowerCase().indexOf(_uOsr[ii].toLowerCase()) > -1) {=0A=
   if ((i=3D_ur.indexOf("?"+_uOkw[ii]+"=3D")) > -1 || =
(i=3D_ur.indexOf("&"+_uOkw[ii]+"=3D")) > -1) {=0A=
    k=3D_ur.substring(i+_uOkw[ii].length+2,_ur.length);=0A=
    if ((i=3Dk.indexOf("&")) > -1) k=3Dk.substring(0,i);=0A=
    for (var yy=3D0;yy<_uOno.length;yy++) {=0A=
     if (_uOno[yy].toLowerCase()=3D=3Dk.toLowerCase()) { _ufno=3D1; =
break; }=0A=
    }=0A=
    if (t) return _uEC(k);=0A=
    else return =
"utmccn=3D(organic)|utmcsr=3D"+_uEC(_uOsr[ii])+"|"+"utmctr=3D"+_uEC(k)+"|=
utmcmd=3Dorganic";=0A=
   }=0A=
  }=0A=
 }=0A=
 return "";=0A=
}=0A=
function _uBInfo() {=0A=
 var sr=3D"-",sc=3D"-",ul=3D"-",fl=3D"-",cs=3D"-",je=3D1;=0A=
 var n=3Dnavigator;=0A=
 if (self.screen) {=0A=
  sr=3Dscreen.width+"x"+screen.height;=0A=
  sc=3Dscreen.colorDepth+"-bit";=0A=
 } else if (self.java) {=0A=
  var j=3Djava.awt.Toolkit.getDefaultToolkit();=0A=
  var s=3Dj.getScreenSize();=0A=
  sr=3Ds.width+"x"+s.height;=0A=
 }=0A=
 if (n.language) { ul=3Dn.language.toLowerCase(); }=0A=
 else if (n.browserLanguage) { ul=3Dn.browserLanguage.toLowerCase(); }=0A=
 je=3Dn.javaEnabled()?1:0;=0A=
 if (_uflash) fl=3D_uFlash();=0A=
 if (_ubd.characterSet) cs=3D_uES(_ubd.characterSet);=0A=
 else if (_ubd.charset) cs=3D_uES(_ubd.charset);=0A=
 return =
"&utmcs=3D"+cs+"&utmsr=3D"+sr+"&utmsc=3D"+sc+"&utmul=3D"+ul+"&utmje=3D"+j=
e+"&utmfl=3D"+fl;=0A=
}=0A=
function __utmSetTrans() {=0A=
 var e;=0A=
 if (_ubd.getElementById) e=3D_ubd.getElementById("utmtrans");=0A=
 else if (_ubd.utmform && _ubd.utmform.utmtrans) =
e=3D_ubd.utmform.utmtrans;=0A=
 if (!e) return;=0A=
 var l=3De.value.split("UTM:");=0A=
 var i,i2,c;=0A=
 if (_userv=3D=3D0 || _userv=3D=3D2) i=3Dnew Array();=0A=
 if (_userv=3D=3D1 || _userv=3D=3D2) { i2=3Dnew Array(); c=3D_uGCS(); }=0A=
=0A=
 for (var ii=3D0;ii<l.length;ii++) {=0A=
  l[ii]=3D_uTrim(l[ii]);=0A=
  if (l[ii].charAt(0)!=3D'T' && l[ii].charAt(0)!=3D'I') continue;=0A=
  var r=3DMath.round(Math.random()*2147483647);=0A=
  if (!_utsp || _utsp=3D=3D"") _utsp=3D"|";=0A=
  var f=3Dl[ii].split(_utsp),s=3D"";=0A=
  if (f[0].charAt(0)=3D=3D'T') {=0A=
   s=3D"&utmt=3Dtran"+"&utmn=3D"+r;=0A=
   f[1]=3D_uTrim(f[1]); if(f[1]&&f[1]!=3D"") =
s+=3D"&utmtid=3D"+_uES(f[1]);=0A=
   f[2]=3D_uTrim(f[2]); if(f[2]&&f[2]!=3D"") =
s+=3D"&utmtst=3D"+_uES(f[2]);=0A=
   f[3]=3D_uTrim(f[3]); if(f[3]&&f[3]!=3D"") =
s+=3D"&utmtto=3D"+_uES(f[3]);=0A=
   f[4]=3D_uTrim(f[4]); if(f[4]&&f[4]!=3D"") =
s+=3D"&utmttx=3D"+_uES(f[4]);=0A=
   f[5]=3D_uTrim(f[5]); if(f[5]&&f[5]!=3D"") =
s+=3D"&utmtsp=3D"+_uES(f[5]);=0A=
   f[6]=3D_uTrim(f[6]); if(f[6]&&f[6]!=3D"") =
s+=3D"&utmtci=3D"+_uES(f[6]);=0A=
   f[7]=3D_uTrim(f[7]); if(f[7]&&f[7]!=3D"") =
s+=3D"&utmtrg=3D"+_uES(f[7]);=0A=
   f[8]=3D_uTrim(f[8]); if(f[8]&&f[8]!=3D"") =
s+=3D"&utmtco=3D"+_uES(f[8]);=0A=
  } else {=0A=
   s=3D"&utmt=3Ditem"+"&utmn=3D"+r;=0A=
   f[1]=3D_uTrim(f[1]); if(f[1]&&f[1]!=3D"") =
s+=3D"&utmtid=3D"+_uES(f[1]);=0A=
   f[2]=3D_uTrim(f[2]); if(f[2]&&f[2]!=3D"") =
s+=3D"&utmipc=3D"+_uES(f[2]);=0A=
   f[3]=3D_uTrim(f[3]); if(f[3]&&f[3]!=3D"") =
s+=3D"&utmipn=3D"+_uES(f[3]);=0A=
   f[4]=3D_uTrim(f[4]); if(f[4]&&f[4]!=3D"") =
s+=3D"&utmiva=3D"+_uES(f[4]);=0A=
   f[5]=3D_uTrim(f[5]); if(f[5]&&f[5]!=3D"") =
s+=3D"&utmipr=3D"+_uES(f[5]);=0A=
   f[6]=3D_uTrim(f[6]); if(f[6]&&f[6]!=3D"") =
s+=3D"&utmiqt=3D"+_uES(f[6]);=0A=
  }=0A=
  if ((_userv=3D=3D0 || _userv=3D=3D2) && _uSP()) {=0A=
   i[ii]=3Dnew Image(1,1);=0A=
   i[ii].src=3D_ugifpath+"?"+"utmwv=3D"+_uwv+s;=0A=
   i[ii].onload=3Dfunction() { _uVoid(); }=0A=
  }=0A=
  if ((_userv=3D=3D1 || _userv=3D=3D2) && _uSP()) {=0A=
   i2[ii]=3Dnew Image(1,1);=0A=
   =
i2[ii].src=3D_ugifpath2+"?"+"utmwv=3D"+_uwv+s+"&utmac=3D"+_uacct+"&utmcc=3D=
"+c;=0A=
   i2[ii].onload=3Dfunction() { _uVoid(); }=0A=
  }=0A=
 }=0A=
 return;=0A=
}=0A=
function _uFlash() {=0A=
 var f=3D"-",n=3Dnavigator;=0A=
 if (n.plugins && n.plugins.length) {=0A=
  for (var ii=3D0;ii<n.plugins.length;ii++) {=0A=
   if (n.plugins[ii].name.indexOf('Shockwave Flash')!=3D-1) {=0A=
    f=3Dn.plugins[ii].description.split('Shockwave Flash ')[1];=0A=
    break;=0A=
   }=0A=
  }=0A=
 } else if (window.ActiveXObject) {=0A=
  for (var ii=3D10;ii>=3D2;ii--) {=0A=
   try {=0A=
    var fl=3Deval("new =
ActiveXObject('ShockwaveFlash.ShockwaveFlash."+ii+"');");=0A=
    if (fl) { f=3Dii + '.0'; break; }=0A=
   }=0A=
   catch(e) {}=0A=
  }=0A=
 }=0A=
 return f;=0A=
}=0A=
function __utmLinker(l,h) {=0A=
 if (!_ulink) return;=0A=
 var p,k,a=3D"-",b=3D"-",c=3D"-",x=3D"-",z=3D"-",v=3D"-";=0A=
 var dc=3D_ubd.cookie;=0A=
 if (!l || l=3D=3D"") return;=0A=
 var iq =3D l.indexOf("?"); =0A=
 var ih =3D l.indexOf("#"); =0A=
 if (dc) {=0A=
  a=3D_uES(_uGC(dc,"__utma=3D"+_udh,";"));=0A=
  b=3D_uES(_uGC(dc,"__utmb=3D"+_udh,";"));=0A=
  c=3D_uES(_uGC(dc,"__utmc=3D"+_udh,";"));=0A=
  x=3D_uES(_uGC(dc,"__utmx=3D"+_udh,";"));=0A=
  z=3D_uES(_uGC(dc,"__utmz=3D"+_udh,";"));=0A=
  v=3D_uES(_uGC(dc,"__utmv=3D"+_udh,";"));=0A=
  k=3D(_uHash(a+b+c+x+z+v)*1)+(_udh*1);=0A=
  =
p=3D"__utma=3D"+a+"&__utmb=3D"+b+"&__utmc=3D"+c+"&__utmx=3D"+x+"&__utmz=3D=
"+z+"&__utmv=3D"+v+"&__utmk=3D"+k;=0A=
 }=0A=
 if (p) {=0A=
  if (h && ih>-1) return;=0A=
  if (h) { _udl.href=3Dl+"#"+p; }=0A=
  else {=0A=
   if (iq=3D=3D-1 && ih=3D=3D-1) _udl.href=3Dl+"?"+p;=0A=
   else if (ih=3D=3D-1) _udl.href=3Dl+"&"+p;=0A=
   else if (iq=3D=3D-1) =
_udl.href=3Dl.substring(0,ih-1)+"?"+p+l.substring(ih);=0A=
   else _udl.href=3Dl.substring(0,ih-1)+"&"+p+l.substring(ih);=0A=
  }=0A=
 } else { _udl.href=3Dl; }=0A=
}=0A=
function __utmLinkPost(f,h) {=0A=
 if (!_ulink) return;=0A=
 var p,k,a=3D"-",b=3D"-",c=3D"-",x=3D"-",z=3D"-",v=3D"-";=0A=
 var dc=3D_ubd.cookie;=0A=
 if (!f || !f.action) return;=0A=
 var iq =3D f.action.indexOf("?"); =0A=
 var ih =3D f.action.indexOf("#"); =0A=
 if (dc) {=0A=
  a=3D_uES(_uGC(dc,"__utma=3D"+_udh,";"));=0A=
  b=3D_uES(_uGC(dc,"__utmb=3D"+_udh,";"));=0A=
  c=3D_uES(_uGC(dc,"__utmc=3D"+_udh,";"));=0A=
  x=3D_uES(_uGC(dc,"__utmx=3D"+_udh,";"));=0A=
  z=3D_uES(_uGC(dc,"__utmz=3D"+_udh,";"));=0A=
  v=3D_uES(_uGC(dc,"__utmv=3D"+_udh,";"));=0A=
  k=3D(_uHash(a+b+c+x+z+v)*1)+(_udh*1);=0A=
  =
p=3D"__utma=3D"+a+"&__utmb=3D"+b+"&__utmc=3D"+c+"&__utmx=3D"+x+"&__utmz=3D=
"+z+"&__utmv=3D"+v+"&__utmk=3D"+k;=0A=
 }=0A=
 if (p) {=0A=
  if (h && ih>-1) return;=0A=
  if (h) { f.action+=3D"#"+p; }=0A=
  else {=0A=
   if (iq=3D=3D-1 && ih=3D=3D-1) f.action+=3D"?"+p;=0A=
   else if (ih=3D=3D-1) f.action+=3D"&"+p;=0A=
   else if (iq=3D=3D-1) =
f.action=3Df.action.substring(0,ih-1)+"?"+p+f.action.substring(ih);=0A=
   else =
f.action=3Df.action.substring(0,ih-1)+"&"+p+f.action.substring(ih);=0A=
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 }=0A=
 return;=0A=
}=0A=
function __utmSetVar(v) {=0A=
 if (!v || v=3D=3D"") return;=0A=
 if (!_udo || _udo =3D=3D "") {=0A=
  _udh=3D_uDomain();=0A=
  if (_udn && _udn!=3D"") { _udo=3D" domain=3D"+_udn+";"; }=0A=
 }=0A=
 if (!_uVG()) return;=0A=
 var r=3DMath.round(Math.random() * 2147483647);=0A=
 _ubd.cookie=3D"__utmv=3D"+_udh+"."+_uES(v)+"; path=3D"+_utcp+"; =
expires=3D"+_uNx()+";"+_udo;=0A=
 var s=3D"&utmt=3Dvar&utmn=3D"+r;=0A=
 if ((_userv=3D=3D0 || _userv=3D=3D2) && _uSP()) {=0A=
  var i=3Dnew Image(1,1);=0A=
  i.src=3D_ugifpath+"?"+"utmwv=3D"+_uwv+s;=0A=
  i.onload=3Dfunction() { _uVoid(); }=0A=
 }=0A=
 if ((_userv=3D=3D1 || _userv=3D=3D2) && _uSP()) {=0A=
  var i2=3Dnew Image(1,1);=0A=
  =
i2.src=3D_ugifpath2+"?"+"utmwv=3D"+_uwv+s+"&utmac=3D"+_uacct+"&utmcc=3D"+=
_uGCS();=0A=
  i2.onload=3Dfunction() { _uVoid(); }=0A=
 }=0A=
}=0A=
function _uGCS() {=0A=
 var t,c=3D"",dc=3D_ubd.cookie;=0A=
 if ((t=3D_uGC(dc,"__utma=3D"+_udh,";"))!=3D"-") =
c+=3D_uES("__utma=3D"+t+";+");=0A=
 if ((t=3D_uGC(dc,"__utmb=3D"+_udh,";"))!=3D"-") =
c+=3D_uES("__utmb=3D"+t+";+");=0A=
 if ((t=3D_uGC(dc,"__utmc=3D"+_udh,";"))!=3D"-") =
c+=3D_uES("__utmc=3D"+t+";+");=0A=
 if ((t=3D_uGC(dc,"__utmx=3D"+_udh,";"))!=3D"-") =
c+=3D_uES("__utmx=3D"+t+";+");=0A=
 if ((t=3D_uGC(dc,"__utmz=3D"+_udh,";"))!=3D"-") =
c+=3D_uES("__utmz=3D"+t+";+");=0A=
 if ((t=3D_uGC(dc,"__utmv=3D"+_udh,";"))!=3D"-") =
c+=3D_uES("__utmv=3D"+t+";");=0A=
 if (c.charAt(c.length-1)=3D=3D"+") c=3Dc.substring(0,c.length-1);=0A=
 return c;=0A=
}=0A=
function _uGC(l,n,s) {=0A=
 if (!l || l=3D=3D"" || !n || n=3D=3D"" || !s || s=3D=3D"") return "-";=0A=
 var i,i2,i3,c=3D"-";=0A=
 i=3Dl.indexOf(n);=0A=
 i3=3Dn.indexOf("=3D")+1;=0A=
 if (i > -1) {=0A=
  i2=3Dl.indexOf(s,i); if (i2 < 0) { i2=3Dl.length; }=0A=
  c=3Dl.substring((i+i3),i2);=0A=
 }=0A=
 return c;=0A=
}=0A=
function _uDomain() {=0A=
 if (!_udn || _udn=3D=3D"" || _udn=3D=3D"none") { _udn=3D""; return 1; }=0A=
 if (_udn=3D=3D"auto") {=0A=
  var d=3D_ubd.domain;=0A=
  if (d.substring(0,4)=3D=3D"www.") {=0A=
   d=3Dd.substring(4,d.length);=0A=
  }=0A=
  _udn=3Dd;=0A=
 }=0A=
 _udn =3D _udn.toLowerCase(); =0A=
 if (_uhash=3D=3D"off") return 1;=0A=
 return _uHash(_udn);=0A=
}=0A=
function _uHash(d) {=0A=
 if (!d || d=3D=3D"") return 1;=0A=
 var h=3D0,g=3D0;=0A=
 for (var i=3Dd.length-1;i>=3D0;i--) {=0A=
  var c=3DparseInt(d.charCodeAt(i));=0A=
  h=3D((h << 6) & 0xfffffff) + c + (c << 14);=0A=
  if ((g=3Dh & 0xfe00000)!=3D0) h=3D(h ^ (g >> 21));=0A=
 }=0A=
 return h;=0A=
}=0A=
function _uFixA(c,s,t) {=0A=
 if (!c || c=3D=3D"" || !s || s=3D=3D"" || !t || t=3D=3D"") return "-";=0A=
 var a=3D_uGC(c,"__utma=3D"+_udh,s);=0A=
 var lt=3D0,i=3D0;=0A=
 if ((i=3Da.lastIndexOf(".")) > 9) {=0A=
  _uns=3Da.substring(i+1,a.length);=0A=
  _uns=3D(_uns*1)+1;=0A=
  a=3Da.substring(0,i);=0A=
  if ((i=3Da.lastIndexOf(".")) > 7) {=0A=
   lt=3Da.substring(i+1,a.length);=0A=
   a=3Da.substring(0,i);=0A=
  }=0A=
  if ((i=3Da.lastIndexOf(".")) > 5) {=0A=
   a=3Da.substring(0,i);=0A=
  }=0A=
  a+=3D"."+lt+"."+t+"."+_uns;=0A=
 }=0A=
 return a;=0A=
}=0A=
function _uTrim(s) {=0A=
  if (!s || s=3D=3D"") return "";=0A=
  while ((s.charAt(0)=3D=3D' ') || (s.charAt(0)=3D=3D'\n') || =
(s.charAt(0,1)=3D=3D'\r')) s=3Ds.substring(1,s.length);=0A=
  while ((s.charAt(s.length-1)=3D=3D' ') || =
(s.charAt(s.length-1)=3D=3D'\n') || (s.charAt(s.length-1)=3D=3D'\r')) =
s=3Ds.substring(0,s.length-1);=0A=
  return s;=0A=
}=0A=
function _uEC(s) {=0A=
  var n=3D"";=0A=
  if (!s || s=3D=3D"") return "";=0A=
  for (var i=3D0;i<s.length;i++) {if (s.charAt(i)=3D=3D" ") n+=3D"+"; =
else n+=3Ds.charAt(i);}=0A=
  return n;=0A=
}=0A=
function __utmVisitorCode(f) {=0A=
 var r=3D0,t=3D0,i=3D0,i2=3D0,m=3D31;=0A=
 var a=3D_uGC(_ubd.cookie,"__utma=3D"+_udh,";");=0A=
 if ((i=3Da.indexOf(".",0))<0) return;=0A=
 if ((i2=3Da.indexOf(".",i+1))>0) r=3Da.substring(i+1,i2); else return =
"";  =0A=
 if ((i=3Da.indexOf(".",i2+1))>0) t=3Da.substring(i2+1,i); else return =
"";  =0A=
 if (f) {=0A=
  return r;=0A=
 } else {=0A=
  var c=3Dnew =
Array('A','B','C','D','E','F','G','H','J','K','L','M','N','P','R','S','T'=
,'U','V','W','X','Y','Z','1','2','3','4','5','6','7','8','9');=0A=
  return =
c[r>>28&m]+c[r>>23&m]+c[r>>18&m]+c[r>>13&m]+"-"+c[r>>8&m]+c[r>>3&m]+c[((r=
&7)<<2)+(t>>30&3)]+c[t>>25&m]+c[t>>20&m]+"-"+c[t>>15&m]+c[t>>10&m]+c[t>>5=
&m]+c[t&m];=0A=
 }=0A=
}=0A=
function _uIN(n) {=0A=
 if (!n) return false;=0A=
 for (var i=3D0;i<n.length;i++) {=0A=
  var c=3Dn.charAt(i);=0A=
  if ((c<"0" || c>"9") && (c!=3D".")) return false;=0A=
 }=0A=
 return true;=0A=
}=0A=
function _uES(s,u) {=0A=
 if (typeof(encodeURIComponent) =3D=3D 'function') {=0A=
  if (u) return encodeURI(s);=0A=
  else return encodeURIComponent(s);=0A=
 } else {=0A=
  return escape(s);=0A=
 }=0A=
}=0A=
function _uUES(s) {=0A=
 if (typeof(decodeURIComponent) =3D=3D 'function') {=0A=
  return decodeURIComponent(s);=0A=
 } else {=0A=
  return unescape(s);=0A=
 }=0A=
}=0A=
function _uVG() {=0A=
 if((_udn.indexOf("www.google.") =3D=3D 0 || _udn.indexOf(".google.") =
=3D=3D 0 || _udn.indexOf("google.") =3D=3D 0) && _utcp=3D=3D'/' && =
_udn.indexOf("google.org")=3D=3D-1) {=0A=
  return false;=0A=
 }=0A=
 return true;=0A=
}=0A=
function _uSP() {=0A=
 var s=3D100;=0A=
 if (_usample) s=3D_usample;=0A=
 if(s>=3D100 || s<=3D0) return true;=0A=
 return ((__utmVisitorCode(1)%10000)<(s*100));=0A=
}=0A=
function urchinPathCopy(p){=0A=
 var d=3Ddocument,nx,tx,sx,i,c,cs,t,h,o;=0A=
 cs=3Dnew Array("a","b","c","v","x","z");=0A=
 h=3D_uDomain(); if (_udn && _udn!=3D"") o=3D" domain=3D"+_udn+";";=0A=
 nx=3D_uNx()+";";=0A=
 tx=3Dnew Date(); tx.setTime(tx.getTime()+(_utimeout*1000));=0A=
 tx=3Dtx.toGMTString()+";";=0A=
 sx=3Dnew Date(); sx.setTime(sx.getTime()+(_ucto*1000));=0A=
 sx=3Dsx.toGMTString()+";";=0A=
 for (i=3D0;i<6;i++){=0A=
  t=3D" expires=3D";=0A=
  if (i=3D=3D1) t+=3Dtx; else if (i=3D=3D2) t=3D""; else if (i=3D=3D5) =
t+=3Dsx; else t+=3Dnx;=0A=
  c=3D_uGC(d.cookie,"__utm"+cs[i]+"=3D"+h,";");=0A=
  if (c!=3D"-") d.cookie=3D"__utm"+cs[i]+"=3D"+c+"; path=3D"+p+";"+t+o;=0A=
 }=0A=
}=0A=
function _uCO() {=0A=
 if (!_utk || _utk=3D=3D"" || _utk.length<10) return;=0A=
 var d=3D'www.google.com';=0A=
 if (_utk.charAt(0)=3D=3D'!') d=3D'analytics.corp.google.com';=0A=
 _ubd.cookie=3D"GASO=3D"+_utk+"; path=3D"+_utcp+";"+_udo;=0A=
 var sc=3Ddocument.createElement('script');=0A=
 sc.type=3D'text/javascript';=0A=
 sc.id=3D"_gasojs";=0A=
 =
sc.src=3D'https://'+d+'/analytics/reporting/overlay_js?gaso=3D'+_utk+'&'+=
Math.random();=0A=
 document.getElementsByTagName('head')[0].appendChild(sc);  =0A=
}=0A=
function _uGT() {=0A=
 var h=3Dlocation.hash, a;=0A=
 if (h && h!=3D"" && h.indexOf("#gaso=3D")=3D=3D0) {=0A=
  a=3D_uGC(h,"gaso=3D","&");=0A=
 } else {=0A=
  a=3D_uGC(_ubd.cookie,"GASO=3D",";");=0A=
 }=0A=
 return a;=0A=
}=0A=
var _utk=3D_uGT();=0A=
if (_utk && _utk!=3D"" && _utk.length>10) {=0A=
 if (window.addEventListener) {=0A=
  window.addEventListener('load', _uCO, false); =0A=
 } else if (window.attachEvent) { =0A=
  window.attachEvent('onload', _uCO);=0A=
 }=0A=
}=0A=
=0A=
function _uNx() {=0A=
  return (new Date((new Date()).getTime()+63072000000)).toGMTString();=0A=
}=0A=

------=_NextPart_000_0241_01C85B7B.BC962280--

